WO2010033969A1 - Amniotic-fluid-derived pluripotent cells - Google Patents

Abstract

The present invention encompasses methods of inducing pluripotency in cells, methods of identifying pluripotent cells and methods of culturing pluripotent cells. The present invention further encompasses methods of making pluripotent, autologous, patient-specific, cell banks derived from amniotic fluid cells.

Description

AMNIOTIC-FLUID-DERIVED PLURIPOTENT CELLS

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. application Serial No. 61/098,960 filed September 22, 2008, the disclosure of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention is generally related to the field of cell biology. In particular, it encompasses methods of inducing pluripotency in cells and methods of making pluripotent, autologous, patient-specific cell banks derived from amniotic fluid cells.

BACKGROUND OF THE INVENTION

Each year millions of people suffer from potentially irreparable organ damage due to genetic conditions, aging, or trauma. The option of organ transplant is often limited as there is a shortage of available organs. Cellular therapy has shown potential for improving the lives of individuals stricken with organ damage.

While cellular therapy for regenerative medicine is an emerging field, it is not without its drawbacks. One such drawback, tumor formation, has greatly hindered the general acceptance of cellular therapy. As such, there is a need for improved methods of cellular therapy which target damaged cells and organs with the patient's own cells, thereby avoiding any immunological incompatibility and/or tumor formation.

Amniotic fluid contains cell types derived from the developing fetus (Priest et al. 1978; Polgar et al. 1984; Polgar et al. 1989; De Coppi et al. 2007a; Atala 2008). Approximately 1% of the cells in cultures of human amniocentesis specimens obtained for prenatal genetic diagnosis express the surface antigen c-Kit (CDl 17) (De Coppi et al. 2007a), the receptor for stem cell factor (Zsebo et al. 1990). This 1% of the cells present in the amniotic fluid can be immunoselected with magnetic microspheres to isolate the c-Kit- positive population, reported to be induced for differentiation in vitro and to be amniotic fluid stem cells (AFS) (De Coppi et al. 2007b; Atala 2008). The c-kit positive AFS cells do not show the full complement of markers expressed by embryonic stem cells, and as such they cannot be called true stem cells. This demonstrates that these 1% AFS cells are not as primitive as embryonic stem cells (De Coppi et al. 2007b) and only represent an intermediate stage between embryonic stem cells and lineage-restricted adult progenitor cells (Siegel et al. 2007; Siegel et al. 2008)

It has been shown that normal human skin cells can be reprogrammed to an embryonic state, producing induced pluripotent stem (iPS) cells (Takahashi et al. 2007; Yu et al. 2007). The generation of iPS cells with characteristics similar to those of embryonic stem (ES) cells from non-embryonic tissue can be achieved by the transduction with retroviruses carrying four transcription factors associated with pluripotency (Wernig et al. 2007; Lengner et al. 2008; Park et al. 2008b). Establishing human iPS cells from somatic cells may offer an alternative way to establish human stem cells without the need to use human embryos. This technology raises the possibility of establishing a patient-specific human ES-like stem cells, which would carry the same genetic mutations as the patients, for research and therapeutic purposes.

The current techniques of identifying and then inducing cells into pluripotency are complicated and time consuming. Thus, there exists a need for safe, reliable, simple, and rapid methods for identifying cells that can be induced into pluripotency and then rapidly inducing those cells into pluripotency.

SUMMARY OF THE INVENTION

The present invention provides novel, safe, reliable, relatively simple, and rapid methods for inducing pluripotency in cells isolated from amniotic fluid (AF).

One embodiment of the invention encompasses methods of making pluripotent, autologous, patient-specific cell banks derived from amniotic fluid cells.

In one embodiment, the present invention provides a method of producing cells having induced pluripotency comprising isolating cells from a sample of amniotic fluid; delivering to the cells at least one transcription factor associated with pluripotency; and selecting cells having induced pluripotency.

