HK1071771B - Human cord blood derived unrestricted somatic stem cells (ussc) - Google Patents

Description

Human cord blood-derived Unrestricted Somatic Stem Cells (USSC)

The present invention relates to somatic stem cells, various stem cells, pharmaceutical products comprising the stem cells of the present invention, and methods for purifying and isolating the stem cells of the present invention, as well as the unique differentiation potential of the stem cells of the present invention.

Although stem cells are permanently self-renewing, such cells are typically slowly circulating. It is believed that such cells produce a transiently expanded cell population that has limited self-renewal capacity in order to increase the number of differentiated cells. To date, the challenge has been to locate stem cells in adults, and therefore, a variety of alternative markers (e.g., colony formation assays for hematopoietic cell lines) have been used in the literature.

There are several U.S. patents relating to Mesenchymal Stem Cells (MSCs) that can differentiate into several progenitor cells, such as muscle progenitor cells, connective tissue progenitor cells, or oval cells, as follows: US 5,486, 359; 5,591, 625; 5,736,396; 5,811, 094; 5, 827, 740; 5,837, 539; 5,908, 782; 5,908, 784; 5, 942, 225; 5,965, 436; 6,010, 696; 6,022, 540; 6,087, 113; 5,858,390; 5, 804, 446; 5,846, 796; 5, 654, 186; 6,054, 121; 5, 827, 735; 5,906, 934. Muscle progenitor cells further differentiate into cardiac muscle, skeletal muscle, and smooth muscle cells, while connective tissue cell progenitor cells can differentiate into bone, cartilage, and fat. Oval cells can differentiate into hepatocytes or pancreatic cells (Grompe et al, 2001).

The presence of non-hematopoietic stem cells in cord blood is still under discussion (Mayani et al 2000, Mareschi et al 2001). German patent application DE 19803267A 1 discloses for the first time osteoblastic progenitor cells and bone formation from human umbilical cord blood.

However, the use of prior art mesenchymal progenitor cells is often limited because they must be fully developed in order to be used as a tool for preparing organs or tissues. In other words, they appear to have been too shaped and specialized to produce a functional regenerating organ or tissue.

Accordingly, it is an object of the present invention to provide a stem cell capable of differentiating into various progenitor cells such as mesenchymal cells, neural cells, blood cells or endothelial cells.

It is another object of the present invention to provide a stem cell free from defects of embryonic stem cells.

It has been found that a newly identified somatic stem cell can achieve the above-mentioned objects. The somatic stem cells of the present invention are derived from human umbilical cord blood, placental blood, and/or blood from a newborn. The somatic stem cell is distinct from, but capable of differentiating into a mesenchymal stem or progenitor cell, a hematopoietic stem or progenitor cell, a neural stem or progenitor cell or an endothelial stem cell or a hepatic progenitor cell. The cells are progenitor cells, mesenchymal stem cells and neural stem cells of hematopoietic cell lines. This unique multifunctional capability, as well as the method of expanding non-restricted somatic stem cells (USSCs) derived from Cord Blood (CB) as said somatic stem cells or as committed cells according to a unique differentiation process, can be precisely characterized, standardized, and used for the production and implementation of stem cell therapy in the form of regenerative medicine.

FIG. 1 shows photomicrographs of primary USSC cultured cells plated at low density.

FIG. 2 shows photomicrographs of confluent USSC cultures.

FIG. 3 shows FACS analysis of CD45 antigen during in vitro culture.

FIG. 4 shows FACS analysis of SSEA4 embryonic markers.

FIG. 5 shows FACS analysis of HLA-type I (A, B, C), HL ADR and CD 14.

FIG. 6 shows FACS kinetics of surface labeling of CD 34.

FIG. 7 shows photomicrographs of USSC cells after neuronal induction.

FIG. 8 shows USSCs anti-nestin immunostaining of the present invention expressing neural stem cell marker nestin.

FIG. 9 shows USSCs producing cells of neuronal lineage.

FIG. 10 shows USSCs producing cells of the glial lineage.

FIG. 11 shows mineralized bone nodule formation after osteogenesis induction and staining with alizarin Red (B).

FIG. 12 shows Alcian Blue staining of USSC-derived particle cultures.

FIG. 13 shows type II collagen staining of USSC cultures after chondrogenic differentiation (green).

FIG. 14 shows adipogenic differentiation of USSC cultures confirmed by Oil Red staining.

FIG. 15 shows photomicrographs of USSC cultures before and after myoblast differentiation.

Figure 16 shows the immunocytochemistry of slow-start myosin after azacytidine treatment.

Figure 17 shows the oval cell phenotype of USSC derivatives.

FIG. 18 shows USSC culture survival and integration following injection of SCID mouse liver parenchyma.

The somatic stem cells of the present invention can be isolated and purified by several methods, including the steps of: density gradient separation, adherent cells cultured using growth factors as described in example 1 and subcultured. After a confluent cell layer has formed, the isolation process is controlled by conventional methods by morphological (fibroblast-like morphology) and phenotypic analysis using antibodies against the surface antigens of CD13 (positive), CD14 (negative), CD45 (negative), and CD29 (positive; see example 2) in order to obtain the cells of the invention.

The somatic stem cells of the present invention are capable of reacting negatively with a marker specific for a hematopoietic cell line such as CD45 and thus are distinct from hematopoietic stem cells which may also be isolated from placental cord blood. CD14 is another surface antigen that is not detectable on USSCs. In addition, the stem cells of the present invention are characterized by a group of antigens such as CD13, CD29, CD44, and CD49e present on the cell surface. Additional features of USSC formulations are the presence of mRNA transcripts of certain receptor molecules, such as epidermal growth factor (EGF-R), placenta derived growth factor receptor alpha (PDGF-RA), and insulin growth factor receptor (IGF-R). In general, the cells are also capable of expressing transcription factors such as YB1 (Y-box transcription factor 1), Runxl (dwarf-associated transcription factor 1) and AML1C (acute myeloid leukemia 1 transcription factor), which are detected by RT-PCR. However, USSC preparations are generally negative for the chondrogenic transcription factor Cart-1 and transcripts of neural markers such as neurofilament, synaptic vesicle protein, Tyrosine Hydroxylase (TH) and Glial Fibrillary Acidic Protein (GFAP).

