TW200813227A - Use of adipose-derived stem cells for treatment of leukodystrophies - Google Patents

Abstract

The present invention relates to a treatment of a leukodystrophy by administration of an adipose-derived stem cell. Specifically, the present invention relates to the treatment of Krabbe disease with an adipose derived stem cell differentiated to express galactocerebrosidase.

Description

200813227 IX. Description of the invention: [Prior Art] Hereditary metabolic disorders include eight identified white matter lesions: metachromatic white matter lesions, Refsum's disease, adrenal white matter lesions, Krabbe disease Phenylketonuria, Canavan disease, Pelizaeus-Merzbacher disease, and Alexander's disease 〇 hereditary demyelinating disease often tends to be in infants or It occurs in young children and its clinical process is destructive. Previously normal children lost vision, hearing, speech and walking skills in rapid progression. The prognosis is death within a few years. Crabbe disease, also known as spheroidal white matter lesions, was first described as an autosomal recessive trait in humans and has subsequently been identified in mice, dogs, cats, sheep, and rhesus monkeys (Baskin, 1989). , Lab Invest. 60:7A; Baskin, 1998, Lab Anim. Sci. 48(5): 476-482; Suzuki, 1985, Neurochem. Pathol. 3(l): 53-68; Wenger, 2000, Mol· Med Today 6(1 1): 449-451). Crabbe disease is a lysosomal reservoir caused by mutation of galactococinase, a lysosomal hydrolase that catabolizes galactoside-based neuropterin (the lipid component of myelin). Lack of galactoceretosidase (GALC) activity results in insufficient myelin formation and similar morphological changes in all species. The histopathological features of this disease are that spherical cells appear in the white matter of the sacral nervous system, which is mainly located around the blood vessels. Spherical cells are composed of macrophages that have accumulated large amounts of glycolipids in their cytoplasm. In addition to the formation of spherical cells, a large amount of myelin loss and astrocytic hyperplasia occur in the central nervous system, which affects the central and peripheral nerves. 120934.doc 200813227

system. In peripheral nerves, there is often axonal degeneration, fibrosis, and giant cell immersion>'闰 (Suzuki et al., 1983, In: Stanbury JW, JB; Fredrickson, DS; Goldstein, JI; Brown, MS, ed. The Metabolic Basis of Inherited Disease: McGraw Hill 1983: 857-880). Characteristic tubular or crystalline inclusions in the cytoplasm of cells of the brain and kidney have been identified by transmission electron microscopy (Andrews et al., 1970, Arch Pathol. 89(1): 53-55). There are also characteristic changes observed by magnetic resonance imaging (MRI) and computed tomography (CT) (Baram et al., 1986, Neurology 36(1): 111-115; Farley et al., 1992, Pediatr. Neurol· 8(6): 455-458; Barone et al., 1996 Am. J. Med. Genet 63(1): 209-217; Percy et al., 1994, Acta. Neuropathol. (Berl) 88(l):26 -32; Demaerel et al., 1991, Neuroradiology 33(4): 368-371; Sasaki et al., 1991, Pediatr. Neurol. 7(4): 283_288; Zafeiriou et al., 1996, Pediatr· Neurol. 15(3) :240-244). Typical MRI findings in people with Krabbe's disease include central and cerebral cortical atrophy, ventricular dilatation, decreased white matter volume, and focal dense lesion. Although the results vary from case to case, MRI is an extremely effective technique for mapping lesions and tracking disease progression in life. One of the most important and unique features of Kraber's disease is elevated white matter of sphingosine (galactosyl sphingosine) (Kobayashi et al., 1988, Ann. Neurol. 24(4) :517-522) Sphingosine galactoside is usually formed in oligodendrocytes by the addition of galactose to sphingosine during active myelination and in normal 120934.doc 200813227

Rapid turnover in the body (Svennerholm et al., 1980, J. Lipid Res. 21(1): 53-64); however, sphingosine galactoside degradation is impaired in patients with Krabbe disease, such patients The brain contains 10 to 100 times the normal amount of this lipid (Wenger, 2000, Mol. Med. Today 6(11): 449-451; Miyataki et al., 1972, BBRC. 48: 538-543; Svennerholm et al. , 1980, J. Lipid Res. 21(l): 53-64; Wenger, 2000, Mol. Med. Today 6(11): 449-451; Vanier et al., 1976, Adv. Exp. Med. Biol· 68 :115-126). Even if sphingosine galactoside accounts for less than 0.1% of galactoside-based neuropterin in the white matter of patients with Krabbe disease, it still significantly toxic cells (Suzuki et al., 1976, In: Volk BS, L, Edit Current Trends in Sphingolipidosis and Allied Disorders. New York: Plenum Press; Taketomi et al., 1964, Jpn J. Exp. Med. 34: 255-265). As sphingosine galactoside accumulates, myelination stops prematurely due to destruction of the oligodendrocyte glial cells (Wenger, 2000, Mol. Med. Today 6(11): 449-451). Therefore, the lack of GALC activity induces the main features of Krabbe disease, including loss of myelin, loss of glial cells, formation of spheroid cells, and sphingosine galactose production without GALC. A large amount of amines accumulate. Most clinical cases of Crabbe disease occur during infancy and rapidly progress to death during childhood. Human infants with Krabbe's disease show multiple signs of behavior, including agitation, excessive crying, loss of motor skills, hypersensitivity to external stimuli, muscle stiffness, arm and leg extensions, clenched fingers, hypotonia, blindness and deafness ( Suzuki, 1985, Neurochem. Pathol. 120934.doc 200813227 3(l): 53-68; Gullotta et al., 1979, Neuropadiatrie. 10(4): 395-400; D'Angostino et al., 1963, Arch Neurol. :82-112; Hagberg et al., 1963, J. Neurol· Neurosurg. Psychiatry 26:195-198; Suzuki et al., 1983, In: Stanbury JW, JB; Fredrickson, DS; Goldstein, JI; Brown, MS, Edit The Metabolic Basis of Inherited Disease: McGraw Hill 1983:8 57-88 0). There is phenotypic variability in seizures and clinical signs in infants with cerebral white matter lesions such as Krabbe disease. Clinical signs in human infants with B-Clinbury disease include growth arrest, progressive microcephaly, and severe growth dysplasia (Zlotogora et al., 1986, Acta. Paediatr Scand 75(2): 251-254). The diseased individual can be clearly diagnosed by confirming insufficient GALC activity in leukocytes or cultured skin fibroblasts. Prenatal diagnosis can be performed using a chorionic villus sample or cultured amniotic fluid cells. Carrier diagnosis is more problematic because obligate heterozygote has extensive enzymatic activity that overlaps with unrelated normal individuals (Wenger et al., 1993, Boston: Butterworth-

Heinemann; Wenger et al., 1991, New York: Wiley-Liss) 迄今为止 To date, treatment options for Crabbe disease have been limited. Although enzyme replacement therapy reduces the rate of disease progression, it does not prevent early death. Although transplantation of bone marrow or umbilical cord cells (including hematopoietic stem cells and mesenchymal stem cells) can reverse disease progression, these transplants often have significant consequences for graft versus host disease. It has recently been demonstrated that human adipose tissue is a rich source of stromal adult stem cells, and based on the original method described by Hauner and others, an effective method for reproducibility has been developed for use in human liposuction tissue 120934 .doc 200813227

From adult stem cells (Hauner et al., 1989, J. Clin. Invest. 84(5): 1663-1670; Hauner et al., 1988, Horm. Metab. Res. Suppl. 19: 35-39; Gimble et al. 2003, Curr· Top Dev. Biol. 58:137-160; Aust et al., 2004, Cytotherapy 6(1): 7-14; Awad et al., 2003, Tissue Eng. 9(6): 1301-1312; Awad Et al., 2004, Biomaterials 25(16): 321 1-3222; Elmslie et al., 2000, J. Clin. Psychiatry 61(3): 179_184; Delany et al., 2005, Mol. Cell Proteomics 4: 731-740; Gronthos et al., 2001, J. Cell Physiol. 189(l): 54-63; Halvorsen et al., 2000, Int. J. Obes. Relat. Metab. Disord. 24 Suppl. 4: S41-44; Halvorsen et al. , 2001, Metabolism 50(4): 407-413; Halvorsen et al., 2001, Tissue Eng. 7(6): 729.741; Hicok et al., 2004, Tissue Eng. 10(3-4): 371. Safford et al., 2002, Biochem. Biophys. Res, Commun. 294(2): 371-379; Safford et al., 2004, Exp. Neurol. 187(2): 319-328; Sen et al., 2001, J; · Cell Biochem 81(2): 312-319; Wickham et al., 2003, Clin. Orthop. (412): 196-212; Guilak et al., 200 5, J. Cell Physiol; Mitchell et al, Stem Cells online January 12, 2006: 2005-0235; Aust et al, 2004, Cytotherapy 6(1): 7-14; Halvorsen et al, 2001, Metabolism 50 (4 ): 407-413). Thus, adipose-derived adult stem cells (ASC) provide another in vitro model for the treatment of white matter lesions such as Crabbe disease (Gimble, 2003, Expert Opinion in Biological Therapy 3: 705-713; Gimble and Guilac, 2003, Current Topics in Developmental Biology, 58: 137-160), because these stems are readily available, sufficient, and do not produce a graft-versus-host immune response.

ASC can be replicated from liposuction extracts via a procedure including collagenase digestion, differential centrifugation, and expansion in culture to replicate ASC from liposuction extracts (Aust et al., 2004, Cytotherapy). 6: 1-8). Based on flow cytometry, undifferentiated human adipocytes exhibit different immunophenotypes and produce additional adipocyte-specific proteins upon induction (Aust et al., 2004, Cytotherapy 6: 1-8; 2001, J. Cell Physiol) ·, 189: 54-63; Halvorsen et al., 2001, Metabolism 50: 407-413; Sen, 2001, J. Cell. Biochem. 81: 312-319; Zuk et al., 2002, Mol. Biol· Cell. : 4279-4295). Human adipose-derived adult stem cells (huASC) show multipotential differentiation and can differentiate into adipocytes, chondrocytes, myogenic cell lines, neuronal cell lines, and osteoblasts (Aust et al., 2004, Cytotherapy). 6: 1-8; 2001, J. Cell Physiol, 189: 54-63; Halvorsen et al., 2001, Metabolism 50: 407-413; Sen, 2001, J. Cell. Biochem. 81: 312-319; Zuk Et al., 2002, Mol·Biol· Cell· 13: 4279-4295; Ashjian et al., 2003, Plast·Reconstr. Surg., 111: 1922-19231; Awad et al., 2003,

Tissue Engineering, 9: 1301-1312; Awad et al., 2004,

Biomaterials 25: 321 1-3222; Halvorsen et al., 2001, Tissue Eng., 7: 729-741; Hicok et al., 2004, Tissue Engineering 10: 371-380; Mizuno et al., 2002, Plast. Reconstr. 109: 199-209; Safford^ A 5 2002, Biochem. Biophys. Res. Commun., 294: 371-379; Safford et al., 2004, Experimental 120934.doc 200813227