In another embodiment, the present invention provides pluripotent cells produced by a method comprising isolating cells from a sample of amniotic fluid; delivering to the cells at least one transcription factor associated with pluripotency; and selecting cells having induced pluripotency.

In a further embodiment, the present invention provides a method of making a pluripotent, autologous, patient-specific cell bank comprising obtaining a sample of amniotic fluid from a pregnant subject; isolating cells from the sample of amniotic fluid; delivering to the cells at least one transcription factor associated with pluripotency; selecting cells having induced pluripotency; and maintaining the cells having induced pluripotency. Pluripotent autologous, patient-specific cell banks produced by such a method, and methods of treating disease comprising administering cells derived from such cell banks are also provided.

In yet another embodiment, the present invention provides a method of increasing the relative number of cells in a cell population expressing one or more transcription factor associated with pluripotency comprising: isolating cells from a sample of amniotic fluid; delivering to the cells one or more transcription factors associated with pluripotency; and selecting cells exhibiting embryonic stem cell morphology, wherein cells exhibiting embryonic stem cell morphology express one or more transcription factors associated with pluripotency. Cells produced by such a method are also provided.

In a further embodiment, the present invention provides a method of providing therapeutic treatment for an individual comprising: obtaining a sample of amniotic fluid from the mother of a patient prior to the birth of the patient; isolating cells from the sample of amniotic fluid; delivering to the cells at least one transcription factor associated with pluripotency; selecting cells having induced pluripotency; causing the pluripotent cells to differentiate into a differentiated cell for therapeutic treatment; and administering the differentiated cells to the patient.

Other embodiments of the invention will be apparent from the description that follows.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows that amniotic fluid cells form about 10-fold more colonies as compared to skin cells obtained from foreskin fibroblasts;

Figure 2 shows successfully reprogrammed human AFC have a typical ES cell-like morphology similar to those generated from foreskin fibroblasts (BJ);

Figure 3 show that AFC express lower endogenous levels of 'sternness' genes oct3/4 and Sox-2 than embryonic stem cells, while c-myc and Klf-4 are expressed in comparative levels;

Figure 4 shows that in AFC the expression of Oct3/4, Sox-2, c-Myc and Klf-4 from the endogenous gene loci are reactivated and reach ES-like levels at an early iPS stage;

Figure 5 shows that foreskin fibroblasts exhibit a slower activation of their endogenous gene expression during reprogramming;

Figure 6 shows that AFC derived iPS colonies show a higher telomerase activity than human ES cells;

Figures 7a-n illustrate the induction of iPS cells from cultured human amniotic fluid (AF) cells, a) Time course of the generation of iPS colonies from AF cells (AF), neonatal (BJ) and adult skin fibroblasts (Skin); b) Frequency of AF-iPS, BJ-iPS and iPS colonies from adult skin generated 28 days after infection; c, d, e) Morphology of cultured AF cells: (c) BJ fibroblasts (d) and primary skin fibroblasts (e); f, g, h) Morphology of an established AF-iPS colony: (f) feeder-dependent (fl) or feeder independent plated onto Matrigel (£2), BJ-iPS colonies (g), iPS cells derived from adult skin (h); i) Typical image of the HES-2 human ES cell line; j, k, 1, m, n) Immunohistochemistry of selected AF-iPS colonies: Cells were stained with alkaline phoshatase (j), OCT 3/4 (k), SOX2 (1), SSEA4 (m) and NANOG (n);

Figures 8a-c depict expression profiles and telomerase activity, a) Gene expression profile of human AF-iPS: RT-PCR was used for analysis of key hES cell-specific markers (OCT3/4, SOX2, KLF4, C-MYC, TERT, NANOG, FGF4, REXl, GDF3, DPP A5) in AF cells, AF- iPS clones (AF-iPS-2, AF-iPS-3) and hES cell line, HES-2. Primers for OCT3/4, S0X2, KLF4 and c-MYC were specific for the 3' untranslated region and designed to specifically amplify the endogenous genes. ACTB internal control is shown as a positive amplification and loading control; b) Expression of exogenous factors: PCR was used for retroviral expression of the four factors. Transgene-specific primers recognize the viral- encoded transcripts of OCT3/4, SOX2, KLF4, C-MYC. The AF-iPS clones do not suppress the transgene expression; c) Telomerase activity: Each of the AF-iPS clones (AF-iPS-2, AF- iPS-3) expressed high telomerase activity, which was comparable to that of hES cell line, HES-2, whereas the cultured parental AF cells showed negligible activity;