Table 1: analysis of transcriptional characteristics of USSCs by RT-PCR

RT-PCR results obtained with putative oligonucleotide primers and mRNA from USSCs and positive control mRNAs from other tissues such as bone, cartilage, brain or cord blood mononuclear cells.

Name (R)	PCR-result USSC	PCR-results (other tissues)
			PDGFR alpha	+	+ (adult skeleton)
IGFR	+	+ (adult skeleton)
			Nerve fiber	-	+ (adult liver)
CD105	+	+ (monocytes from CB)
			GFAP	-	+ (fetal brain)
Synaptic vesicle proteins	-	+ (fetal brain)
			Tyrosine hydroxylase	-	+ (fetal brain)
YB1	+	+ (fetal brain)

Runxl	+	+ (adult skeleton)
			AML1c	+	+ (adult skeleton)
BMPR II	+	+ (adult cartilage)
			Type I collagen	+	+ (adult skeleton)
Cart-1	-	+ (monocytes from CB)
			Cartilage adhesin	-	+ (adult skeleton)
CD49e	+	+ (adult skeleton)

By using a quantitative Affymetrix GeneChip^TMMicroarray, direct comparison of RNA expression of USSC preparations with bone marrow derived MSCs (Caplan, 1991). Transcripts of the Fibulin-2 gene (gene library No. X82494) were detected at high expression levels in USSCs, but were not detected in MSCs. Fibulin-2 production was previously demonstrated in fibroblasts (Pan et al, 1993). Northern blot analysis of mRNA from various human tissues revealed a large number of 4.5-kb transcripts in heart, placenta and ovary tissues (Zhang et al, 1994). The protein has been mapped on human embryos 4-10 weeks gestation at the light microscopic level using polyclonal antibodies. Fibulin-2 was detected mainly in neuropithium, the spinal ganglia and the peripheral nerves (Miosge et al, 1996).

In a rat animal model, rat liver myofibroblasts (rMF) were co-localized with fibulin-2. The cells are located in the portal vein region, the central vein wall, and only occasionally appear in the liver parenchyma. In the early stages of fibrosis, rMF was detected in the developing scar. In the late stages of fibrosis, rMF occupies the majority of the cells in the scar (Knittel et al, 1999). In other animal models, the mouse Fibulin-2 protein is expressed in the endocardial pad matrix during embryonic cardiac development, during epithelial-mesenchymal transition. Fibulin-2 is also synthesized by developing smooth muscle precursor cells of aortic arch vessels and by coronary endothelial cells derived from neural crest and epicardial cells, respectively (Tsuda et al, 2001).

The hyaluronic acid synthase gene (D84424), the transcript of the fibromodulin gene (U05291) and the transcript 1NFLS (WO3846) were not detectable in USSCs, but were present at higher levels in MSCs. Northern blot analysis indicated that hyaluronan synthase is ubiquitously expressed in human tissues (Itano and Kimata, 1996). The product of this enzyme, hyaluronic acid, has a variety of functions, including filling the interstices, lubricating the joints, and providing a matrix through which cells can migrate (Hall et al, 1995). Fibromodulin is a member of the small family of proteoglycans. This protein has a broad tissue distribution with the greatest content occurring in articular cartilage, tendons and ligaments (Sztolovics et al, 1994). Transcript 1NFLS was cloned from human fetal liver.

The expression level of the CD24 gene (L33930) in USSCs was very low compared to the expression level in MSCs. CD24 is expressed in many B-lineage cells as well as on mature granulocytes (Van der Schoot et al, 1989).

In contrast to MSCs, the somatic cells of the invention differ according to the tissue source from which they were isolated. In addition, USSCs are characterized by the absence of expression of human white blood cell type I antigen (HLA-type I). Unlike the somatic stem cells of the present invention, previously disclosed MSCs isolated from bone marrow and muscle tissue express high levels of HLA-type I antigens on their cell surfaces. The cells of the invention are also capable of expressing stage-specific early antigen 4(SSEA4) (see FIG. 4).

Typically, somatic stem cells of the invention exhibit a fibroblast-like cell morphology and proliferate adherently.

In a preferred embodiment of the invention, the somatic stem cells (USSCs) of the invention are present in a mixture of various precursors representing other somatic stem cells, such as hematopoietic cell lines preferentially expressing AC133 and CD34, mesenchymal progenitor somatic stem cells, neuronal progenitor somatic stem cells or combinations thereof. This embodiment is preferred because it includes a high degree of regenerative potential based on the ability to differentiate into other different somatic stem cells or the presence of somatic stem cells as described in the preferred embodiments of the present invention. Preferably the mesenchymal or neuronal progenitor somatic stem cells are derived from the stem cell differentiation of the invention.

According to the present invention, there is provided a pharmaceutical product (regenerative therapeutic agent) comprising the somatic stem cell of the present invention and a plurality of the somatic stem cells of the present invention or a mixture thereof. The pharmaceutical product may further contain carrier substances or auxiliary substances, which are pharmaceutically and pharmacologically acceptable. The invention also relates to methods of using the USSC of the invention or a plurality of stem cells or mixtures thereof for gene therapy, organ transplantation, drug testing, in vitro growth of blood vessels, treatment of vascular, skeletal, hepatic, pancreatic and neurological diseases.

For example, the USSCs of the present invention may be applied topically to a desired location, e.g., with or without biological material.

Depending on the type of disease, local and/or systemic use of USSCs is appropriate. The USSCs can be used directly or with pharmaceutically acceptable carriers or adjuvants. It may be advantageous to add other substances that can promote healing of the associated disease. For example, in orthopedic applications, substances that improve bone regeneration may be used in conjunction with USSCs.