Neurology, 187: 319_328; Wickham et al., 2003, Clin·

Orthop·, 412: 196-212; Winter et al., 2003, Arthritis

Rheum, 48: 418-429; Zuk et al., 2001, Tissue Eng. 7: 211-28). Dexamethasone, insulin, isobutylmethylxanthine and dexamethasone, as evidenced by the fact that 30% to 80% of the cells accumulate lipid vacuoles (using Neu Red Ο dye to stain neutral lipids) based on flow cytometry In the presence of thiazolidinediones, undifferentiated human fat cells undergo fat formation (Halvorsen et al., 2001, Metabolism 50: 407-413; Sen et al., 2001, J. Cell Biochem, 81: 312-319). . There is still a need in the art for methods to identify and characterize differentiated ASCs. The present invention addresses this need by providing a means of identifying and characterizing ASCs that exhibit GALC, which are suitable for treating Crabbe disease. SUMMARY OF THE INVENTION The present invention encompasses a method of treating at least one symptom of a white matter lesion in a mammal. The mammal is preferably a primate. The mammal is more preferably a monkey. The mammal is best human. The method comprises administering an isolated adipose derived stem cell (ASC) exhibiting non-immunogenic characteristics to a mammal. ASC preferably exhibits galactocerebrosidase. In one aspect, the white matter lesion is selected from the group consisting of: Crabbe disease, adrenal white matter lesions/adrenal myelin neuropathy, Aicardi-Goutieres syndrome, Alexander Symptoms, children with ataxia (CACH) with reduced central nervous system myelination (CACH), autosomal dominant cerebral artery disease (CADASIL) with subcortical infarction and leukoencephalopathy, carrefour disease, cerebral palsy Neoplasia, metachromatic brain 120934.doc •12- 200813227 White shell disease, neonatal adrenal white matter lesion, ovarian white matter lesion syndrome, chronic childhood brain sclerosis, Lefsum's disease, Nap (van der Knaap syndrome and ZeUweger syndrome. The white matter lesion is preferably Crabbe disease. In another aspect, galactocerebrosidase is expressed in ASC in an amount effective to reduce the amount of sphingosine galactosidase in the white matter of the mammal. In another aspect, the galactocerebrosidase is self-differentiating ASC in an amount effective to reduce the sphingosine galactosidase content in the white matter of the mammal. The present invention also includes at least one symptom for treating white matter lesions. The method wherein the symptom is selected from the group consisting of axonal degeneration, fibrosis, macrophage infiltration, astrocytosis, myelination, agitation, excessive crying, loss of motor skills, external stimulation Hypersensitivity, muscle stiffness, stretching of arms and legs, gripping fingers, decreased tension, blindness and deafness. In one aspect, the ASC is administered intravenously to the mammal. For mammals, ASC can be allogeneic or autologous. In another aspect, the ASC further comprises a biocompatible matrix. The biocompatible matrix is selected from the group consisting of alginic acid, agarose, fibrin, collagen, laminin, fibronectin, glycosaminoglycan (£17〇). 03&]1^11〇§13^&11), hyaluronic acid, heparin sulfate, chondroitin sulfate A, dermatan sulfate and bone matrix are in a state where ASC is incubated in vitro before being administered to a mammal. 120934.doc -13- 200813227 Time but does not induce differentiation. The invention also encompasses a method of identifying ASCs that exhibit galactocerebrosidase in a population of adipose tissue derived cells. The method comprises providing a matrix specific for galactosporin, wherein the matrix is degraded when galactosidase is present in Asc, thereby identifying the cell population; ASC 〇. In one aspect, the matrix is galactosyl sphingosine or galactosamine-based neuropterin. In another aspect, the ASC is differentiated into cells exhibiting at least one characteristic selected from the group consisting of: white blood cells, fibroblasts, chondrocytes, osteoblasts, Schwann cells, oligos Glial glial cells and neurons. The invention also encompasses a method of increasing the level of galactose brain enzyme in a tissue or mammal. The method comprises administering a divided ASC exhibiting non-immunogenic characteristics to a mammal, wherein the ASC differentiates into cells expressing galactocerebrosidase in vivo or ex vivo. In another aspect, the ASC is differentiated into cells exhibiting at least one characteristic selected from the group consisting of: white blood cells, fibroblasts, cells, chondrocytes, osteoblasts, Schwann cells, oligodendrocytes Glial cells, and neurons. The invention also encompasses isolated ASCs that exhibit non-immunogenic features, wherein ASC exhibits galactocretosidase and is identified by providing a matrix specific for galactocerebrosidase to a population of cells, wherein half of the ASC is present. The matrix is degraded by galactoceretosidase, thereby identifying ASC in the cell population. The isolated ASC is preferably human cells by 120934.doc -14· 200813227. In another aspect, the invention provides a substantially homogeneous isolated ASC population, wherein the isolated ASC exhibits a non-immunogenic profile. In one aspect, the ASC exhibits galactoceretosidase and is identified by providing a matrix specific for galactocerebrosidase to the cell population, wherein the matrix degrades when a galactoceretosidase is present in the ASC Thereby identifying ASCs in the cell population. • In another aspect, the isolated ASC is genetically modified to express galactose brain enzymes. In another aspect, the isolated ASC is transfected with a vector that exhibits galactoceretosidase. In some aspects, galactocerebrosidase is derived from humans, wolves, mice or rats. [Embodiment] ASC has great potential in transplantation and disease treatment. The present invention provides methods and compositions for obtaining ASCs that differentiate to exhibit at least one characteristic of non-fatty tissue derived cells. In one embodiment, Asc is differentiated into cells that exhibit galactoceretosidase. The invention further comprises a method of identifying differentiated adipose-derived stem cells exhibiting galactoceretosidase in a population of adipose tissue-derived cells. The invention further provides a method of treating leukocyte lesions in a mammal by administering isolated adipose derived stem cells. Mammals are better than humans. The white matter lesion is preferably Crabbe disease. THAT DEFINITIONS Unless otherwise defined, all of the techniques used herein are generally understood to have the meaning commonly understood by those skilled in the art. Generally, the terminology and cell culture, molecular genetics, organic chemistry, nucleic acid chemistry, and hybridization laboratory procedures are well known and commonly used in the art. 120934.doc -15. 200813227 Procedure.

Nucleic acid and peptide synthesis use standard techniques. The techniques and procedures provided herein are generally implemented in accordance with the techniques and conventional methods in various comprehensive literature (e.g., Sambrook and Russell, 2001, Molecular Cloning, A).

Laboratory Approach, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY and Ausubel et al., 2002, Current

Protocols in Molecular Biology, John Wiley & Sons, New York, NY). Abbreviations of the invention are used throughout the application. ASC: adipose-derived adult stem cells; BMI: body mass index; 2D-PAGE: two-dimensional polyacrylamide gel electrophoresis; D: differentiation; DMEM: Dulbecco's Modified Eagles Medium; hu: human ; PBS: phosphate buffered saline; pr: primate; U: undifferentiated. The following terms as used herein have the meaning associated with this section.

The article "a" as used herein refers to one or more (i.e., at least one) of the grammatical objectives of the article. By way of example, "an element" means one element or more. The term π about π is known to the average person and varies to some extent based on the context in which it is used. The term "adipose tissue-derived cells" refers to cells derived from adipose tissue. The starting cell population isolated from adipose tissue is a heterogeneous population of cells including, but not limited to, stromal vascular fraction (SVF) fine 120934 .doc -16- 200813227. Moon fat refers to any adipose tissue. The adipose tissue may be brown or white adipose tissue. The adipose tissue is preferably subcutaneous white adipose tissue. The adipose tissue may be derived from any organism having adipose tissue. The adipose tissue is preferably derived from the Nanli animal, preferably from humans. A convenient source of human adipose tissue, A is derived from adipose tissue of liposuction surgery. However, adipose tissue. Source or adipose tissue separation method for the present invention It is not critical. _ The term "fat-derived adult stem cells (ASC) 1 as used herein refers to stromal cells derived from adipose tissue, which can be used as a variety of different cell types (such as, but not limited to) adipocytes. Stem cell precursors such as bone cells, chondrocytes, muscles, and neurons/glial cell lines. Adipose-derived adult stem cells constitute a subpopulation derived from adipose tissue and can be isolated from other components of adipose tissue using standard culture procedures or other methods not disclosed herein. Further, cell surface markers disclosed herein can be used to make fat. The source adult stem cells are isolated from the cell mixture. _ The term ''fat cells'' as used herein refers to any type of adipose tissue, including undifferentiated adipose-derived adult stem cells and differentiated fat-derived adult bodies. Stem cell. ^ The term "allogan" as used herein is intended to mean any substance derived from different mammals of the same species. As used herein, the term "autologous, is intended to refer to Any substance of the same individual as the individual introduced later. The term "phenotypic characteristic" as used herein means at least one of the following characteristics: morphological appearance, expression of specific protein, type of dyeing or use 120934.doc -17- 200813227 The ability to dye a substance. The term "applicator" as used herein means any device for administering the compound of the invention: a composition, including but not limited to a hypodermic syringe, a pipette, and the like. It should be considered as used herein. "The central nervous system and / or spinal cord. In some cases, the term is also 0

'Including the brain of a mammal, including the eye and the eye used in this article,' means that the end point of the mutation has been reached so that the cell has been highly specific to the environment and/or function. Or adaptive cells. Generally, differentiated cells are characterized by the expression of a base encoding a protein associated with differentiation in the cell. For example, GALC in white blood cells is a typical example of the finally differentiated white blood cells. "Differentiation medium" means stem cells, adipose tissue-derived stromal cells, embryonic stem cells, ES-like cells, MSCs, neurospheres, nsc^ other progenitor cells that contain additives or lack of additives such that they are not fully differentiated. A cell growth medium that develops into cells having some or all of the characteristics of differentiated cells when cultured in culture. When the cell is as used herein, the term is "differentiating," the cell is in the process of differentiation. "Differentiated fat-derived adult stem cells "Different adipose-derived adult stem cells as defined herein as isolated from any adipose tissue." Undifferentiated Adipose-derived adult stem cells are cells that have been isolated from adipose tissue and cultured to promote proliferation, but which do not have detectable expression proteins or other phenotypic characteristics indicative of biospecificity and/or adaptability. 120934.doc - 18-200813227 'Disease' is the state of animal health in which the animal cannot maintain constancy and in which the animal health continues to deteriorate if the disease does not improve. In contrast, an animal's condition is a state of health in which the animal can remain conserved but in which the animal's state of health is worse than the state of health in the absence of the condition. If left untreated, the condition does not necessarily cause further deterioration in the animal's health. The term "a disease, disorder or condition of the central nervous system as used herein," refers to one of the results of a mutation caused by a gene mutation in a gene expressed by a cell of the central nervous system or a cell affecting the central nervous system. A disease, disorder, or condition characterized by structural and/or functional abnormalities of the central nervous system, such as defective myelin. These genetic defects may be mutated genes, non-functional genes, or repressed genes in cells of the central nervous system. The term 'endogenous' as used herein refers to any substance produced from within an organism, cell or system, or within an organism, cell or system. ''Exogenous'' means an organism, cell Or any substance produced outside the system or outside the organism, cell or system. "isolated cell" refers to a cell that is isolated from other components and/or cells of the tissue or mammal that naturally accompany the isolated cell. As used herein, "graft" refers to transplantation to an individual that is often used instead, Correcting or overcoming defective cells, tissues or organs. The graft may additionally comprise a scaffold. The tissue or organ may be composed of cells derived from the same individual; herein the graft may be used interchangeably with the following terms: "autograft" And "autologous grafts." Grafts comprising cells from genetically distinct individuals of the same species may be used interchangeably with the following terms: "allografts" and 'allografts. Grafts from the individual to the individual's identical twins 120934.doc -19- 200813227 (identical twin) are referred to herein as "isogenic grafts" or "homologous grafts"" xenografts " or "Allogeneic graft" refers to a graft from an individual to another individual of a different species. As used herein, the "immune phenotype" refers to a cellular phenotype based on the surface protein profile of the cell. The term "white matter lesion" as used herein refers to a disease or condition characterized by progressive degeneration of white matter due to incomplete growth or development of the myelin sheath (which acts as a protective layer of the insulator surrounding the nerve fibers). "White matter lesions" is a group of disorders caused by genetic defects in the metabolic process of myelin production or a chemical component thereof. Each of the white matter lesions is the result of genetic defects in one of these chemicals. Specific white matter lesions include (but are not limited to) Crabbe disease, adrenal white matter lesions / adrenal myelin neuropathy, Aicardi-Goutieres syndrome, Alexandria, with diffuse central nervous system Childhood ataxia (CACH) with reduced sheath formation, autosomal dominant cerebral artery disease (CADASIL) with subcortical infarction and leukoencephalopathy, carrefour disease, cerebral palsy xanthomas, metachromatic white matter lesions , neonatal adrenal white matter lesions, ovarian white matter lesions syndrome, chronic childhood brain sclerosis, Lefsum's disease, Nap (Van der Knaa) p) Syndrome and Zellweger syndrome. "Polypeptide" means a composition consisting of an amino acid residue, a related naturally occurring structural variant, and a synthetic non-naturally occurring analog linked via a peptide bond. Polymers, their associated naturally occurring structural variants, and synthetic non-naturally occurring analogs. For example, synthetic polypeptides can be synthesized using automated polypeptide synthesizers. 120934.doc -20- 200813227 Terminology "Precursor Cell" , "source ancestors" and "stem cells" are used interchangeably in this technology and herein and refer to progenitor cells that are pluripotent or lineage unrestricted, potentially capable of performing an unlimited number of mitosis to renew themselves Or produce progeny cells that will differentiate into the desired cell type. Compared to pluripotent stem cells, lineage-restricted progenitor cells are generally considered to be unable to produce a large number of cell types with different phenotypes. Instead, progenitor cells produce one or possibly Two lineage-restricted cell types. The term "multi-energy" or "multi-directional differentiation potential" as used herein refers to stem cell differentiation The ability to form more than one cell type. The term "late passage adipose tissue-derived stromal cells, as used herein, refers to cells that exhibit less immunogenicity when compared to early passage cells. The immunogenicity of the adipose tissue-derived stromal cells corresponds to the number of passages. The cells are preferably subcultured to at least the second generation, more preferably at least the third generation, and most preferably at least the fourth generation. The term "protein" generally refers to a large polypeptide. The term "peptide," generally refers to a short polypeptide. "Therapeutic" treatment is to reduce or eliminate such signs and/or reduce or reduce the frequency, duration, and intensity of the symptoms to patients exhibiting pathological signs. The treatment of the purpose. The "therapeutically effective amount" of the compound is an amount of the compound sufficient to provide a beneficial effect to the subject to which the compound is administered. As used herein, a therapeutically effective amount " is also sufficient to provide a beneficial bolus amount to a subject to whom the cells are administered. The term "treatment" as used herein means reducing the frequency of a disease or condition, i.e., reducing the frequency or symptoms of a disease or condition experienced by an animal. 120934.doc 21-200813227 The frequency of a symptom. ''Herb'' refers to any substance derived from a mammal of a different species. DESCRIPTION The present invention relates to the discovery that ASC can be used to treat white matter lesions, preferably Klapgar's disease. The invention also relates to the discovery that adipose derived stem cells can differentiate into cells expressing galactoceretosidase (GALC). The ability of ASC to treat Krabbe disease can be identified in vivo or in vitro in a variety of animal model systems including, but not limited to, monkeys and canines. The invention also facilitates the identification of such GALC-expressing cells from heterogeneously differentiated, undifferentiated or mixed adipocyte populations. Cells that exhibit GALC include, but are not limited to, white blood cells, fibroblasts, chondrocytes, osteoblasts, Schwann cells, oligodendrocytes or neurons. The advantage of using ASC for this purpose is that Asc is sufficient, readily available, and does not produce a graft-versus-host immune response. The subject may be a mammal, preferably a human or a monkey. Method for isolation and differentiation of L adipose stem cells (ASC) In one aspect, any animal (preferably human or monkey) can be used.