Figures 9 a-c show quantitative real-time PCR assay for expression of OCT3/4, SOX2, KLF4, C-MYC, TERT, NANOG, GDF3 and DPP A5 in human iPS and parental AF cells: a) AF and AF-iPS cells; b) human adult skin fibroblasts and iPS cells from adult skin; c) human foreskin fibroblasts (BJ line) and iPS cells from BJ fibroblasts. Individual PCR reactions were normalized against an internal control (ACTB) and plotted relative to the expression level in the parental AF cells. Figures 9 d and e depict relative expression levels of the endogenous four transcription factors (OCT4, SOX2, KLF4, MYC) relative to the hES cell line, HES-2, over the course of reprogramming;

Figures 10 a-d show differentiation of AF-iPS into the three germ layers, a) Floating embryoid bodies (EB) derived from AF-iPS at day 7; b) and c) After EB formation, aggregates were transferred onto gelatin-coated plates and allowed to spontaneously differentiate for 10 days. Resulting cells had differentiated into neurons (b) and epithelial-like cells (c). d) After EB differentiation AF-iPS (AF), iPS cells from adult skin (Skin) and control hES (HES) were stained with anti-nestin (an ectodermal marker), anti-α-smooth muscle actin and anti-troponin I (mesodermal markers), and anti-α-fetoprotein (AFP) (an endodermal marker); and

Figures 1 la-c show the analysis of human ES cells for differentiation potential, a) Teratomas were analyzed for the presence of markers for ectoderm (Tujl), mesoderm (alpha-actinin) and endoderm (AFP). For reference, nuclei are stained with DAPI. Antibody reactivity was detected for derivatives of all three germ layers. b,c) AF-iPS produce teratomas (b) containing derivatives of different lineages in immuno-compromised mice (H&E staining) (c).

DETAILED DESCRIPTION OF THE INVENTION

For simplicity and illustrative purposes, the principles of the present invention are described by referring to various exemplary embodiments thereof. Although the preferred embodiments of the invention are particularly disclosed herein, one of ordinary skill in the art will readily recognize that the same principles are equally applicable to, and can be implicated in other compositions and methods, and that any such variation would be within such modifications that do not part from the scope of the present invention. Before explaining the disclosed embodiments of the present invention in detail, it is to be understood that the invention is not limited in its application to the details of any particular embodiment shown, since of course the invention is capable of other embodiments. The terminology used herein is for the purpose of description and not of limitation. Further, although certain methods are described with reference to certain steps that are presented herein in certain order, in many instances, these steps may be performed in any order as may be appreciated by one skilled in the art, and the methods are not limited to the particular arrangement of steps disclosed herein.

The present invention provides reliable and rapid methods for inducing cells into pluripotency. The present invention further encompasses the use of amniotic fluid epithelial cells for reprogrammed pluripotency and the creation of a patient-specific cell banks for personalized medicine. The present invention is based on the discovery that normal amniotic fluid cells (AFC) behave differently than other somatic cells when being induced to pluripotency. It has been found that amniotic fluid cells form approximately 10-fold more colonies as compared to skin cells obtained from adults when induced, and the time for colony formation is significantly shorter. Typically, amniotic fluid cells need approximately half the time to form a pluripotent colony compared to newborn foreskin fibroblasts. Further, it has been found that the Oct3/4, Sox2, Klf4 and c-Myc markers, which characterize pluripotency in stem cells, are the same as those found in human embryonic stem cells. Immunocytochemistry showed strong Oct-4 staining in the AF cell derived iPS colony and that Oct3/4 is strongly expressed (as measured by PCR) in the amniotic fluid cell derived iPS colonies. In addition, another feature of "sternness", telomerase activity, is much higher at day 12 compared to BJ cells.