In using USSCs, it can be used in a manner substantially similar to known methods of using MSCs. In addition, the use of stem cells is disclosed in the following documents: strauer et al, "Intrakoronare, humane autologe Stammzelltransplantation zur Myokarrregenation nach Herzinfarkt", Dtsch med Wochenschr 2001; 126: 932-; quarto r, et al, "repair of large bone defects using autologous bone marrow somatic cells", N Engl J Med 2001; 344: 385-; vacatti c. a., "brief: replacing the torn phalanx "N Engl J Med 2001 with tissue engineered bone; 344: 1511-1514, May 17, 2001; hentz v.r., "tissue engineering to reconstruct the thumb", N Engl J Med 2001; 344: 15471548, respectively; brittberg m., "treatment of deep cartilage defects in the knee by autologous chondrocyte transplantation", N Engl J Med 1994; 331: 889-; freed c.r., "transplantation of embryonic dopamine neurons for the treatment of parkinson's disease", N Engi J Med 2001; 344: 710, 719; shin' oka t., "tissue engineered pulmonary artery graft", N Engl J Med 2001; 344: 532-533.shapiroa.m.j., "transplant islets for seven patients with type I diabetes by a sugarless cortin immunosuppression method", N Engl J Med 2000; 343: 230-238. The above documents are incorporated herein by reference.

The stem cells of the present invention are further described in more detail.

The stem cells of the invention are adherent cells with a fibroblast-like cell shape and two or three nucleoli (see fig. 1), obtained after EDTA-treatment with trypsin and re-seeding under suitable culture conditions (example 1) rapidly expanding to a confluency with long stretched morphology (fig. 2). FIG. 1 shows a micrograph of a primary USSC culture. The low density plated cells confirmed the fibroblast-like morphology of the USSCs. The cells can easily grow for more than 14 culture passages. FIG. 2 shows a photomicrograph of a confluent USSCs culture. The nearly confluent layer of cellular USSCs showed parallel orientation of the cells.

The surface marker phenotype of primary adherent cell layers and all derivatives of subsequent generations thereof was negative for the CD45 marker and this negative was retained. FIG. 3 shows FACS analysis of CD45 antigen during in vitro culture. The marker antigen CD45 specific for hematopoietic cells was barely detectable in USSCs from later generations (fig. 3, days 48, 54, 82).

After in vitro culture with method a (example 1), the USSC preparation became positive for stage-specific early antigen 4(SSEA4) and showed uniform expression of this embryonic marker. FIG. 4 shows FACS analysis of SSEA4 embryonic markers. Cells expanded by method a (example 1) clearly showed expression of stage-specific early antigen 4(SSEA 4). At the same time, USSC cultures were negative for HLA-type I surface antigen expression (fig. 5A), HLA-DR antigen expression (fig. 5B), and CD14 negative (fig. 5C). FIG. 5 shows FACS analysis of HLA type I (A, B, C), HLA DR and CD 14. Following in vitro expansion, USSC cultures of the invention were HLA-type I antigen negative (panel A). The cells were also negative for HLA-DR (panel B) and CD14 (panel C) specific for antigen presenting cells (HLA-DR) and monocytes (CD 14).

FIG. 6 shows FACS kinetics of surface labeling of CD 34. USSCs were grown in H5100/PEI for more than 10 generations. During this culture phase, a significant enhancement of CD34 antigen expression was observed. With respect to the hematopoietic stem cell marker CD34, fig. 6 reveals that no CD34 positive cells were detected from passage 3 until day 54. In contrast, at day 82, passage 7, a new CD 34-positive sub-population appeared. On the other hand, if the CD34 and/or FIK1 positive progenitor cells are cultured in a medium that is conditioned by cytokines specific for hematopoietic differentiation, the typical mixed or hematopoietic colonies of red blood cells and white blood cell precursors (CFU-GM and BFU-E) develop into CD45⁺Hematopoietic progenitor cells were comparable (example 9).

On the other hand, cord blood mononuclear cells lacking CD14 appear to be typical of neural stem cells if they are cultured in high glucose content medium. FIG. 7 shows photomicrographs of USSC cells after neuronal induction. USSCs of the present invention cultured in Dulbecco's Modified Eagle Medium (DMEM) containing high glucose content exhibited astrocyte-like morphology. FIG. 7 shows an example of the cultured cells, which exhibited the glial morphology obtained after 13 days of culture in culture (example 6). USSCs express the neural stem cell marker nestin after amplification with PEI. Initial observations indicated that nestin staining was less pronounced following stimulation of cells with neuronal inducing agents such as Retinoic Acid (RA), basic fibroblast growth factor bFGF, and nerve growth factor beta (NGF-beta) (McKay, 1997).

FIG. 8 shows USSCs expressing neural stem cell marker nestin in detail. (A) USSCs were cultured in H5100/PEI medium for 7 days and standard anti-nestin immunohistochemistry. (B) Cells were induced in H5100 containing RA, bFGF and NGF for 9 days, and then cultured in H5100/PEI for 7 days. Note that nestin staining was reduced compared to cells grown under conditions in (A).