120934.doc is preferably separated from the same subject by the fat group. Or the organization to which it is administered may be the same species -22-200813227. In a separation method of ASCs, the concentration is between 25·〇ι% to 0.5% at a temperature between 25 ° C and 50 ° C, preferably between 33 ° C and 40 ° C, at an optimum temperature of 37 ° C. Between 4% and 0.2%, preferably between about 4% and 0.2%, the optimum concentration of collagenase is between 0.01% and 0.5%, preferably 0.04%, and most preferably about 〇_2%. Trypsin and/or a concentration of 0.5 ng/ml to 10 ng/ml of lysate (dispase) and/or an effective concentration of hyaluronidase or deoxyribonuclease (DNase) and a concentration of about 〇·〇ι mM to 2.0 mM, Approximately 1 to 0 mM, preferably 〇53 mM ethylenediaminetetraacetic acid (edTA) is used to treat adipose tissue for 10 minutes to 3 hours, preferably 30 minutes to 1 hour, preferably 4 5 minute. The cells are passed through a transition network of nylon or cheesecloth from 20 microns to 8 microns, more preferably from 40 microns to 4 microns, and most preferably at 70 microns. The cells are then subjected to differential centrifugation directly in culture medium or via Ficoll or perC〇ll or other particle gradients. It is from 4 ° C to 50 ° C, preferably from 20 ° C to 40. (:, preferably at a temperature of about 25 ° C, centrifuge the cells at a speed of 1 〇〇 to 3 〇〇〇 X g, preferably 2 〇〇 to 15 〇〇χ g, optimal 500 X g for 1 minute to 1 hour Preferably, 2 minutes to 15 minutes, preferably 5 minutes. After separation, the ASCs are incubated in the stromal cell culture medium in the culture device for a period of time or until the cells reach a confluency, and then the cells are transferred to another culture device. The culture device may be any culture device commonly used for in vitro culture of cells. The degree of full disk is preferably greater than 70% before the cells are transferred to another culture device. The degree of full plate is better than 9〇%. Any time outside the culture of the cells. The stromal cell culture medium can be replaced at any time during the culture of the ASCs. The stromal cell culture medium is preferably replaced every 3 120934.doc -23-200813227 to 4 days. Then eight 8 (^, It can be used immediately or cryopreserved for later use. ASCs can be harvested by trypsinization, EDTA treatment, or any other procedure used to harvest cells from the culture device. Cells. Cell cultures generally refer to cells obtained from living organisms and then grown under controlled conditions. Primary cell cultures are cells, tissues or organs obtained directly from the organism and prior to the first subculture. Cultures. Cells are expanded in culture when placed in growth media under conditions that facilitate cell growth and/or division, resulting in a larger population of cells. When cells are expanded in culture, usually by The amount of time required to multiply the number of cells (also known as doubling time) measures the rate of cell proliferation. Each round of subculture is called a subculture. Therefore, when the cells are subcultured, they are said to have been subcultured. Or the cell line is sometimes referred to or characterized as the number of times it has been subcultured. For example, a population of cultured cells that have been subcultured ten times may be referred to as a P10 culture. Primary culture (ie, self-tissue separation of fine Φ cells) The latter first culture) is denoted as P0. After the first subculture, the cells are described as secondary cultures (P1 or subculture 1). After the second subculture, the cells are referred to as tertiary cultures ( P2 or subsequent 2) Those skilled in the art should be aware that there may be many population doublings during the passage; therefore, the population of cultures is greater than the number of sub-algebras. Cell expansion during the time between generations (ie, population doublings) Depending on a number of factors, including but not limited to, the seeding density, the time between the substrate, the medium, and the subculture. In another aspect, the invention provides non-immunogenic features and exhibits galactoceretosidase (GALC) Isolation of ASC. Accordingly, the present invention encompasses a method of treating 120934.doc -24 - 200813227 ASC to induce differentiation of ASC into cells expressing GALC. In one embodiment, the cells expressing GALC are leukocytes, fibroblasts, Chondrocytes, osteoblasts, Schwann cells, oligodendrocytes or neurons. In another aspect, the ASC additionally comprises a biocompatible matrix. The biocompatible matrix is preferably calcium alginate, agarose, fibrin, collagen, laminin, fibronectin, glycosaminoglycan, hyaluronic acid, heparin sulfate, chondroitin sulfate A, dermatan sulfate, and bone matrix. gelatin. Although the invention is not limited by any theory of operation, it is believed to contain serum, embryo extracts (preferably non-human embryo extracts), purified or recombinant growth factors, cytokine in a two- or three-dimensional microenvironment. The medium of the hormone and/or chemical agent pre-treatment of the preadipocytes induces differentiation. The immunophenotype of ASC changes gradually depending on the culture procedure (i.e., following algebra). Following the plasticity of human ASC and subsequent amplification, a cell population that is relatively homogeneous compared to the heterogeneity of the original stromal vascular layer is enriched with cells that exhibit "stromal" immune expression. ASC also exhibits stem cell-related markers including, but not limited to, human multidrug transporters (ABCG2) and aldehyde dehydrogenase (ALDH). The immunophenotype of ASC can be used as a unique identification marker for ASC. Specific cell subpopulations can also be isolated from a mixed cell population derived from adipose tissue using unique cell surface markers on the relevant cells. Cell surface markers of BMSC have been characterized (e.g., Meinel et al, 2004, Ann Biomm. Eng. 32: 112-122 and Meinel et al, 2004, J Biomed Mater Res. A. 71: 25-35-4, each The full text is incorporated herein). As exemplified in the present invention, huASC shows differentiation potential comparable to BMSC, including the following cell surface markers 120934.doc -25-200813227 Notes: CD9, CD10, CD13, CD29, CD44, CD49d, CD54, CD55, CD59 , CD71, CD73, CD90, CD105, CD106, CD 146, CD166, α-smooth muscle actin, collagen type 1, collagen type III, HLA-ABC, nestin (nestin), osteopontin, osteopontin, Osteonectin and vimentin. Those skilled in the art will appreciate that antibodies specific for cell surface markers can bind to a physical support (i.e., streptavidin (81: generation 91 & ¥1 (1111) beads) and thus bind and separate An ASC having such a specific cell surface marker. Examples of antibodies that specifically bind to ASC include, but are not limited to, anti-ABCG2 antibodies. After binding, for example, magnetic beads (including but not limited to) Dynabeads 8 (Dynal) can be used. Biotech, Brown Deer, WI)) magnetic separation to separate the bound ASC from the remaining cells. Further use of Dynabeads®, MACS isolation reagent (Miltenyi Biotec, Auburn, CA) to remove ASC from the mixed cell population. Or, ASC The immunophenotype allows for sorting using a flow cytometry-based cell sorter. As a result of the separation step or cell sorting, an ASC-rich population can be obtained. The ASC population is preferably a purified cell population. The isolated ASC is cultured and expanded in vitro using the methods disclosed herein or by conventional methods. Unrestricted primary medium suitable for use in the methods of the invention Examples include the minimum essential medium Eagle (Minimum Essential Medium Eagle), ADC-1, LPM (no bovine serum albumin), F10 (HAM), F12 (HAM), DCCM1, DCCM2, RPMI 1640, BGJ medium (with or without Fitton) -Jackson Improvement), Basic Medium Eagle (Basal 120934.doc -26- 200813227

Medium Eagle) (BME-added Earle's base), Dulbec's modified basal medium (DMEM_no jk clear), Yamane, IMEM-20, Glasgow Modification Eagle Medium (GMEM), Leibovitz L- 15 medium, McCoy 5A medium, medium M199 (M199E-containing Earle's base), medium M199 (M199H· containing Hank's base), minimum essential medium Eagle (MEM-E-containing Earle's base), minimum necessary Medium Eagle (MEM-H-containing Hank's base) and minimum essential medium Eagle (MEM-NAA-containing non-essential amino acid). A large number of other media include medium 199, CMRL 1415, CMRL 1969, CMRL 1066, NCTC 135, MB 75261, MAB 8713, DM 145, Williams, G, Neuman & Tytell, Higuchi, MCDB 301, MCDB 202, MCDB 501, MCDB 401, MCDB 411, MDBC 153. A preferred medium for use in the present invention is DMEM. These and other suitable media are available from GIBCO (Grand Island, Ν·Υ·, USA) and Biological Industries (Bet HaEmek, Israel), and the like. A number of these media are summarized in Methods in Enzymology (Vol. LVIII, "Cell Culture, pp. 62-72, edited by William B. Jakoby and Ira H. Pastan), published by Academic Press, Inc. Additional non-limiting examples of the medium in the method of the invention may contain fetal serum of bovine or other species at a concentration of at least 1% to about 30%, preferably at least about 5% to 15%, optimally about 10%. Embryonic extracts of other species may be present at a concentration of from about 1 to about 30%, preferably at least about 5% to 15%, optimally about 10%. 120934.doc -27- 200813227 ''Growth factor, cytokines, hormones n refers to the following specific factors, including but not limited to growth hormone, erythrop0eitin, thrombopoietin, interleukin 3, interleukin at a concentration of picograms per milliliter to milligrams per milliliter. 6, interleukin 7, macrophage community stimulating factor, c-kit ligand / stem cell factor, osteoprotegerin, (osteoprotegerin) ligand, insulin, insulin-like growth factor, epidermal growth factor, fibroblast growth Factor, god Growth factors, ciliary _ neurotrophic factors, platelet-derived growth factors, and b〇ne morphogenetic proteins. At these concentrations, in colony forming unit (CFU) assays, suitable for use in the methods of the invention Growth factors, cytokines and hormones can induce up to 1% of adipose-derived stromal cells to form blood cells (lymphoid, erythroid, myeloid or platelet). (1) et al., 1973, J. Natl. Cancer Inst. 50: 603-623; Lee et al., 1989, J. Immunol. 142: 3875-3883; Medina et al., 1993, j. Exp. Med. 178: 1507-1515) 〇_ Further recognition Additional components may be added to the culture medium. These components may be antibiotics, antifungal agents, albumin, amino acids, and other components known in the art for cell culture. In addition, components may be added to enhance Differentiation, such as enhanced differentiation into cells that express GALC. "Chemicals" means including, but not limited to, antioxidant compounds (such as butylated hydroxymethoxybenzene (BHA) or 2-mercaptoethanol) , steroids, retinoids and temptations ASC differentiation of other compounds or agents. In one embodiment, ASC is cultured in insulin, dexamethasone, and isobutylmethylxanthine. In another aspect, the invention provides a method of identifying and/or characterizing ASCs that exhibit GALC in adipose tissue-derived cell populations 120934.doc • 28-200813227. The efficacy of ASC can be characterized by one or more of the methods discussed herein in one of the readily available animal models including, but not limited to, mouse models, canine models, or monkey models. The monkey is preferably a rhesus monkey. "Representation" of a resulting differentiated cell refers to the identification of surface and intracellular proteins, genes, and/or other markers that indicate that the ASC lineage is limited to a particular terminal differentiation state. These methods include, but are not limited to, (a) Screening of cell surface proteins by immunofluorescence using a secondary fluorescent marker _ linked protein-specific monoclonal antibody' includes the use of flow cytometry; protein-specific monoclonal antibodies linked by secondary fluorescent labeling Detection of intracellular proteins by immunofluorescence, including flow cytometry; (c) detection of cellular genes by polymerase chain reaction, in situ hybridization and/or northern blot analysis; and/or (d) Detecting GALC performance; 0) Detecting GALC activity. In a preferred embodiment, the method of identifying cells exhibiting GALC comprises providing a matrix specific for GALC to a population of cells and wherein the matrix is present when GALC is present in # ASC Degradation, thereby identifying ASCs in the cell population. In one aspect, the matrix is galactosyl sphingosine or galactosyl-ceramide. ^ Partial or terminally differentiated cells can be identified by surface and Intracellular proteins, proteins, genes, and/or other markers indicative of ASC lineages that are defined to a particular terminal differentiation state. These methods include, but are not limited to, (a) by immunofluorescence analysis (eg, flow cytometry) Or ASC surface protein (as exemplified herein, such as fatty acid binding protein, adipocyte (3101), HSP20-like protein, statin, strepin, PDZ, Lim domain protein 1 or lean 120934.doc -29- 200813227 Prime, as well as alkaline phosphatase, CD44, CD 146, integrin β1 or osteogenic protein) in situ immunostaining) cell surface protein (Gr〇nth〇s et al, 1994, Blood 84: 4164-4173) (b) Use of specific monoclonal antibodies against peroxisome proliferator-activated receptors, retinoid X receptors, vitamin 1) receptors or Cbfal by immunofluorescence (eg flow cytometry or adipose tissue) Detection of intracellular proteins by in situ immunostaining of stromal cells; detection of lineage-selective mRNA (such as osteocalcin by methods such as polymerase chain reaction, in situ hybridization, and/or other dot analysis) , ppAR γ, halogen Cbfal , interleukin 7, osteoprotegerin ligand and/or mega-cell community stimulating factor, white blood cell marker and growth factor) (see GimMe et al. 1989, Blood 74: 303-31 1). ASCs are also suitable for use in the present invention. For example, genetic modification can result in the expression of a foreign gene ("transgenic gene) or the altered expression of an endogenous gene. The genetic modification can have therapeutic benefit. In one embodiment , asc, seat this modification to express GALC in order to treat Crabbe disease. The GALC used for genetic modification of ASC can be from humans, baboons, mice or rats. Alternatively, the m-passage modification can provide a means of tracing or identifying the modified cell, e.g., after transplanting the composition of the invention into an individual. Tracking cells can include tracking migration, assimilation, and survival of transplanted genetically modified cells. The genetic modification can also include at least one second gene. For example, the second gene can encode an alternative antibiotic gene or another selectable marker. Proteins suitable for tracking cells include, but are not limited to, any of green fluorescent protein (GFP), other fluorescent proteins (eg, enhanced green, cyan blue, yellow, blue, and red fluorescent proteins; Clontech , Palo Alto, CA) or other standard 120934.doc 200813227 protein (eg LacZ, FLAG-tag, Myc, His6 and their analogues). Bromodeoxyuridine is also suitable for tracking cells.