The presently disclosed findings allow for the development of novel strategies to treat incurable diseases with cellular regenerative therapy utilizing fetal epithelial cells abundantly present in the amniotic fluid by directing those cells back to an earlier pluripotent stage and then re-directing them forward to patient-specific cell lineages. By establishing a cellular bank of these specific lineages from a newborn (whose mother underwent amniocentesis), immediate cellular therapy may be applied upon birth, if necessary. Further, these lineages also may function as a patient specific cellular bank ready for future personalized therapies.

It has been found that 99 % of cells present in the amniotic fluid, mainly epithelial cells originating from the skin of the developing fetus, may be used in the presently disclosed methods. These somatic skin cells are targets for induced pluripotency, whereby the internal clock is turned back to an earlier stem cell-like stage for the purpose of regenerative medicine.

The amniotic fluid cells used in the method of the present invention may be isolated from amniotic fluid, for example amniotic fluid from amniocentesis performed for fetal karyotyping or amniotic fluid obtained at term. Amniotic fluid cells are isolated from the amniotic fluid, for example by centrifugation followed by removal of the supernatant. The amniotic fluid cells are reprogrammed to pluripotent cells by delivering at least one transcription factor associated with pluripotency to the amniotic fluid cell. Transcription factors associated with pluripotency are known in the art and include Oct4, Sox2, Klf4 and c- Myc. In a preferred embodiment, the transcription factors used in the present methods are Oct4, Sox2, Klf4 and c-Myc. In another preferred embodiment, the transcription factors are Oct4, Sox2 and Klf4. In another preferred embodiment, the transcription factors are Oct4 and at least one of Klf4 and c-Myc.

Delivery of the transcription factors may be effected by methods known in the art, for example by viral transduction using one or more vectors that contain genes that encode one or more of these factors. For example, delivery of the transcription factors may be accomplished by transduction of the amniotic fluid-derived cells with vectors that contain genes that encode the factors, thereby causing ectopic expression of the factors in the cells. Methods of transduction of at least one of the transcription factors may use retroviral vectors as disclosed by Wernig at al. (2007) Nature 448:318-324 and Park et al. (2008) Nat Protoc. 3:1180-1186; lentiviral vectors as disclosed by Brambrink et al. (2008) Cell Stem Cell 2:151- 159 and Maherali et al. (2008) Cold Spring Harbor Syrnp Quant Biol 73:157-162; or a single polycistronic vector as disclosed by Carey et al. (2009) Proc. Natl. Acad Sci. USA. 106:157- 162, the disclosures of which are all incorporated herein by reference in their entireties. Other methods that may be used to deliver at least one of the transcription factors use non- viral expression such as the piggyBac transposition system disclosed by Woltjen et al. (2009) Nature 458: 776-770 and Kaji et al. (2009) Nature 458:771-775, the disclosures of which are all incorporated herein by reference in their entireties.

Other methods include the use of transient transfection of plasmids as disclosed by Okita et al. (2008) Science 322:949-953; the use of non-integrating adenoviruses as disclosed by Stadtfeld et al. (2008) Science 322:945-949; the use of chemical inhibitors as disclosed by Li et al. (2009) Cell Stem Cell 4:16-19 and Huangfu et al. (2008) Nat Biotechnol. 26: 1269-1275; the use of episomal expression vectors as disclosed by Yu et al. (2009) Science 324:797-801; and the use of Cre-excisable viruses as disclosed by Soldner et al. (2009) Cell 136:964-977, the disclosures of which are all incorporated herein by reference in their entireties. In another embodiment, at least one of the transcription factors may be delivered directly by treating the AFC with proteins comprising a protein transduction domain or cell-penetrating peptide fused to the transcription factor, as disclosed by Zhou et al. (2009) Cell Stem Cell 4:381-384 and Kim et al. (2009) Cell Stem Cell 4:472-476, the disclosures of which are all incorporated herein by reference in their entireties.