Further analysis of the cells revealed that expression of proteins specific to nerve cells, such as gamma-aminobutyric acid (GABA, FIG. 9B), tyrosine hydroxylase (FIG. 9B), synaptic vesicle protein (FIG. 9D), neurofilament (FIG. 9F), or typical glial antigens, such as galactocerebroside (Ga1C, FIG. 10B) and glial fibrillary acidic protein (GFAP, FIG. 10D) were also observed. FIG. 9 shows USSCs of the invention capable of generating neuronal cell lines. USSCs of the invention were grown in H5100/PEI for 7 days and maintained in H5100 containing RA, bFGF and NGF for 27 days. After treatment by standard immobilization methods, neuron-specific antibodies are used. (A, C, D) phase contrast photographs, (B, D, F) fluorescence photographs of the same formulations as A, C, D. The nuclei were stained with the DNA stain DAPI (blue). (B) Double immunofluorescence photographs using anti-GABA (red), and anti-tyrosine hydroxylase (TH, green). (D) Anti-synaptic vesicle protein staining (green). (F) Neuron-specific anti-neurofilament staining (red) is shown. A mixture of antibodies directed against different subtypes of neurofilaments is used. FIG. 10 shows USSCs of the invention generating glial cell lines. The cells were provided with the same cell culture conditions as shown in FIG. 9. DAPI is blue. (A, C) represents a phase difference photograph. (B) The same cells as (A) were immunostained against GalC (red). (D) The same cells as (C) were stained for anti-collagen fibril acid protein (GFAP, red).

However, if the above universal stem cells are obtained from any expanded generation and induced under culture conditions with DAG (dexamethasone, ascorbic acid, beta-glycerophosphate) or in media with fibronectin, differentiation along a bone-forming cell line can be induced (example 3). As shown in Table 2, the bone-specific marker gene (alkaline phosphatase, osteocalcin, type I collagen) can be easily induced and detected by RT-PCR.

Table 2: RT-PCR analysis during bone-forming differentiation of USSCs.

	Control	Day 7	Day 14
				Beta-actin (Positive control)	+	+	+
Alkaline phosphatase	-	+	+
				Type II collagen	-	+	+
Osteocalcin	+	+	-

All three marker genes for osteogenic differentiation showed enhanced mRNA expression at day 7 of DAG induction. Beta-actin was used as a positive control.

Fig. 11 shows the formation of mineralized nodules after osteogenic induction, and after staining with alizarin red (B). By adding dexamethasone to medium H5100, ascorbic acid and β -glycerophosphate induced osteogenic differentiation of the nearly confluent USSC layer. On day 10 of stimulation, a characteristic condyle appeared (fig. 11A). Mineral deposition of the condyle can be confirmed by alizarin red staining (11B). Under the osteogenic induction conditions described above, the cells of the invention underwent a global osteogenic differentiation, as evidenced by the accumulation of mineralized bone in the various bone segments (FIG. 11A), which can be stained with alizarin red (FIG. 11B). In addition, accumulation of hydroxyapatite was detected in cell culture 6 days after staining by yon Kossa.

From the above results, it was confirmed that cord blood contains very early stem cells, which have not been detected so far, and such stem cells can be expanded in large quantities. In addition, it is possible to induce such cells to differentiate into MSCs and differentiate into osteoblasts from MSCs, as confirmed in fig. 11A. After comprehensive induction with DAG, further differentiation into mineralized nodules can be obtained, as demonstrated in fig. 11B with alizarin red staining.

Chondrogenic differentiation demonstrated even greater versatility of the cells of the invention after culture in high glucose DMEM containing dexamethasone, proline, sodium pyruvate, ITS + Premix and TGF-. beta.1 (Johns tone et al, 1998). Cells were harvested on days 0 and 14 of the differentiation experiment and analyzed by RT-PCR (table 3, example 4).

Table 3: RT-PCR analysis during chondrogenic differentiation of USSCs

	Control	Day 14
			Beta-actin (Positive control)	+	+
Cart-1	-	+
			Type II collagen (uncut)	-	+
Cartilage adhesin	-	+

Three unique marker genes for ongoing cartilage formation were expressed 14 days after cartilage stimulation.

The results of the above studies clearly show that Cart-1, a specific chondrogenic transcription factor, is up-regulated 14 days after chondrogenic stimulation. In addition, mRNA transcripts for two typical cartilage extracellular proteins (type II collagen and chondroadhesin) were also up-regulated. In addition, the cells of the invention are clearly able to produce extracellular proteoglycan molecules characteristic of chondrocyte differentiation, as demonstrated by alizarin blue staining. FIG. 12 shows alizarin blue staining of USSC-derived particle cultures. USSCs were grown in sedimented cultures in chondrogenic differentiation medium. After 6 days of culture in the induction medium, a large amount of Proteoglycan (PG), a marker unique to chondrogenic differentiation, was not detected by alizarin blue staining (panel a). In contrast, PGs were easily detectable as evidenced by blue/green color (panel B).

In addition, the presence of cartilage-specific type II collagen can be demonstrated at the protein level. FIG. 13: type II collagen staining of USSC cultures after chondrogenic differentiation (green).

USSCs were cultured in chondrogenic differentiation medium. Expression of extracellular matrix protein type II collagen at day 14 was confirmed by fluorescence microscopy using anti-type II collagen primary antibody and FITC anti-mouse secondary antibody (fig. 13B).

Further versatility of non-limiting stem cells was demonstrated herein from the aforementioned stem cell differentiation into adipocytes under PEI-method expanded culture conditions with high concentrations of dexamethasone (example 5).

FIG. 14 shows adipocytes that can be specifically stained with oil red (Sigma). Adipocytes are characterized by a large number of intracellular vesicles and specific red staining by oil red.

In addition, favorable evidence of muscle differentiation occurred when USSCs were cultured in H5100 containing 10. mu.M 5' -azacytidine for 24 hours, followed by culture with H5100 containing 100ng/ml bFGF. The change in cell morphology was accompanied by expression of slow-agonistic myosins (FIGS. 15 and 16).

In addition, when reading from CD34 as shown in FIG. 6⁺When sub-populations subcloning PEI-induced USSCs, typical oval cells and oval cells usually appear in later generationsProliferation of cells (FIG. 17) (example 8). The cells expressed the enzyme dipeptidyl peptidase IV to varying degrees, indicating that the oval cells can be further differentiated into hepatocytes.