The ASC can be genetically modified using any method known to those skilled in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A

Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) and Ausubel et al., eds. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY). For example, ASC can be exposed to include packages

I. A expression vector comprising a nucleic acid of a transgenic gene such that the nucleic acid is introduced into the cell under conditions suitable for the expression of the transgene in the cell. The transgenic gene is typically an expression cassette comprising a polynucleotide operably linked to a suitable promoter. A polynucleotide may encode a protein, or it may encode a biologically active RNA (e.g., an antisense RNA or a ribonuclease). Thus, for example, a polynucleotide can encode a pair of toxins, hormones (such as peptide growth hormone, hormone releasing factor, sex hormones, adrenocorticotropic hormone, cytokines (eg, p interferon, interleukin, lymphokine), etc. a cell-surface-incorporated intracellular signaling moiety (eg, cell attachment molecule, hormone receptor, etc.), a gene that promotes resistance to a given lineage differentiation factor (eg, bone morphogenetic protein (BMP)). ^ Within the performance ,, the coding polynucleotide is operably linked to a suitable promoter. Examples of suitable promoters include prokaryotic promoters and viral promoters (e.g., retroviral ITR, LTR, immediate early viral promoter (ΙΕρ) (such as herpesvirus ΙΕρ (e.g., ICP4-IEp and ICPO-IEEp), cellular giant viruses ( CMV) IEp)) and other viral promoters (such as Lloyd's sarcoma virus (Rous 120934.doc -31- 200813227

Sarcoma Virus, RSV) promoter and murine leukemia virus (MLV) promoter)). Other suitable promoters are eukaryotic promoters (eg, enhancers (eg, rabbit (P-globin regulatory elements))), active promoters (eg, beta-actin promoters, etc.), signal-specific promoters (eg, An inducible promoter, such as a promoter in response to RU486, etc.) and a tissue-specific promoter. It is well within the skill of the art to select promoters suitable for driving gene expression in a predetermined cellular environment. Performance 匣 may include more than one coding polynucleotide and, if desired,

Other elements can then be included (e.g., polyadenylation sequences, sequences encoding membrane insertion signals or secretion guides, ribosome entry sequences, transcriptional regulatory elements (e.g., enhancers, silencers, etc.) and analogs thereof). The expression containing the transgene should be incorporated into a gene vector suitable for delivery of the transgene into the cell. Depending on the desired end application, any such vector can be used to genetically modify the cells (eg, plastid, naked dna, virus (such as adenovirus, adeno-associated virus, herpes virus) lentivirus, papilloma Viruses, retroviruses, etc.)). Any method of constructing the desired expression in such vectors can be used, many of which are well known in the art (e.g., direct selection, homologous recombination, etc.). The choice of vector largely determines the method used to introduce the vector into the cell (eg = by protoplast fusion, calcium phosphate precipitation, gene grab, preparation ^ electroporation, DEAE dextran or lipid carrier mediated Transfection, viral vector infection, etc., such methods are generally known in the art. II. Method of treatment In a case of a disease, the present invention provides a method for treating leukoencephalopathy in animals

A method of at least one symptom of Sl, which method comprises a non-immunogenicity I20934.doc-32-200813227. The isolated ASC is administered to the mammal. In one embodiment, the white matter lesions are Crabbe disease, adrenal white matter lesions/adrenal myelin neuropathy i:, and Aicar (ji_G〇utieres syndrome), Alexandria, with a diffuse center Cerebral arterial disease (CADASIL), carinavon disease White matter lesions, neonatal adrenal white matter lesions, ovarian white matter lesions _ syndrome, spastic cerebral sclerosis, Lefsum's disease, Van der Knaap syndrome or Zellweger Syndrome. White matter lesions are preferably Krabbe disease. In another aspect, the invention provides an increase in tissue or mammal by administering a non-immunogenically isolated ASC to the mammal. Method for GALC content. The ASC of the present invention can be administered at various time points. For example, cells can be administered at the onset of symptoms of white matter lesions. In one embodiment, the onset of symptoms is about 1 day, preferably 2 Day, more The cells are administered for 3 days, preferably 4 days and more preferably for 7 days. In another embodiment, the cells can be administered to the mammal several weeks after the onset of symptoms. The cells can be administered to the host in a variety of ways. Modes are parenteral, - intraperitoneal, intravenous, intradermal, epidural, intraspinal, intracranial, joint, internal, synovial, intrathecal, intraarterial, intracardiac, intramuscular, subcutaneous, local , transdermal, surgical implant, internal surgical paint or infusion pump. In one embodiment, the agent and carrier are administered in the form of a sustained release formulation, such as a direct tissue injection or A bolus, an implant, a microparticle, a microsphere, a nanoparticle, or a nanosphere. Although the method of injecting a differentiated ASC intravenously by tail vein injection is exemplified herein, the present invention does not Limited to such methods. The differentiated cells of the invention can be detected in a subject by a variety of techniques including, but not limited to, flow cytometry, immunohistochemistry, in situ hybridization, and/or tissue or cell biotechnology. Existence. See for example 〇ρ印等,, 1999, Proc Natl Acad Sci 96: 1071 1-10716 移植 • The transplantation of the cells of the present invention can be accomplished using techniques well known in the art and techniques described herein or in the future. A method of transplanting, implanting, infusing, or otherwise introducing a cell into a mammal is included. Methods of bone grafting are also well known in the art and are described, for example, in U.S. Patent No. 4,678,470, a description of pancreatic cell transplantation. A method of transplanting cells into any anatomical location in the body is taught in U.S. Patent No. 6,342,479, and U.S. Patent No. 5,571, filed on Jan. To transplant cells of the invention into humans, cells are prepared as described herein. In one embodiment, the cell line is derived from a patient into which the cell is implanted (autologous φ homologous transplantation). In another embodiment, the cell line is from a non-human primate, such as a rhesus monkey. A preferred mode of participation is as follows. In the case where the cell line is not derived from the patient (allograft), the gold-type or hapiotype compatibility between the donor cell and the patient should be determined to a minimum. Surgery was performed using a Br〇wn-R〇berts-Wells computed tomography (CT) stereotactic guide. Local anesthesia was performed on the scalp area of the patient and midazolam was administered intravenously. The patient undergoes a CT scan to determine the coordinates of the region to receive the graft. The injection sleeve is often composed of a 17 gauge stainless steel outer casing and a 19 gauge internal probe. Insert it into the brain to the correct 120934.doc -34- 200813227 coordinates, then remove and replace with an i9 size infusion cannula that has been preloaded with approximately 30 μl of tissue suspension. When the cannula was withdrawn, the cells were slowly infused at a rate of about 3"/Μη. A plurality of locating needle channels were made throughout the relevant area, approximately 4 mm apart. The patient was examined for bleeding or edema by CT scan after surgery. Neurological assessment and ρΕτ scans were performed at different time intervals after surgery to determine the metabolic activity of the implanted cells. About 1 to 5 and about 10 π cells per 100 kg of humans were administered to humans. In certain embodiments, Inoculates 1 to 6 χ 1 〇 12 cells per 100 kgA L5 X. In some embodiments, 1 X 1 〇 9 to $ X 1011 cells per 1 〇〇 0 person. In some embodiments, 4 〇 1 〇 9 to 2 x 1011 cells per 100 kg of human are administered. In other embodiments, 1 〇〇 kg, 5 χ 10 to 1 X 1 〇 1 G cells are administered. Cells can be administered to humans by a variety of methods including, but not limited to, infusion and intravenous administration. In certain embodiments, the cells are administered a single time. In some embodiments, multiple administrations are performed. In some embodiments, the process is administered multiple times for 3-7 consecutive days. In some embodiments, the process is continued for 3-7 days. 7 administrations. In other embodiments, the procedure is administered 5 times for 5 consecutive days. In some embodiments, a single dose of about 1 to 5 and about 1 to 13 per 1 kg person is administered. The cells in between. In some embodiments, a single dose of between about 1. 5 X 108 and about L5 X 1〇i2 cells per 1 〇 () kg of human. In some embodiments, A single dose of cells between about 1 x 1 and about 5 χ 1 〇 per 100 kg of a person is administered in a single dose. In some embodiments, a single dose of about 5 每 1 per 1 kg person is administered. 〇10 cells. In some embodiments, a single dose of 1 χ 1 〇 1 细胞 cells per 1 〇〇 ^ human 0 120934.doc -35 - 200813227 In some embodiments, multiple doses per 100 1 kg 5 to 10 u cells in kg. In some embodiments, 15 x 108 to 108 cells per 100 kg A. In some embodiments, for 3-7 consecutive days. The process is repeated for 9 to 5 χ 1〇11 cells per 100kg of M x 1 . In a certain case, the process of 3 consecutive days of 3-7 days is repeated for 4 X μ per 1〇〇^ person. Cells. In some embodiments, '2 x 1011 per 100 kg of people are administered multiple times over a continuous 3-7 day course In certain embodiments, 3.5 X 1 () 9 cells are administered 5 times in 5 consecutive days. In certain embodiments, the process of sputum is administered 5 times to 4 X 109 cells 5 times. In certain embodiments, β &amp; 5_human &amp; and 13 χ 1〇11 cells are connected. In some embodiments, 2 〇 1〇11 are administered 5 times in 5 consecutive days. cell. In an embodiment of the invention, the cells of the invention are administered to a patient suffering from a disease, disorder or condition (e.g., Crabbe disease) involving the cells of (10) to increase or replace the diseased or damaged cells. ASC is preferred for humans who are enrolled in diseases, disorders or conditions of white matter lesions. The precise location of cell recruitment depends on a number of factors including, but not limited to, the area to be treated, the type of disease being treated, the age of the person and the severity of the disease, and similar causes. The determination of the site of administration is entirely within the skill of the technicians skilled in the administration of such cells. According to the present invention, cells can be administered to a patient via an intravenous route. There are several ways in which ASC can be used in mammals (more Cui humans) to treat white matter lesions. For example, the cells can be used to drive cells prior to introduction into a patient or to serve, for example, cells that have differentiated into leukocytes or fibroblasts prior to introduction into a patient. In either case, the cells can be differentiated to exhibit at least one protein characteristic of leukocytes or fibroblasts, such as, but not limited to, GALC, at 120934.doc-36-200813227. In one embodiment, the ASC is differentiated in vivo into cells expressing GALC, as appropriate. In another embodiment, the ASC is cultured in vivo for a period of time without being induced to differentiate prior to its administration to the mammal. The invention will now be more fully described by the following examples. However, the present invention may be embodied in many different forms and should not be construed as being limited to the embodiments described herein; rather, those skilled in the <RTIgt; The scope of the invention. EXAMPLES Example 1: Isolation and differentiation of adipose stromal cells (ASC) to determine whether ASC undergoes adipogenesis, cultured in the presence of dexamethasone, insulin, isobutylmethylxanthine, and thiazolidinedione followed by dexamethasone and insulin The culture system is established in the presence of adult stromal cells cultured. The materials and methods used in the experiments presented in this example are now described. Isolation of adipose stem cells: Human adipose tissue has been shown to be a sufficient source of stromal adult stem cells (Gimble et al., 2003, Curr. Top Dev. Biol. 58: 137-160; Aust et al., 2004, Cytotherapy 6(1) : 7-14; Awad et al., 2003, Tissue Eng. 9(6): 1301-1312; Awad et al., 2004, Biomaterials 25(16): 3211-3222; Elmslie et al., 2000, J. Clin Psychiatry 61(3): 179-184; Delany et al., 2005, Mol. Cell Proteomics 4: 731-740; Gronthos et al., 2001, J. Cell Physiol 189(l): 54-63; Halvorsen et al. Person, 2000, Int. J. Obes. Relat. Metab. Disord. 24 Suppl. 4: S41-44; 120934.doc -37- 200813227

Halvorsen et al, 2001, Metabolism 50(4): 407-413; Halvorsen et al, 2001, Tissue Eng. 7(6): 729-741; Hicok et al, 2004, Tissue Eng 10 (3-4): 371-380; Safford et al., 2002, Biochem. Biophys. Res. Commun. 294(2): 371-379; Safford et al., 2004, Exp. Neurol. 187(2): 319-328; Sen et al. 2001, J. Cell Biochem 81(2): 312-319; Wickham et al., 2003, Clin. Orthop. (412): 196-212; Guilak et al., 2005, J. Cell Physiol.; Mitchell et al. Stem Cells online January 12, 2006:2005-0235) ° Based on Hauner and others (Hauner et al., 1989, J. Clin. Invest 84(5): 1663·1670; Hauner et al., 1988, Horm. Metab· Res. Suppl. 19:35-39) The initial method described has developed a reproducible and effective method for isolating adult stem cells from human liposuction tissue (Aust et al., 2004, Cytotherapy 6 (1) ): 7·14; Halvorsen et al., 2001, Metabolism 50(4): 407-413; Mitchell et al., Stem Cells online January 12, 2006: 2005-0235). The procedure involves tissue collagenase digestion, differential centrifugation, and amplification in culture. In the analysis of samples obtained from 42 individual donors, an average of 247,401 soil 136,514 human adipose-derived stem cells (huASC) 'tissue donor populations were recovered from 1 ml of liposuction waste during a 2.4-day 2.4-day expansion period ( The demographic profile of n = 120 patients) is summarized in Table 1. 120934.doc -38- 200813227 Table 1: Donor demographics ί25 25&lt;BMI&lt;30 30&lt;ΒΜΙ &lt;35 ΒΜΙ&gt;35 f sentence ΒΜΙ士 SD 22.3 soil 1.4 27.5±1.4 32.3 ±1.3 36.9 ±1.3 ΒΜΙ range 19.9-24.7 25.1-29.9 30.1-34.2 353-39.2 Total number of subjects 57 73 13 7 Number of Caucasians 51 34 11 5 Number of Africans / Americas 5 2 0 0 Number of Asians 1 5 2 2 Number of Spanish 0 2 0 0 Female 98% (56) 86% (37) 85% (11) 71% (5) Male 2% (1) 14% (6) 15% (2) 29% (2) Average age 38.9 ± 8.4 41.1 ± 11.4 42.7 ± 14.8 34.1 Soil 10.7 Age range 24-62 18-64 25-63 22-50

After in vitro passage, these cells identified as ASC showed comparable differentiation potential to stem cell stem cells (MSCs) from bone marrow sources (Table 2) (Aust et al., 2004, Cytotherapy 6(1):7-14 Halvorsen et al., 2001, Metabolism 50(4): 407-413; Mitchell et al., Stem Cells online January 12, 2006: 2005-023 5) Other similar groups have reported similar findings 78-85. Flow cytometry has been used as the initial proteome research method to define the SC immunophenotype. Table 2: Characteristics of huASC (passing 2) Surface positive marker surface negative marker differentiation potential CD9, CD10, CD13, CD29, CD44, CD1 CD14, fat fine ^ = CD49d, CD54, CD55, CD59, CD7 CD CD16, CD18 , chondrocytes CD73, CD90, CD105, CD106, CD3 CD45, gold CD146, CD166, α·smooth muscle CD50, CD56, support actin, collagen type I, collagen CD62, CD104, myocyte type III, HLA-ABC, Nestin, Osteogenic Protein, Factor VIII-related Ag, (Heart, Bone) Osteonectin, Vimentin HLA-DR Myofibroblast Neurons Osteoblastic Fat Formation: Dexamethasone, Insulin, Isobutyl The stromal cell cultures were induced for 3 days by thiopurine and sputum ketone, followed by dexamethasone 120934.doc -39 - 200813227 in the presence of pine and insulin. After 14 days of culture, cells were fixed and neutral lipids were stained with Oil Red(R) (Fig. 1, left) and the leptin content of the modified medium was determined by ELISA day (Fig. 1, right). The results of the experiments presented in this example are now described. In the presence of dexamethasone, insulin, isobutylmethylxanthine and thiazolidinediones, huASC undergoes adipogenesis (Fig. 1). The cells accumulate lipid vacuoles (staining neutral lipids with Oil Red sputum dye (Figure 1A)) and exhibit adipocyte-specific markers, including the secreted cytokine leptin (Figure 1B) and the fatty acid binding protein aP2 (Halvorsen et al, 2001, Metabolism 50(4): 407-413; Sen et al, 2001, J. Cell Biochem. 81(2): 312-319). In addition, the cells display a lipolytic response to adrenergic compounds which is a biochemical feature of mature primary adipocytes (Halvorsen et al., 2001, Metabolism 50(4): 407-413). These results show that adult stromal cells can undergo fat formation. Example 2: Multi-directional differentiation potential of ASC To determine whether non-human primate ASCs can multi-differentiate, non-human primate ASCs are cultured under the same conditions as described for huASC and then based on morphology, proliferative potential And differentiation potential to study. The materials and methods used in the experiments presented in this example are now described. Multi-directional differentiation potential of huASC: The differentiation potential of huASC is not limited to adipocyte lines. Conditions have been developed to promote the differentiation of huASC along chondrocytes and osteoblasts (Awad et al., 2003, Tissue Eng. 9(6): 1301-13 12; Awad et al., 2004, Biomaterials 25(16):3211- 3222; Wickham et al., 2003, Clin· Orthop. (412): 196-212; Guilak 120934.doc -40- 200813227

Et al., 2005, J. Cell Physiol, Erickson et al, 2002, Biochem Biophys. Res. Comnum. 290(2): 763-769; Wang et al, 2005, J. Cell Physiol). When suspended in calcium alginate and incubated in the presence of ascorbate, dexamethasone, and transforming growth factor alpha, huASC shows markers of induced chondrogenesis, including collagen sputum and type VI and proteoglycans (Awad et al. 2003, Tissue Eng. 9(6): 1301-13 12; Awad et al., 2004, Biomaterials 25(16): 321 1-3222; Wickham et al., 2003, Clin. Orthop. (412): 196-212; Erickson et al., 2002, Biochem. Biophys. Res. Commun. 290(2): 763-769). When cultured in the presence of 1,25 dihydroxyvitamin D3, dexamethasone, ascorbate, and alpha-phosphoglyceride, huASC secretes osteocalcin and mineralizes its extracellular matrix (characteristics of osteoblast function) (Halvorsen et al) Human, 2001, Tissue Eng. 7(6): 729-741; Hicok et al., 2004, Tissue Eng 10 (3-4): 371-380; Guilak et al., 2005, J. Cell Physiol). When implanted in immunodeficient mice subcutaneously, huASC is combined with hydroxyapatite biomaterials to synthesize bone-like substrates in vivo (Hicok et al., 2004, Tissue Eng 10 (3-4): 371.380; Justesen et al. 2004, Tissue Eng. 10(3-4): 3S1-391) A large body of data confirms that huASC and murine ASC cultured in the presence of antioxidants undergo morphological and phenotypic changes consistent with neuronal differentiation (Safford et al. , 2002, Biochem. Biophys. Res. Commun. 294(2): 371-379; Safford et al., 2004, Exp. Neurol. 187(2): 319-328; Ashjian et al., 2003, Plast. Reconstr. · 111(6): 1922-1931). The list of neuronal markers represented by huASC has been 120934.doc -41- 200813227 Extension includes nestin, GFAP, S-100, NeuN, MAP2, GABA, NR-1 and 2 subunits of glutamate receptors and voltage Gating and channeling (Safford et al., 2004, Exp. NeuroL 187(2): 319-328). It has also been found that huASC also secretes large amounts of cytokines in vitro and supports hematopoiesis (RW Storms, JM Gimble, MS preparation). In addition, it has been demonstrated that the pure huASC retains its multi-directional differentiation potential. The results of the experiments presented in this example are now described. The stromal vascular portion of adipose tissue has been found to contain high frequency lineage specific colony forming units (CFU) (Mitchell et al, Stem Cells online January 12, 2006: 2005-0235). The average of η = 7 to 12 donors is as follows: CFU-F (fibroblast), 1/30 cells; CFU-ALP (alkaline phosphatase), 1/285 cells; CFU-Ad (fat cells) , 1/40 cells; and CFU-Ob (osteocytes), 1/12 cells. With the gradual progression, the frequency of individual lines is about 10 times stronger. These values exceed the 2-3 orders of magnitude of the values assessed by bone marrow-derived MSCs. Figure 2 shows the morphology, proliferation and differentiation potential of non-human primate ASCs (pASCs). The culture-amplified non-human primate ATSCs showed typical spindle-shaped fibroblast morphology (Fig. 2A is low density and Fig. 2B is high density). Compared to pATSCs, primate bone marrow stem cells (pBMSCs) are more heterogeneous and have fibroblast morphology (C). Single cells were expanded to a pure lineage and colony forming units (CFUs) were generated, which were evident after 0丨61118&amp; staining (Fig. 2D). Subatomic 3-4 times of pATSCs retained multilineage differentiation and experienced adipogenesis (Fig. 2E), bone formation (Fig. 2F) and cartilage formation (Fig. 2G). Subcutaneous fat from non-human primates 120934.doc -42- 200813227 Tissue-separated ASCs (prASCs) have pluripotency similar to that of huASCs. Pure passages of prASCs differentiated along fat cells, chondrocytes, osteoblasts, and neuronal pathways (Fig. 2). These results indicate that ASCs from non-human primates and other large animal models can be used as human replacements in preclinical testing. Example 3: Proteomic analysis of adipose stem cells To determine the performance profile of differentiated huASCs compared to undifferentiated huASCs, proteome analysis was performed on these cells. The materials and methods used in the experiments presented in this example are now described. Two-dimensional-polyacrylamide gel electrophoresis: Protein analysis relies on traditional two-dimensional electrophoresis to initially separate complex mixtures of proteins. The sample was dissolved in a solution containing 8 Μ veins, 4% CHAPS, 65 mM DTT, 40 mM Tris. After centrifugation to remove insoluble material, 333-500 pg of protein is mixed with rehydration buffer (8 guanidine, 4% CHAPS, 1% IPG buffer, 0.3% DTT) and under active rehydration conditions (eg throughout the strip) The tape was applied with a slight pressure) and introduced into a dry IPG strip (usually 18 cm, pH 4-10NL). The protein was concentrated at a maximum of 10,000 V for a total of 90,000 v-h. After completion of the first-dimensional electrophoresis, the IPG strips were directly subjected to second-dimensional SDS-PAGE or frozen at -80 °C for later analysis. For the second dimension, the IPG bands were first equilibrated with 50 mM Tris-HCL (pH 8.8), 6 guanidine, 30% glycerol, 2% SDS, 1% DTT for 15 minutes, followed by 50 mM Tris-HCL ( pH 8·8), 6 guanidine, 30% glycerol, 2% SDS, 5% iodoacetamide were equilibrated for the second time for 15 minutes. The strip was washed with running buffer (25 mM Tris, 190 mM glycine, 0.1% SDS) and then embedded in agarose 120934.doc -43-200813227 at a low melting temperature, placed at 25 x 20 cm 12% On a acrylamide gel. Run the gel at a constant voltage until the bromophenol blue dye front reaches the bottom of the gel. Protein staining and quantification: After two-dimensional electrophoresis, the gel was stained with Sypro Ruby. The stained gel was scanned with Molecular Imager FX (its data was directly imported into PDQuest). For each gel, the relative abundance of each of the analytical protein features was quantified by mathematical fit of a two-dimensional Gaussian curve. Each data was normalized (expressed as a percentage of total abundance or relative to a group of housekeeping proteins) and routine statistical analysis (identification of unique points, lack of points, or up- or down-regulation under specific conditions) was obtained from the packaged software. However, the statistical analysis data is usually output in the Excel test form. Mass spectrometric analysis of proteins: according to the guidelines proposed by the editor of Molecular &amp; Cellular Proteomics 101 and the proteome research techniques well known in the art as described, for example, in the following book, the contents of which are incorporated herein by reference. Implement all proteomics research: Proteome Research: New Frontiers in Functional Genomics (Principles and Practice), MR Wilkins, Editor,