Expanded USSCs in vitro survive and survive after injection into the regenerated liver of SCID mice with 50% partially hepatectomized and non-hepatectomized liver, while cord blood-derived monocytes are undetectable even with a 25-fold higher number of cells transplanted. FIG. 18 shows survival and integration of USSC cultures after injection into the liver parenchyma of SCID mice. FIG. 18A: red fluorescence 7 days after transplantation, indicating the survival and integration of PKH 26-labeled human USSCs of the invention into mouse liver tissue (without hepatectomy). In contrast, no red fluorescence indicating integration of human MNCs was detected after transplantation of cord blood-derived Monocytes (MNCs). FIG. 18B: cryosection of mouse liver tissue equivalent to a: transmitted light micrographs of mouse liver tissue with integrated human USSCs.

Since the precursors of liver and pancreatic β -islet cells are identical, the CB-derived oval cells are also able to differentiate into insulin-producing β -islet cells, making it a useful tool for cell therapy in diabetic patients or patients with liver disease.

In addition to the above-noted apparent clinical utility, any well characterized and expanded stem cell component and progeny thereof under standardized conditions can be used to monitor and determine the effects and molecular and cellular effects of newly developed pharmaceutical agents, and thus can also be used to replace certain animal-based experiments.

Thus, the stem cells and differentiated cells derived from human cord blood cultures disclosed herein, which have been standardized, are useful as valuable test reagents in the pharmaceutical and biomaterial industries.

The USSC preparation was able to develop into multiple colonies of different hematopoietic cell lines under appropriate culture conditions, thereby providing evidence that such cells could be used for hematopoiesis.

The mixed colonies formed by the cells also had cells labeled positive for FLK1+ and AC133+, Tiel, and Tie2 in appropriately conditioned medium containing specific concentrations of VEGF, Flt3L, SCGF (stem cell growth factor), and in methylcellulose. After further differentiation, the markers appear characteristic of endothelial cells: AC133 negative, CD31⁺、CD54⁺、VWF⁺、VE-Catherin⁺。

The use of the in vitro growth of said endothelial cells of autologous and allogeneic blood vessels for the treatment of vascular diseases is disclosed.

At the same time, it is clear that all the above-mentioned in vitro prepared and uniformly expanded progenitor cells and their differentiated cells can be used at the clonal level as extremely important tools for determining the role of specific genes and their products for cell biology and all medical uses based on cell or molecule mediated therapy.

Only a small fraction of cells of this particular type are sufficient to produce the adherently growing USSCs of the invention in large quantities, as well as more differentiated mesenchymal stem cells for the production of regenerative cell types useful for medical purposes.

A completely new aspect of this knowledge is the fact that the progenitor cells may develop asymmetrically into two or more different differentiated cell types. Thus, novel biological principles including the regulation of common components even in functionally targeted cell regeneration performed in vitro are revealed.

As a result of the present invention, stem cell-based therapeutics must be designed according to this principle, and cannot include only one clonal cell type. The invention will be further illustrated in the following non-limiting examples.

Example 1

Collection of Cord Blood (CB)

Cord blood collection in the obstetrical department is performed with consent from the situation-conscious mother. After the infant is born, the placenta remains in the uterus, where the umbilical cord is double clamped 7-10 cm from the infant's navel and cut off laterally. After the umbilical cord is sterilized, the umbilical vein is incised, and CB is collected in a collection bag containing glucose Citrate Phosphate (CPD) as an anticoagulant.

Isolation of monocytes from umbilical cord blood

The cord blood was carefully loaded into a Ficoll solution (density 1.077 g/cm)³) The above. Density gradient centrifugation (450g, room temperature, 25 min) was performed. Mononuclear cells (MNC) at the interface of the two phases were collected and washed 2 times with phosphate buffered saline, ph7.3 (PBS).

Preparation of adherent cell layer with fibroblast-like morphology

At about 5 × 10³Cells/cm²Plating mononuclear cells in T25 culture flasks (Nunclon) [ A.), B.), C.)]. Four different culture methods were used to initiate the growth of adherent stem cells:

a.) first of all in a solution containing 10^-7CB-derived MNCs were cultured in Myelocult H5100 medium (StemShell Technologies, Vancouver/Canada) with M dexamethasone.

B.) first of all in a solution containing 10^-7CB-derived MNCs were cultured in Mesencult (StemShell technologies, Vancouver/Canada) with M dexamethasone.

C.) first of all in a solution containing 10^-7CB-derived MNCs were cultured in low glucose dmem (bio whittaker) with 30% FCS with M dexamethasone.

D.) at 5X 10⁶Density of/ml CB-derived MNCs were plated into 10ml of dexamethasone-free Myelocult H5100 medium (StemShell Technologies, Vancouver, Canada) in 50ml culture flasks (Nunclon).

All cultures were grown at 37 ℃ in 5% carbon dioxide in completely humid air and fed 1 time per week, including removing all medium containing non-adherent cells and adding 10ml of fresh medium. After several time points adherent spindle cells were removed by treatment with 0.05% trypsin and 0.53mM EDTA for 2 minutes, rinsed with medium containing 50% serum, collected by centrifugation at 780g, and analyzed by flow cytometry or RT-PCR. Adherent cells with fibroblast-like morphology appeared in about 30% of all cell cultures after 2-3 weeks.

Culture conditions for amplification of USSCs of the invention

USSCs of the present invention can be amplified in H5100 medium (PEI medium) containing 10ng/ml IGF I (insulin-like growth factor-I), 10ng/ml PDGF-BB (platelet-derived growth factor-BB) and 10ng/ml rh-human EGF (recombinant human epidermal growth factor) at a density of 1X 10⁴And 1X 10⁵Between cells/ml (amplification method A). Additionally, the USSC preparation can be expanded in initiation growth media A, B and C.