Springer Verlag, 1007; 2-D Proteome Analysis Protocols, Andrew L Link, ed., Humana Press, 1999; Proteome

Research: Two-Dimensional Gel Electrophoresis and

Identification Methods (Principles and Practice), edited by T. Rabilloud, Springer Verlag, 2000; Proteome Research: Mass Spectrometry (Principles and Practice), P. Jame, Springer Verlag, 2001; Introduction to Proteomics, DC Liebler, Edit, Humana Press, 2002; Proteomics in 120934.doc -44- 200813227

Practice: A Laboratory Manual of Proteome Analysis, R. Westermeier et al., ed., John Wiley &amp; Sons, 2002. After electrophoresis, staining, scanning, spot detection, and match set preparation, select the relevant protein and enter its standard number into the &quot;Cut List." By Protein Point Glue Meter (at PDQuest 7·2· Under Control 0) Use this &quot;Cut List&quot; to automatically select and remove the minimum abundance to maximum abundance of protein characteristics from one or more gels. Precipitate the excised gel into a 96-well plate, where Sample tracking was maintained by PDQuest 7.2.0 and ProteinLynx Global Server. The plates were transferred to a MassPrep table where the proteins in the gel plugs were automatically faded, reduced, alkylated, dehydrated, rehydrated and trypsinized. The resulting peptide was extracted, cleaned and then deposited onto a MALDI plate and in a 96-well plate (for Q-TOF). The peptide fingerprint was determined by matrix-assisted laser desorption free/time of flight (MALDITOF) mass spectrometry. The resulting peptide mass fingerprint is used to interrogate the SwissProt, TREMBL, or NCBI database to experimentally identify known proteins. If MALDITOF is unable to identify protein spots or if protein is specified There are some uncertainties, and the peptides are separated by capillary liquid chromatography coupled to an ESI-MS/MS MicroMass Q-TOF mass spectrometer. Partially derived sequences derived from peptides are used to interrogate protein, genome or EST databases. Clearly identify proteins. As long as multiple gel features are identified to identify the same protein with one or more names, the protein is only entered into the database as a single entity. PDQuest 7·2·0 software and WorksBase data management system tracked from initial identification To all samples and individual points of statistical analysis, protein spotting, mass spectrometry and protein identification. The data obtained by mass spectrometry is reversed. 120934.doc -45- 200813227 Annotate the starting gel image so that the click-related point is displayed. Its identity, peptide mass spectrometry, derived amino acid sequences, and pre-selected data downloaded from public databases. To facilitate the analysis of the vast amounts of data generated in these experiments, GenMAPP (Gene MicroArray Pathway Profiler) and MapppFinder (genmapp.org/) were used. ) to check the data. GenMAPP is designed to view the gene list on a map representing biological pathways and gene groupings. Computer application of the data. The results of the experiments presented in this example are now described. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and tandem mass spectrometry were used to compare the protein bodies of huASC in undifferentiated and adipocyte differentiated states ( Figure 3) (Delany et al., 2005, Mol. Cell Proteomics 4: 731-740). More than 430 Sypro staining spots on 2DPAGE gel were distinguished from undifferentiated and adipose tissue huASC (288 of which were shared) and more than 170 individual proteins were identified by mass spectrometry and expressed by undifferentiated huASC (Table 1) (Table 1) Delany et al., 2005, Mol. Cell Proteomics 4: 731-740). After fat formation, more than 40 proteins were up-regulated by more than 2 fold, while an additional 13 proteins were reduced by more than 3 fold (Figure 4 and Table 2) (Delany et al., 2005, Mol. Cell Proteomics 4: 731 _740) Figure 4 Describe the individual protein characteristics of undifferentiated (U) and adipocyte differentiation (D) huASC. When 2D-PAGE analysis of total huASC lysates, differentiation-dependent changes were made by targeting fatty acid binding proteins, adipocytes (3 101 ), HSP2-like prion protein (7204), cytosolic phosphoprotein (3107), and elfin/PDZ and Lim. Identification of the arrow of domain protein 1 (6521). These results indicate that differentiated huASCs have a different performance profile of 120934.doc -46 - 200813227 compared to undifferentiated huASCs. This further supports the multipotential differentiation potential properties of cultured huASCs. Example 4: Characterization of animals with Krabbe disease To determine whether a rhesus monkey is a suitable model for studying human Clape disease, thoroughly examine the diseased animal and compare the results to the clinical course of the disease in humans. The materials and methods used in the experiments presented in this example. Animal growth: Ultrasound is used monthly during pregnancy to monitor the growth of a growing primate fetus (Macaca mulatta) primate fetus to assess any growth delays or changes during pregnancy and to monitor abortion The rate, the month of death, and or may cause problems with dystocia. In general, animals are naturally given birth. After the baby is born, blood is drawn to determine the genetic status (wild type, heterozygous or homozygous disease) and the sex is checked and all children's health and maternal health are checked. The genomic DNA is extracted from peripheral blood mononuclear cells and a diagnostic PCR protocol (including restriction digestion) is performed to determine the Krab status (sick or normal heterozygous carrier, homozygous carrier). Infants who maintain heterozygous zygotes and infants of the same type are babies with their mothers and normal animals are released from the program. Neuroimaging: All MRIs were used monthly to study all affected infants, as well as age-matched carriers and non-carrying controls. The MRI sequence included a sagittal T1-weighted scan, a sagittal T2-weighted scan, an axial T1-weighted scan, an axial proton density, an axial D2-weighted scan, a coronal T2-weighted scan, and a comparative axial and coronal Τ1 weighted scan. When necessary, Prohance (Gadoteridol) was administered to the animals for comparison with mM·〗 mM/kg (intravenous). 120934.doc -47- 200813227 MRU field description includes the location, extent and morphology of leukoencephalopathy and related cranial endothelium and ventricular changes.

Data from early nuclear magnetic resonance (MR) image analysis showed no clear abnormalities observed. The diseased animals did not show abnormal changes in gray matter or white matter when compared with the age-matched control group. In addition, the ventricular system and the skin are not significant. Important 疋 / / main thinking, the animal is thoroughly analyzed! You must have a post-mortem. The change in CNS seems reasonable in the later stages of life. As a support of this theory, it is noted that one of the 22-month-old Krab animals has a T2 signal zone associated with a triangular region of the posterior horn of the lateral ventricle. In humans, this is often described as an early discovery of Crabbe disease. Perform an EMG nerve conduction study and perform an infant behavior test. In addition, videos of such infants are recorded to record the behavior, movement and difficulties of such infants in everyday situations. Animals were also monitored for weight and assessed for their diet and general health. After significant weight loss and/or severe breathing difficulties were measured, the animals were euthanized and a complete autopsy performed. Physiology: Neurotransmission studies were performed as previously described (England et al. 1997, Ann. Neur et al. 41 (3): 375-384). If any sick baby is born, they are monitored by magnetic resonance imaging (MRI). Conducted a continuous study of the transmission of oracles, which began within two months of the beginning of the life of four homozygous zygotes and two heterozygous pheasants to characterize peripheral neuropathy. Because there was no significant combination between the carrier and the normal group to produce a non-affected comparison group for all subsequent two: ^ mother one (four), the significant age of the ageing material group, 丄.F(1,57)= 24.06, P&lt;〇·0001; calendar nerve··,58)=26.44, 120934.doc -48- 200813227 p&lt;〇._; ulnar nerve·· F(1,59)=28 68, (d) age Under the fourth, the average conduction velocity of the median nerve, the ulnar nerve and the phrenic nerve in the diseased monkey was significantly lower.&gt;&lt;0.__/ Wehner et al. '2005'M(4)leNerve32(7):185 call. On the string

The degree of conduction slowing is very uniform along all segments of each nerve and is extremely versatile between the nerves. When compared to unaffected monkeys, the continuous conduction velocity indicates that the diseased monkey is _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ This = was found to be characteristic of severe primary demyelinating polyneuropathy and was similar to the expected disease in the monkey, although bilateral motor, ulnar and sacral motor conduction (IV) showed normal composite muscle action potential amplitude, but all The nerve showed a severe prolongation of the distal latency and a severe slowing of the conduction velocity. In the diseased monkey, although the F wave is well formed and can be replicated, the incubation period is severely prolonged. When the monkey is getting older, the difference in conduction velocity between the diseased and unaffected animals is more pronounced. There is no evidence that excessive time is scattered in any nerve. GLD electrophysiology phenotype in diseased monkeys. Motor neurotransmission _ velocity diffusion and homologous slowing are characteristic of hereditary demyelinating (myelin dysplasia) neuropathy secondary to defects in myelination in the peripheral nervous system. Lipid Analysis··Measured from two diseased babies (AA54 and V539) and from three control rhesus monkeys using the method described by Fujita (Fujita et al., 1996, Hum. Mol. Genet 5(6): 7U_725) Lipid profile of brain and kidney samples. Samples of gray matter and white matter were separated from each other as much as possible before extraction. The neurosteroid galactose was analyzed on the tissue samples of 2〇_4〇 mg by high pressure liquid chromatography using reverse phase column separation and fluorescence detection. 120934.doc •49- 200813227 Galactocerebrosidase activity: Biochemical assays for galactocerebrosidase activity with undifferentiated pRASC and huASC, with subculture changes. The degree of enzyme activity is compared to the degree of enzyme activity detected in peripheral blood mononuclear cells obtained from primates and humans. Infant behavioral assessment: Infant neurobehavioral assessment of 14 and 30 day old animals (early infant assessment; tool description see (Schneider et al. '1991, Am. J. Primatol 25: 137-155)).

A typical protocol for implementing the Infant Neurobehav (6) al Assessment Scaie (NBAS) and the Barry Infant Development Scale is as follows: At least two trained individuals participated in the test. One person is responsible for catching the baby and another person is testing and recording the response to the test item. Arrange all the items required for the test, then bring the baby into the room and keep it within the reach of the examiner. All tests were carried out in a quiet, light (four) bright area. Place a label on the door and declare &quot;Do not disturb, test in progress&quot;. 1 Infants and their mothers are at the same time - the trained animal care or veterinary armor is injected intramuscularly with chloramine hydrochloride _ (9) she - ". two de_ mg / kg) to anesthetize the mother. Remove the baby from the mother 'Tray with a towel and bring it to the examiner. The examiner keeps the baby from the waist down and wraps around the v, 4 in the towel, so that each arm is free to move ^ to the / cognitive project followed by the neuromotor function project And the project is implemented in the order of the project. The project and the test period are in a quiet and vigilant state. Because: the baby is in the whole time, the examiner can introduce the (4) baby. Today, if necessary and appropriate 120934.doc 200813227 NBAS takes about 2 minutes and can be used on infants up to 3 days old (recommended test days 7, 14, and 28). It takes about 10 minutes for the Barry Infant Development Scale and can be used for 2 to 12 Between the months. After the test is completed, the baby is returned to his mother. The results of the experiments presented in this example are now described and summarized in Table 3: Table 3 · Clinical results of rhesus monkey babies with clinical disease Overview Animal No. H463 Λ Life 76 days --- --- - The clinical signal of the disease in the absence of fresh cut-off disease. The autopsy of the starting animal - V5jy 159 days of the Ningshu nervous system (CNS) signal, which consists of severe muscle fibrillation, ataxia and overextension of the head and limbs. AA54 12 days consists of CNs signal CF36, which is especially involved in the muscle fibrillation and ataxia of the head. The stillbirth is diagnosed by prevalence of chorionic villus sampling _ C180 190 days especially involving severe muscle fibrillation and ataxia in the head. Gradually worsening twice as much as inspiratory force DG51 103 days _ obvious muscle tremors, difficulty in moving, difficulty in kicking the squad DH31 21 months of severe body tremors, difficulty moving, ataxia and overstretching and exogenous eyes (exopthalmia) EA75 22 months Whole body tremor, CNS symptoms, facial paralysis EJ72 52 days f tight hand and foot, can not support the head, can not use the legs / arms, no sound ----- __ super sound monitoring fetal animals confirmed in normal, carriers and suffering There is no significant change in fetal growth or development between diseased animals. However, due to the limited number of diseased animals produced so far, speculations about developmental abnormalities and fetal deaths Basically, it is necessary to continue the supersonic examination of the snails. Because the Crab animals are born in the community, their weight is monitored as part of the regular animal feeding. Figure 5 shows the weight of each diseased animal and the unaffected/normal male and Comparison of the weight of females. In general, sick animals cannot increase their weight at the same rate as normal animals of the same age and gender. 120934.doc •51- 200813227 Chorionic villus samples (CVS) often from pregnant monkeys The placenta is obtained and this type of sample is part of the natural fertility programme. Techniques for performing PCR-based molecular diagnostics for routinely detecting Krabo mutations (two base pair deletions) have been developed. Every disease track the disease progression of all diseased animals. Table 4 provides an overview of the clinical observations of infants with Klub's disease. Table 4: Unique characteristics of rhesus monkey animal models with Kappa disease 1 The development of fetal monkeys approximates the development of human fetuses 2 Similar to human CNS development and mutations 3 Similar to human CNS organization 4 Myelin formation during development The timing is very similar to the similarity of human 5 T1 and T2 weighted MRI images. 6 Results of gene therapy studies indicating results in humans. 7 Perform bone marrow transplantation and apheresis procedures. 8 Study the efficacy of therapeutic interventions, as well as safety and toxicity. Opportunity 9 is based on the application of human memory, motor function, cognition and behavioral testing

Content of galactosyl sphingosine (sphingosine galactoside) in rhesus monkeys: lipid analysis showed a significant increase in sphingosine galactoside content in the brain and kidney of affected infants (Table) 5). The content of sphingosine galactosides in the white matter of the brain is 20 times higher than normal, and is increased to about 3 500 pmol per milligram of protein, while the concentration of other myelin lipids is decreased. Although the concentration of galactosyl-ceramide is less than normal, the ratio of galactosyl-ceramide/sulfate cerebroside is normal and the sphingomyelin having long-chain fatty acids is significantly reduced. On the other hand, cerebral cortical gray matter showed a normal pattern of major lipids and a small increase in sphingosine galactoside content (this may be due to a small amount of contaminating white matter). In the kidney of the diseased monkey, although the concentration of galactosyl-ceramide was not significantly increased, on the thin-layer chromatography, the hydroxy-fatty acid solution 120934.doc -52-200813227 detached was found in the control group. Lack of spectrum. The amount of sphingosine galactosidase was found to be relatively large compared to the undetectable content of the control kidney obtained from normal animals (0·1 nmol per milligram of protein). Table 5: Content of galactosyl sphingosine (sphingosine galactoside) in rhesus monkeys * Gray matter white matter AA54 (12 days) 20 840 V539 (158 days) 115 3500 Normal control (new birth) &lt;2 normal (4.5 years) 25* 160 carriers (5.4 years) 3 85 * pmol per milligram of protein; ** as judged by gal-cer content may contain certain white matter GALC enzyme activity: previously used The GALC activity in the platinum sphere was measured to screen the colonies. Although some white blood cells of monkeys contain less GALC activity than other monkeys, it is not possible to ultimately identify carriers by enzymatic analysis. _ GALC activity in 2 homozygous diseased monkeys, 21 normal monkeys, and 20 wild monkeys was measured as previously described (Wenger et al., 1991, New York: Wiley-Liss). In leukocytes and cultured skin fibroblasts, 2* diseased infants have less than 2% of GALC activity in normal infants. 21-PCR confirmed that the average GALC activity of non-bearing rhesus monkeys was 0·94 nmol/h per mg of protein, while the average GALC activity of 20 PCR-identified carriers was 0.52 nmol/h per mg of protein. As predicted, the average amount of GALC activity in the animals carried is approximately 120934.doc -53 - 200813227 half of the average amount of GALC activity in wild-type animals, since the animals carry only one functional gene. Similar to the situation in humans, a wide range of values for normal and carrying GALC activity in rhesus monkeys was observed. Normal individuals have GALC activity values ranging from 0.39 to 1.6 nm/i/mg per mg of protein, while carriers have GALC activity values ranging from 0. 2 to 1·ι nmol/h per milligram of protein. In fact, 13 of the monkeys showed a higher GALC activity than the lowest in normal animals. This makes it possible to identify the problem of carrying monkeys only by biochemical discrimination and to illustrate the value of the carrier-based test based on mutations in the inbred mating population. Infant behavioral assessment: Infant neurobehavioral assessment of 14 and 30-day-old diseased animals (early infant assessment; see description (Schneider et al., 1991, Am J. Primatol. 25: 137_155). Animals were identified as sick until later, so these babies (DG5丨 and DH3 1) were tested only at 30 days of age. At each time point, 20 normal mothers bred as a control group were also tested. The composite scores of the test groups at 3 days are shown in Figure 6. When compared with normal monkeys, the composite scores of the motor groups and active groups of animals CI80, DG5 and EJ72 were much lower (more than one at all time points). The standard deviation is below.) In terms of orientation and state control, all animals are in the normal range at all time points tested. Also check the measured individual test items for exercise (Figure 6, right). Shows poor coordination, lacks the ability to maintain balance, and cannot confirm the normal degree of spontaneous motor activity'. It has resistance to passive bending and stretching of the limbs that cannot be distinguished and cannot withstand moderate resistance. That is, muscle strength decreases when active contraction. Crabbe infants also show a significant amount of tremor. Based on the measure of temperament, the sick baby tends to respond more strongly, of which 120934.doc -54- 200813227 vocalization per minute Higher. When the sick girl ^ ^ ^ is left alone or when pulled up or hugged, 'it tends to show that it can't calm itself. At the beginning of 2 months, use the modified Bally The test coffee was called 继' Am' J. pnmat〇1 22:61 67) to test the sick baby and the Zhao group. The revised Barry Scale includes three subtests: Cognition, Exercise, and Behavior. Cognitive subtests contain problem-solving programs that examine sensory-perceptual sensitivity, recognition, and the ability to respond to them. After two months, with

Often the monkeys scored lower on the sports subtest than at all other test time points. In the measurement behavior/social orientation project, the most significant difference between the diseased monkey and the normal monkey at all time points was the degree of agitation. These two neurobehavioral assessment tools detect differences between diseased monkeys and normal monkeys. Neurophysiology··Performed continuous nerve conduction studies, which began within two months of the start of life of 4 homozygous, 2 heterozygous, and 2 normal rhesus monkeys (Henghe macaque) to characterize peripheral neuropathy. Since there was no significant difference between the carrier and the normal group, these were combined to produce a comparison group that was not diseased for all subsequent analyses. For each nerve, a significant interaction between age and group was found (median nerve: ρ&lt;0·0001; sacral nerve: F(1,58)=26.44, ρ&lt;〇·〇〇〇ι; ulnar nerve: F (l, 59) = 28.68, ρ &lt; 0·0001). At all ages, the average conduction velocity of the median nerve, ulnar nerve, and phrenic nerve in the diseased monkey was significantly lower than that of the unaffected monkey (ρ &lt; 0·0001) (Fig. 7, Weimer et al., 2005, Muscle Nerve 32). (2)·· 185-190). In the diseased monkey, although the motor nerve conduction study of the bilateral median, ulna and iliac crest showed normal composite muscle action potential vibration 120934.doc -55- 200813227 field, all nerves showed severe prolongation of distal latency and severe conduction velocity. Reduce 〖again. In the diseased monkey, although the F wave is well formed and reproducible, the stagnation period is severely prolonged. When the monkey ages, the difference in conduction velocity between the diseased monkey and the unaffected monkey is more pronounced. There is no evidence of excessive time dispersion in any nerve. In the diseased monkey, the degree of conduction slowing along all regions of the nerve is not uniform and is extremely consistent between the nerves. When compared to the unaffected sputum, the continuous conduction rate indicates demyelination followed by myelin dysfunction in the diseased monkey. These findings are characteristic of severe primary demyelinating multiple neuropathy and are consistent with the expected GLD electrophysiological phenotype. The diffusion and uniform slowing of the conduction velocity of the motor is a characteristic of the hereditary demyelinating (myelin dysplasia) neuropathy secondary to the defect of the medullary formation of the peripheral nervous system. A similar study on monkeys was within the normal limits of rhesus monkeys. Neuroimaging: All MRIs were used monthly to study all affected infants, as well as age-matched carriers and non-carrying controls. MRI sequences include sagittal τ 1 weighted scan, sagittal T2 weighted scan, axial T1 weighted scan, axial proton density, axial T2 weighted scan, coronal T2 weighted scan, and comparative axial and coronal τι weighted scans. When necessary, ubiquinone (intravenous) was administered to the animals at 0 mM mM/kg for comparison. MRI descriptions include the location, privacy, and sorrow of white matter, as well as related endothelial and ventricular changes. Data from early MR imaging analysis showed no clear abnormalities observed. The diseased animals did not show abnormal changes in gray matter or white matter when compared to the age-matched control group. In addition, the ventricular system and the cortical groove are not significant. It is important to note that an thoroughly analyzed animal must undergo an autopsy at approximately 1 day. 120934.doc -56- 200813227 The change in CNS seems reasonable in the later stages of life. As a support for this theory, it was noted that a Clape animal that survived for more than 22 months had an increased T2 signal region associated with the triangle of the posterior horn of the lateral ventricle. In humans, this is often described as an early discovery of Crabbe disease. Pathology: After pathological examination, all euthanized monkeys (η = 7) had spheroid cells in the CNS white matter. The peripheral nerves are enlarged and firm. PAS-positive multinucleated spheroid cells and smaller PAS-positive macrophages are highly permeable to the brain, cerebellum, and spinal cord. It has severe diffuse demyelination in both the brain and nerves. The fibers of the peripheral nerve are widely separated and contain loose fibrous fibrous connective tissue and fine granular to uniform eosinophilic material in the gap. There is no obvious myelin around the nerve fibers, resulting in a loss of normal CNS architecture. Many animals have mild to moderate inflammation of the lungs. Two females also have acute cervical/uterine inflammation. In a single stillborn infant, there is a significant slight demyelinating lesion in the CNS and peripheral nerves and spheroid cells in the CNS. Example 5: ASC transplantation - in vitro and in vivo analysis in canines and spirits Autologous transplantation of ASC performed in elongate animals. The materials and methods used in the experiments presented in this example are now described. In vitro analysis: All studies were performed with a minimum of five canine donors and five primate donors per gender. Therefore, a minimum of ten canines and ten rhesus monkeys were processed. ASC isolation: according to the surgical protocol reviewed and approved by the Institutional Animal Care and Use Committees from the Cairn Terrier (LSU-School of 120934.doc -57- 200813227)

Veterinary Medicine) or Tulane Primate Center obtained subcutaneous adipose tissue. Isolation of ASC by treatment of canine and rhesus subcutaneous adipose tissue in a manner similar to that developed for the isolation of human ASC from lipoaspirate (Dubois et al, 2005, Adipocytes 1 (3): 139-144) . Tissue treatment, isolation, and culture are performed using selected serogroups selected to support huASC proliferation and adipogenic differentiation. Similarly, tissue culture media, plastic articles, and other reagents are standardized throughout the species to remove any potential bias or artifact sources in the manufacturing process. The tissue was minced, digested with type I collagenase at 37 ° C, and separated by differential centrifugation at room temperature. A pelleted basal vascular layer (SVF) was inoculated at a constant density of 0.156 gm tissue area per square centimeter of surface area during initial coating and at a density of 500 or 5000 cells per square centimeter of surface area during subsequent passages. Additional coating conditions for specific assays are outlined below. Cell yield per unit weight of tissue was determined. In addition, the cell proliferation rate in the cultures of each stage was calculated. If necessary, the cells are stored in accordance with the parameters optimized for huASC (Thirumala et al., 2005, September-October; 21(5): 1511-24). These steps ensure that the “manufacturing” procedures for canine eight 8 € (caASC) and primate ASC (pASC) do not deviate from the manufacturing process of huASC. These steps also simplify the comparison of any cross species of ASC properties in future steps. Analysis of Colony Formation Units: Determination of the frequency of colony forming units of a particular lineage or phenotype by limiting dilution based on poisson distribution (Mitchell et al., Stem Cells online January 120934.doc -58- 200813227 12 , 2006·2005-0235). Determine the density of nucleated cells in the stromal vascular layer (SVF). Start with a density of 104 cells per well and inoculate 2-fold serially diluted nucleated SVF cells on a 96-well plate. Hold for 9 days. At this point, the plate was directly obtained for staining with indoleamine blue (CFU-fibroblast assay) or alkaline phosphatase (CFU-ALP assay). Additional plates were induced with adipogenic or osteogenic differentiation medium and It was maintained in the culture for 9 or 21 days, followed by histochemical staining with Oil Red 0 (CFU-fat cell assay) or Alizarin Red (CFU-osteocyte assay). The presence or absence of cell colonies in the wells at cell density and the calculation of lineage-specific CFU frequencies. These analyses determine the frequency of CFU in SVF. A comparative study was performed with ASCs after passage 2 or 4 to determine adhesion and expansion. Whether any enrichment or loss of a particular lineage occurs after the increase process. Differentiation potential: Use open protocols and detection methods for the following lineage pathways (Guilak et al., 2006, Journal of Cellular Physiology, Jan; 206(1): 229- 37)), assessing the differentiation potential of prASCs under augmented generations 2, 4 and 6: adipogenesis, chondrogenesis, neurons and bone formation. Based on morphology, histochemistry and/or immunohistochemical staining and lineage PCR detection of specific gene markers to determine differentiation (Halvorsen et al, 2001, Metabolism 50 (4): 407-413; Halvorsen et al, 2001, Tissue Eng 7 (6): 729-741; Safford et al, 2002, Biochem. Biophys. Res. Commun. 294(2): 371-379; Safford et al., 2004, Exp. Neurol. 187(2): 319-328; Sen et al., 2001, J. Cell Biochem. (2): 312-319; Wickham et al., 2003, Clin· Orthop. (412): 196_212; Erickson et al., 2002, Biochem. Biophys. 120934.doc -59- 200813227