Example 2

Immunophenotypic classification of cells by cytofluorimetric assays

To determine the immunophenotype of USSCs, FITC-conjugated anti-CD 45(Becton Dickinson, Coulter), PE-conjugated anti-CD 14(PharMingen, Coulter), goat F (ab')₂Anti-mouse IgG + IgM (H + L) -FITC (Coulter) -labeled anti-SSEA-4 (MC-813-70), anti-CD 10-PE (CALLA, PharMingen), goat F (ab')₂Anti-mouse IgG + IgM (H + L) -FITC labeled anti-HLA-type I (Coulter), anti-CD 13-PE (Becton Dickinson, Coulter); cells were stained for anti-CD 29(Coulter), anti-CD 44(Coulter), anti-CD 49e (Coulter), anti-CD 90(Coulter), anti-HLA-II-FITC (Coulter). Cells were analyzed using EPICS XL (Coulter or FACS analyzer (Becton Dickinson).

Example 3

Confirmation of osteogenic differentiation potential of USSCs

USSCs obtained as described in example 1 were cultured in standard medium until a 70% confluency was reached. By adding 10^-7M dexamethasone, 50. mu.g/ml ascorbic acid and 10mM beta-glycerophosphate induced osteogenic differentiation of the cells (Bruder et al, 1994, Jaiswal et al, 1997). On day 10 of stimulation, the cells that showed calcium phosphate deposition produced the condyle. Mineralized condyles were detected by alizarin red staining as follows: adherent cells in the culture were washed 2 times with PBS ph7.3 and stained with 5ml of 0.1% alizarin red solution at room temperature for 1 hour, followed by washing with 0.1% acetic acid and absolute alcohol and PBS. Alizarin red and von Kossa staining of calcium confirmed the mineralizing potential of the cells (Stanford et al, 1995, Rungby et al, 1993). Osteogenic differentiation was also confirmed by RT-PCR using the bone-specific differentiation markers osteocalcin (0C), Osteopontin (OP), bone-specific Alkaline Phosphatase (AP), Bone Sialoprotein (BSP), platelet-derived growth factor receptor alpha (PDGF-Ra), Epidermal Growth Factor Receptor (EGFR), and type I collagen.

Example 4

Demonstration of the chondrogenic differentiation potential of USSCs

For chondrogenic differentiation, 2X 10 cells were prepared⁵Adherent stem cells were placed in sedimented cultures in 15ml polypropylene tubes. High glucose DMEM containing dexamethasone, proline, sodium pyruvate, ITS + Premix and TGF-. beta.1 was used as the cell culture medium (Johnstone et al, 1998, Yoo et al, 1998). Cell fractions were analyzed by RT-PCR on days 7, 14 and 21 for cartilage-specific gene products encoding Cart-1, type II collagen and chondroadhesin. In addition, USSCs were used in the sediment culture. After 2 weeks, the deparaffinized sections were fixed with 4% paraformaldehyde for 15 minutes at room temperature and washed with ethanol. Sections were stained in 1% alizarin blue/3% acetic acid, ph2.5(Sigma) for 5 minutes and washed with distilled water. They clearly show a positive staining of specific proteoglycans as confirmed by alizarin blue staining (FIG. 12) (Chao, G. et al, 1993). After 14 days of chondrogenic induction, consolidation was performed according to standard methodsCells were fixed and the presence of collagen type II specific cell matrix was confirmed by fluorescence microscopy analysis (Rosenbaum et al, 1998) (FIG. 13B).

Example 5

Confirmation of USSCs adipogenic differentiation potential

As confirmed by oil red staining, in a solution containing 10^-6Culturing USSCs in H5100 with M dexamethasone, 50. mu.g/ml ascorbic acid, and 10mM β -glycerophosphate resulted in partial differentiation of USSCs into adipocytes (Ramirez Zacaraias et al, 1992).

Example 6

Confirmation of the neuro-differentiation potential of USSCs

Cell isolation and culture conditions for glial cells

The mononuclear cord blood cells obtained as described above were depleted of CD14+ cells by CD 14/Magnetically Activated Cell Sorting (MACS) separation system using a VS + separation column according to the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach). Monocyte with CD14 eliminated at 2X 10⁶The density of the cells was determined by culturing in T25 culture flasks (Nunclon) containing 10ml of high glucose medium (Dulbecco's MEM containing 4500G/L glucose) at 37 ℃ in 5% CO₂The culture was carried out in a completely humid atmosphere. After 10-15 days of culture, cells in the shape of glia were examined.

Differentiation into neuronal cells

A) Cells were expanded for 7 days in H5100 medium itself or in H5100 medium containing 40pg/ml PDGFB, 10pg/ml EGF, 10pg/ml IGF-I. Cells were trypsinized at approximately 3.5X 10³Cells/cm²Was plated onto 24-well plates on coverslips covered with poly-D-lysine (PDL) and laminin (PDL/lam). Then, by adding e.g. all-trans retinoic acid (10)^-5Induction formulations of M), bFGF (20ng/ml) and NGF-beta (50ng/ml) initiate neuronal differentiation.

Fluorescence microscopy

After the induction period (27 days), cells were fixed according to standard methods (Rosenbaum et al, 1998) and stained with antibodies against neuron specific antigens. Samples were analyzed by fluorescence and transmission light microscopy.

Example 7

Demonstration of differentiation potential in muscle cell lines

In H5100 medium supplemented with 10ng/ml PDGFBB, 10ng/ml EGF, 10ng/ml IGF (StemCell Technology), 5% CO at 37 ℃₂Medium culture contains 1X 10⁴USSCs until the cells reached approximately 70% confluence. Cells were then cultured with 10 μ M5' -azacytidine (Sigma) for 24 hours, washed 2 times with PBS, and cultured in H5100 medium supplemented with 100ng/ml FGF (Sigma). After 1 week of culture in differentiation medium, the morphology of the cells changed (FIG. 15). After 10 days, the cells were trypsinized and transferred to fibronectin-covered glass chamber slides for immunostaining.