Res· Commun· 290(2): 763_769; Guilak et al., Journal of

Cellular Physiology, in press). Galactocerebrosidase activity • Biochemical assays for galactocerebrosidase activity with undifferentiated 2prASC &amp; huASC, with subculture changes. The degree of enzyme activity was compared to the degree of enzymatic activity detected by lambda in peripheral blood mononuclear cells obtained from primates and humans. In vivo analysis · Intravenous transplantation protocol: During culture, ASC was labeled with bromine-derived deoxyuridine (BrdU) to achieve tracking purposes. The ASC of subculture 2 or subculture 6 was harvested by trypsin/EDTA digestion, and washed at room temperature and suspended in serum-free medium at a concentration of no more than 1 〇6 cells per ml. Cell formation assays/chromosome spreading were performed on aliquots of prASC to confirm any signs of non-integer ploidy. The cells were administered to immunodeficient mice (n〇d/scid) by tail vein injection at a dose of up to 4 X 1 7 cells per kg body weight or about ι 6 cells per animal. The mouse control group was injected only with an equal volume of medium. After 2, 8 or 26 weeks, the hidden animals were killed and the tail was dissected to determine signs of tumor formation. Immunohistochemistry was performed on a continuous section of the main tissues (brain, liver, kidney, heart, lung, adipose tissue) in the Dendrobium sinensis fixed with formalin and embedded with antirdU antibody to detect Transplantation, labeling of ASC migration and survival. Flow Cytometry: All studies were performed on pRASC with a minimum of 5 donor isolates per sex. Flow cytometry was performed on rhesus adipose tissue-derived cells at different stages of isolation and amplification (from svf to passages 1 to 4). Cells are fixed according to published procedures and stained with a panel of antibodies (see below); other antigens may be included. If necessary, huASC was used as a control group. 120934.doc -60- 200813227 Rhesus peripheral blood mononuclear cells are used as positive controls for hematopoietic and other antigens (expected to be absent on the surface of prASC). Initial analysis was performed on both fresh and frozen pRASC. If it is determined that the frozen unaltered results, continue to collect additional information on the frozen material to increase the flexibility of the experiment and reduce the cost of flow cytometry. If cryopreservation significantly alters the surface immunophenotype, only fresh prASC data is collected. Additional studies to examine the extent of performance of ALDH in rhesus ASCs at different stages of isolation and amplification were performed. Each research department employed a kit of kits purchased from StemCo (Durham, NC) according to published methods (Mitchell et al, Stem Cells online January 12, 2006: 2005-0235). The ability to inhibit enzyme activity using the matrix analog DEAB was used as a negative control, while ALDH was used as a positive control by huASC. Proteomics: All studies were performed on pRASCs with a minimum of 5 donor isolates per sex. Initial studies were performed using the sub-1 prASC to match the existing data set obtained for huASC (Delany et al, 2005, Mol. Cell Proteomics 4: 731-7). Repeat 2D gels were performed on the protein extracts from each donor and a total of 10 gels were examined. A "mother" gel is produced for each gender and both genders. For mass spectrometry and protein identification, select up to 200 features/points of the &quot;mother&quot; gel of both genders. The pRASC &quot;parental gel is directly associated with the annotated ''parent' gel prepared for huASC In comparison, according to this analysis, the percentage of protein characteristics preserved between species is determined. The characteristics/points unique to the female or male pRASC "mother" gel obtained by mass spectrometry are identified. Those skilled in the art should be aware of Various modifications and other embodiments of the present invention have the benefit of the above-described teachings and the teachings of the related teachings in the related drawings. 120934.doc 61-200813227. Accordingly, it is understood that the invention is not limited to the specific embodiments disclosed and modified And other embodiments are intended to be included within the scope of the accompanying claims, and are intended to be The disclosure of the case is hereby incorporated by reference in its entirety. FIG. 1 , including FIG. 1A and FIG. 1B, illustrating dexamethasone Insulin, isobutylphosphonium xanthine and thiazolidinedione were induced for adipogenesis in a plated stromal cell culture cultured for 3 days in the presence of dexamethasone and insulin. After 14 days of culture, cells were fixed and oil red was used. 〇 stain neutral lipids (Fig. ία) and determine the leptin content of the modified medium by ELISA days (Fig. 1B). Figure 2 'includes Figures 2A to 2G, to describe the morphology of primate adipose stem cells (prASC), A series of images of proliferation and differentiation potential. Figures 2 and 2B are images of low-density and high-density cultures of non-human primate amplified asC, respectively, showing spindle-shaped fibroblast morphology. Figure 2C shows Shows a more heterogeneous image of primate bone marrow stem cells (prBMSC) compared to pASC with fibroblast morphology. Figure 2D depicts clonal expansion into a pure population and can produce colony formation as confirmed by Giemsa staining Image of a single cell of unit (CFU). Figure 2E is an image showing the ability of multiple generations of 3-4 pATSC to retain multilineage differentiation during adipogenesis. Figure 2F shows the retention of prATSC in 3-4 of bone formation. Image of differentiation ability. Figure 2G is an image showing the ability of multiple generations of 3-4 prATSC to retain multilineage differentiation during cartilage formation. 120934.doc -62- 200813227 Figure 3 shows undifferentiated (Undiff) and adipocytes after 9 days of induction Two-dimensional polyacrylamide gel electrophoresis performed with protein lysates prepared from human ADAS cells under differentiation (Diff) conditions. The gel was stained with Sypr〇Ruby. The figure shows representative gels from various conditions and based on A parent compound prepared by the feature preserved on a repeating gel prepared from protein extracts of four individual donors. Figure 4 is a series of images showing the characteristics of individual proteins from undifferentiated (U) and adipocyte differentiated (D) ihuASC2. In the 2D_Page analysis of total huASC lysates, 'differentiation-dependent changes are marked by fatty acid-binding proteins, adipocytes (3101), HSP20-like proteins (7204), cytosolic phosphoproteins (3107), and also 丨11/卩1^ and [丨] 11 domain protein 1 (6521) arrow identification. Figure 5 is a graph showing the increase in body weight of sick infants compared to the mean and standard deviation of normal male and female infants (20 animals in total). Figure 6 is a combination of Figures 6A and 6B to show the behavioral assessment factor scores of newborns who are 3 days old compared with normal infants (Figure 6A) and the average and standard deviation of normal infants. Two images of the neurological activity scores (Fig. 6B) of the neonatal behavior assessment.囷 is a graph showing the mean and standard deviation conduction velocity in the ulnar nerve based on group size and age. 120934.doc •63.

Claims (1)

200813227 十 'Application Patent Range: A method for treating at least one symptom of a leukodystrophy in a mammal, the method comprising administering an isolated adipose-derived stem cell (ASC) exhibiting a non-immunogenic characteristic to the mammal . 2. The method of claim 1, wherein the white matter lesion is selected from the group consisting of: Krabbe disease, adrenal white matter lesion/adrenal sheath sheath neuropathy, Aikadi-Gottris ( Aicardi-Goutieres) Syndrome, Aiexan (jers disease), Affiliated Mi ~ [Autumn Ataxia (CACH) with reduced central nervous system Müller formation, autosomal dominant brain with subcortical infarction and leukoencephalopathy Arterial disease (CADASIL), Canavan disease, cerebral vasospasm, metachromatic white matter lesions, neonatal adrenal white matter lesions, ovarian white matter lesions, chronic childhood cerebral sclerosis (PeHZaeUS) -Merzbacha disease), Refsum disease, Van der Knaap syndrome, and ZeU_ weger syndrome. 3. The method of claim 2, wherein the white matter lesion For Crabbe disease. 4. The method of claim 1, wherein the non-immunogenic feature is galactocerebrosidase expression. As in the method of the monthly reference 1, the Asc is differentiated into a galactosyl brain enzyme. 6. The method of claim 4, wherein the galactose brain (4) is expressed in an amount effective to reduce the content of serotonin galactose (psych〇_sme) in the white matter of the mammal. The method of claim 5, wherein the galactocerebrosidase is expressed in an amount effective to reduce the sphingosine galactoside content in the white matter of the ritual animal. The method of claim 1, wherein the at least one symptom is selected from the group consisting of axonal degeneration, fibrosis, macrophage infiltration, star opening y, , field cell: 3⁄4 birth, reduced sheath, IrritabiHty, excessive crying, loss of motor skills b, hypersensitivity to external stimuli, muscle stiffness, stretching of arms and legs, gripping fingers, hypotonia, blindness and deafness. The method of claim 1, wherein the ASC is administered intravenously to the mammal. The method of claim 1, wherein the ASC is selected from the group consisting of allogeneic and autologous (aut〇l〇g〇us) of the mammal. The method of claim 1, wherein the ASC further comprises a biocompatible matrix. 12. The method of claim 11, wherein the biocompatible matrix is selected from the group consisting of calcium alginate, agarose, fibrin, collagen, laminin, fibronectin ( Fibronectin), glucosamine (glye〇samin〇glycan), hyaluronic acid, heparin sulfate, - &amp; sacralin A, dermatan sulfate and bone matrix gelatin. η. The method of claim 1, wherein the mammal is a primate. 14. The method of claim 13, wherein the primate is selected from the group consisting of humans and monkeys. 15. The method of claim 13, wherein the primate is a human. 16. The method of claim 7, wherein the ASC is cultured in vitro for a period of time before the cells are administered to the mammal to induce differentiation. 17·—A method for identifying ASC that expresses galactoceretosidase in a population of cells in the month 1:1, and in the truncated source cell population, #^#胃This method includes galactocerebrosidase specificity The base 2 provides, and, the cell population, wherein the galactocerebrosidase is degraded in the ASC, thereby identifying the ASC in the cell population. 18. If a gentleman, 丄^, &lt; 10,000 is applied, wherein the substrate is galactosyl sphingosine or galactosyl ceramide. 19. The method of claim 17, wherein the ASC differentiates into a cell exhibiting at least one characteristic of a cell selected from the group consisting of: white blood cells, 'David's cells, chondrocytes, osteoblasts, Xuwang Cells (Schwann) I oligodendrocytes and neurons. 20. A method for increasing the amount of galactocerebrosidase in a tissue or mammal, &quot;Hai method comprising administering an isolated ASC exhibiting non-immunogenic characteristics to the animal, wherein the ASC is in vivo or in vivo External differentiation into a cell that exhibits galactoceretosidase. 21. The method of claim 20, wherein the mammal is a primate. The method of claim 21, wherein the primate is selected from the group consisting of humans and monkeys. 23. The method of claim 21, wherein the primate is a human. 24. The method of claim 20, wherein the differentiated ASC is a cell that exhibits at least one characteristic selected from the group consisting of white blood cells, fibroblasts, chondrocytes, osteoblasts, Schwann cells, and oligo Glial glial cells and neurons. 25. The method of claim 20, wherein the ASC is cultured in vitro for a period of time without inducing differentiation prior to administration of the mammal to the mammalian 120934.doc 200813227 animal. 26. The method of claim 20, wherein the ASC is an allogeneic to the mammal. The method of claim 20, wherein the ASC is autologous to the mammal. 28. The method of claim 20, wherein the ASC further comprises a biocompatible matrix. 29. The method of claim 28, wherein the biocompatible matrix is selected from the group consisting of calcium alginate, agarose, fibrin, collagen, laminin, fibronectin, glycosaminoglycan, Hyaluronic acid, heparin sulfate, chondroitin sulfate A, dermatan sulfate and bone matrix gelatin. 30. An isolated ASC showing non-immunogenic features, wherein the ASC exhibits galactococinase and is identified by providing a galactococinase specific matrix to the cell population, wherein the ASC is present The galactoceretosidase degrades the matrix thereby identifying the ASC in the population of cells. 31. The isolated ASC of claim 30, wherein the ASC is isolated from a primate. 32. The isolated ASC of claim 30, wherein the primate is selected from the group consisting of humans and monkeys. 33. The isolated ASC of claim 31, wherein the primate is a human. 34. The isolated ASC of claim 30, wherein the ASC differentiates into a cell selected from the group consisting of white blood cells, fibroblasts, chondrocytes, osteoblasts, Schwann cells, oligodendrocytes, and nerves. 120934.doc 200813227 yuan. 35. The ASC of claim 30, wherein the ASC is an allogeneic recipient. 36. The separate ASC of claim 30, wherein the ASC is the recipient's own. 3 7. A substantially homogeneous isolated ASCs population, wherein the isolated ASC exhibits a non-immunogenic profile, wherein the ASC exhibits galactocretosidase and is identified as follows: a galactocerebrosidase-specific substrate is provided The cell population is degraded by the presence of the galactoceretosidase in the ASC, thereby identifying the ASC in the cell population. 3 8. An isolated ASC genetically modified to exhibit galactocerebrosidase 0 39. The isolated ASC of claim 38, wherein the galactocerebrosidase is derived from humans, monkeys, mice or Rat. 40. The isolated ASC of claim 38, wherein the ASC is transfected with a vector that exhibits galactoceretosidase. 41. The isolated ASC of claim 38, wherein the ASC is a human cell. 120934.doc