Immunohistochemistry

Cells were fixed with 5% formaldehyde/PBS for 15 min and washed 2 times with PBS, pH 7.3. Cells were cultured using standard methods with an anti-skeletal myosin (slow) specific primary antibody (clone NOQ7.5.4D, 1: 400) (shown in green) and either an anti-CD 13 primary antibody (shown in red) or a monoclonal anti-skeletal myosin primary antibody (clone MY-32, 1: 4000). Staining was positive for USSCs cultured under the above culture conditions (fig. 16).

Example 8

Human USSC cells and cord blood-derived Monocytes (MNCs) were labeled with PKH26 red fluorescent cell adapter kit (Sigma, PKH26 GL). 2 x 10 to⁵USSCs and 5X 10⁶MNCs were injected into the liver parenchyma of SCID mice with and without 50% hepatectomy. Complete liver regeneration in hepatectomized animals was obtained 7 days after transplantation. Liver tissue was analyzed for the presence of red-labeled human cells by fluorescence microscopy of cryosections (fig. 18).

Example 9 demonstration of differentiation potential of USSC hematopoietic cell lines

Three different USSCs formulations (USSCs in DMEM medium with 30% FCS) grown for long periods (passage 5-8) in appropriate expansion medium^KCB55USSC in H5100 Medium containing dexamethasone^KCB12USSC in MesenCult Medium with dexamethasone^KCB13And USSC in H5100 Medium containing PEI^GK12) Inoculation into 250ul (2X 10)⁴-2×10⁵Cells) cell suspension, the cells were seeded in hematopoietic special media (methodult 4434) on 24-well plates 3 times repeatedly. More than 50 cell colonies were counted and classified according to established criteria to determine their origin in granulocyte/macrophage (CFU-GM), early erythrocyte (BFU-E) or pluripotent (CFU-GEMM) progenitors. Under differentiating conditions, it was confirmed that colony formation in the different cultures began at week 1 of observation and until week 3 thereafter. The USSC preparation formed multiple colonies of different lineages, thereby providing evidence that such cells were capable of hematopoiesis.

Example 10

Molecular method for analyzing unrestricted somatic stem cells and their continuous differentiation products

The PCR primers used to amplify the specific cDNA sequences from osteocalcin, osteopontin, bone sialoprotein, alkaline phosphatase, PDGFR α and EGF receptors were selected from different corresponding exons, in order to be able to distinguish their correspondingly generated DNA fragments according to size.

The corresponding special cDNA clones were obtained by cloning into the pCRL1 vector (Invitrogen/USA) and subsequently transferred into the E.coli strain TOP 10F and characterized by cycle sequencing on an automated sequencer (Applied Biosystems).

The RT-PCR reaction is carried out in a two-step process. 200ng of total RNA from the cells were first reverse transcribed in a 20. mu.l reaction volume for 1 hour at 50 ℃ using 10U of AMV reverse transcriptase (Promega, Mannheim), 1.5pmol 3' -gene specific primer, 1mM dNTPs and buffer provided (Promega, Mannheim). The PCR reaction was performed using 2. mu.l of cDNA in 1U HotStarTaq DNA polymerase, buffer and Q-solution (Qiagen, Hilden), 1.5mM dNTPs and 20pmol3 '-and 5' -gene-specific primers. The PCR reaction was carried out as follows: the initial step was 95 ℃ for 15 minutes, 94 ℃ for 30 seconds for 37 cycles, 56 ℃ for 30 seconds, 68 ℃ for 1 minute, and the final polymerization step was 68 ℃ for 5 minutes.

Table 4: PCR primers for amplification of specific cDNA sequences

The 5 '-and 3' -primer sequences of the genes examined and the expected length of the PCR fragment in bp are shown

Name (R)	5' primer sequence	3' primer sequence	bp
				PDGFR alpha	acagtggagattacgaatgtg	cacarcagtggtgatctcag	251
IGFR	cgagtggagaaatctgcgg	gaccagggcgtagttgtag	272
				EGFR	tgccacaaccagtgtgct	ccacataattacggggacac	205
Nerve fiber	attcgcgcgcagcttgaag	cctggtaggaggcaatgtc	265
				GFAP	ctctccctggctcgaatgc	cctcctgataactggccg	871
Synaptic vesicle proteins	cctgcagaacaagtaccgag	ccttgctgcccatagtcgc	516
				Tyrosine hydroxylase	caccttcgcgcagttctcg	ctgtccagcacgtcgatgg	387
YB1	ggtgaggaggcagcaaatgt	agg9ttggaatactgtggtc	279
				Runxl	gcaagctgaggagcggcg	gaccgacaaacctgaagtc	296
AMLlc	cagtgcttcatgagagaatgc	gaccgacaaacctgaagtc	453
				Cart-1	ggagacgctggacaatgag	ggtagctgtcagtccttggc	560
CD105	cctgccactggacacagg	atggcagctctgtggtgttg	411
				Type I collagen	ggacacaatggattgcaagg	aaccactgctccactctgg	441
Type II collagen	tttcccaggtcaagatggtc	cttcagcacctgtctcacca	377
				Osteocalcin	agtccagcaaaggtgcagc	ggccgtagaagcgccgat	231
Alkaline phosphatase	gcttcagaagctcaacacca	cgttgtctgagtaccagtcc	454
				Beta-actin	gagaaaatcttgcaccacac	ctcggtgaggatcttcat	340

Reference to the literature

Bruder S., Fink DJ., and Caplan AI (1994). mesenchymal stem cells for use in bone formation, bone repair, bone regeneration therapy. Cell. biochem.56: 284.

caplan, ai, mesenchymal stem cells. (1991) J.orttop.res.9: 641-50.

Grompe, M and Finegold M.J. hepatic Stem cells/p.455-497 from Stem cell biology, Cold Spring Harbor Laboratory Press, 2001.

Grompe, m.and Finegold MJ. hepatic stem cells. p.455-497 from StemCell Biology, Cold Spring Harbor Laboratory Press, 2001.

Hall，C.L.；Yang，B.；Yang，X.；Zhang，S.；Turley，M.；Samuel，S.；Lange，L.A.；Wang，C.；Curpen，G.D.；Savani，R.C.；Greenberg，A.H.；Turley，E.A.：Overexpression of thehyaluronan receptor RHAMM is transforming and is also requiredfor H-ras transformation.Cell 82：19-26，1995.

Itano, n.; expression cloning and molecular characterization of Kimata, k. Biol chem.271: 9875 + 9878, 1996 Jaiswal N, Haynesworth SE, Caplan AI, Bruder SP. purified, culture expanded human mesenchymal stem cells were osteogenically differentiated in vitro. J Cell biochem.1997 Feb; 64(2): 295-312.

In vitro chondrogenesis of Johnstone B, Hering TM, Caplan AI, Goldberg VM, Yoo JU. bone marrow-derived mesenchymal progenitor cells. Exp Cell res.1998 Jan 10; 238(1): 265-72.

Different roles of the localized (myo-) fibroblast subpopulation in liver tissue repair of Knittel T, Kobold D, Piscaglia F, Saile B, Neubauer K, MehdeM, Timpl R, Ramadori G. Histochem Cell Biol 1999 Nov; 112(5): 387-401

Kritzik m.r. and Sarvetnick n. pancreatic stem cells. 499-513 fromStem

Cell Biology，Cold Spring Harbor Laboratory Press，2001.

Mareschi K, Biasin E, pinabello W, Aglietta M, Madon E, Fagioli f. 86(10): 1099-100 isolation of human mesenchymal stem cells: bone marrow and umbilical cord blood.

Pan, T. -C.; sasaki, t.; zhang, r. -z.; fassler, r.; timpl, r.; chu, M. -L.: the structure of fibulin-2 is not expressed, a novel extracellular matrix protein with multiple EGF-like repeats and a consensus motif for calcium engagement. Cell biol.123: 1269-1277, 1993.

Ramirez-Zaciais JL, Castro-Munozledo F, Kuri-Harcuch W.Histochemistry 1992 Jul; 97(6): 493-7 fat conversion and triglycerides are quantified by staining the cytoplasmic fat with oil red 0.

Rosenbaum, c., Kluwe, l., Mautner, VF., Friedrich, r.e., Mutter, HW., Hanemann, CO. (1998): enhanced proliferation and potassium conductivity of schwann cells isolated from NF2 schwann tumors can be attenuated with quinidine. Neuroobiol Dis5, 55-64.

Rungby J, kastem M, Eriksen EF, Danscher g.histochem j.1993jun; 25(6): 446-51 von Kossa reaction of calcium deposits: silver lactate staining increases sensitivity and reduces background.

Stanford CM, Jacobson PA, Eanes ED, Lembke LA, Midura RJ.J Biol Chem 1995 Apr 21; 270(16): 9420-8 Rapid formation of hydroxyapatite mineral in osteoblast lines (UMR 106-01 BSP).

Shariro a.m.j., Lakey j.r.t., Ryan e.a., Korbutt g.s., Toth e., Warnock g.l., kneema n.m., Rajotte r.v.n Engl JMed 2000 Jul 27; (343): 230-238 islet transplantation was performed in seven patients with type I diabetes using an immunosuppressive method of aglycosin.

Sztroovics, r.; chen, X. -N.; grover, j.; roughly, p.j.; korenberg, j.r.: localization of the human fibromodulin gene (FMOD) on chromosome 1q32 and completion of the cDNA sequence. Genomics 23: 715-717, 1994.

Tsuda T, Wang H, Timpl R, Chu ML. expressed marker-transformed mesenchymal cells in developing heart valves, aortic arch vessels and coronary vessels. Dev Dyn 2001 Sep; 222(1): 89-100

Van der school, c.e.; huizinga, t.w.j.; gadd, s.k.; ma jdic, 0.; wijmans, r.; knapp, w.; von dem Borne, a.e.g.: three novel PI-linked proteins were identified on granulocytes. In: knapp, w.; dorken, b.; gilks, w.r.; rieber, e.p.; schmidt, r.e.; stein, h.; von dem Borne, a.e.g.k.: leukocyte Typing IV: white cell differentiation antibodies. oxford: oxford Univ.Press (pub.)1989.Pp.887-891

Yoo JU, Barthel TS, Nishimura K, solvaga L, Caplan AI, Goldberg VM, Johnstone b. J Bone Joint Surg am.1998 Dec; 80(12): 174557.

zhang, r. -z.; pan, T. -C.; zhang, z. -y.; mattei, M. -G.; timpl, r.; chu, M. -L.: fibulin-2(FBLN 2): human cDNA sequence, mRNA expression, and mapping of the gene on human and mouse chromosomes. Genomics 22: 425-430, 1994.

Abbreviations

DAG osteogenic differentiation medium containing dexamethasone, ascorbic acid and beta-glycerophosphate

HLA human leukocyte antigens

MSC mesenchymal stem cells

PEI culture medium containing PDGF-BB, EGF and IGF

Stage-specific early antigen 4 of SSEA4

USSC unrestricted somatic stem cells

PG proteoglycan

Sequence listing

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Claims (2)

1. Use of non-restricted somatic stem cells in the manufacture of a medicament for treating a vascular disease, a myocardial or smooth muscle disease, a liver disease, type I diabetes, a neurological disease, parkinson's disease, or a hematologic disease, wherein the non-restricted somatic stem cells are isolated from umbilical cord blood or placental blood and:

(i) negative for CD45 and CD14 surface antigens;

(ii) positive for CD13, CD29, CD44, and CD49e antigens;

(iii) positive reaction to the expression of YB1, AML-1, RUNX-1 and fibulin-2; and is

(iv) Negative response to expression of hyaluronan synthase, fibromodulin and INFLS.

2. The use of claim 1, wherein the medicament further comprises in vitro differentiated progeny of the unrestricted somatic stem cell.