The resulting pluripotent cells may be identified by the expression of hES markers, high telomerase activity and exponential growth, in vitro embryoid body differentiation into three germ layers, and teratoma formation after subcutaneous transplantation into severe combined immune-deficient (SCID) mice.

The pluripotent cells of the invention may be directed towards a cell fate required to repair a damaged organ. For example, a fetus that is diagnosed with a specific prenatal health condition involving a damaged heart may be treated with cells that have been reprogrammed to become cardiac cells.

Pluripotent cells in accordance with the instant invention may also be banked for future use. In cases where the fetus does not have any genetic or multifactorial disease, a pluripotent cell population for autologous general tissue and organ repair may be banked. In cases where the fetus is diagnosed with a genetic or multifactorial disease, a pluripotent cell population for autologous tissue and organ repair for that specific disease may be banked. By way of example, in a case where a fetus has a heart related disease, the amniotic fluid derived pluripotent cells are directed towards cardiovascular lineage and then the cardiovascular progenitors are banked to later target heart specific diseases for future personalized regenerative medicine.

Example 1 Production of Induced Pluripotent Cells

Primary fibroblast culture from AFC

AFC were centrifuged (200 g, 10 min) at room temperature and the pellet was gently resuspended in Chang medium. Cells were plated into 100 mm gelatinized Petri dishes and left undisturbed. Media was changed every 3-4 days. After 2 weeks in culture, they were trypsinized to disperse cells and allow their growth in a monolayer. AFC were cultured at 37 ⁰C in a humified 5% CO₂ atmosphere. Cells were passaged at a ratio 1 :4 every 5 days until they reached 80% confluence, when they were infected. For subsequent passages, the media was aspirated, washed with PBS, detached with 0.05% trypsine for 5 min at 37 ⁰C. Retroviral Infection and iPS Cell Generation

PLAT-A packaging cells were plated at 8 x 10⁶ cells per 100 mm dish and incubated overnight. The next day the cells were transfected with a mixture of equal amounts of Oct4/Sox2/Klf4/c-myc pMXs vectors with Fugene-6 transfection reagent (Takahashi et al. 2007). Forty-eight hours after transfection, the medium was collected as the first virus- containing supernatant and replaced with fresh medium. Twenty-four hours later, the second virus-containing supernatant was collected. The viruses were filtered through a 0.45 μm pore- size filter and supplemented with 8 μg/ml polybrene. The human amniotic fluid cells were infected with the first virus-containing supernatant for 4 hours and medium was replaced. Twenty-four hours later the cells were re-infected with the second supernatant. Six days after transduction fibroblasts were harvested by trypsinization and re-plated on a feeder layer at 5 x lO⁴ cells per 100 mm dish. The medium was replaced with ES cell medium supplemented with 10 ng/ml bFGF and changed every other day. Twenty days later, colonies with human ES cell appearance (with a packed flat morphology, large nucleus, scant cytoplasm and occasionally spontaneous differentiation in the center), were picked and transferred into 0.2 ml of ES cell medium, mechanically dissociated to small clamps and plated on feeder cells (passage 1) for expansion.

Cell culture

Human ES cells and iPS cells were maintained on irradiated mouse embryonic fibroblasts (MEF) in Dulbecco's Modified Eagle Medium (DMEM) culture medium supplemented with 15% Fetal Calf Serum, 0.1 mM non-essential amino acids, 1 mM L- glutamine, 0.1 mM β-mercaptoethanol and 10 ng/ml human recombinant fibroblast growth factor (hbFGF) (all from Invitrogen).

Qualitative Real Time Polymerase Chain Reaction (qRT-PCR) Analysis

Relative gene expression was determined using two-step quantitative real-time PCR. Total RNA was isolated and purified as described in the RNeasy Isolation kit (Qiagen) with on column DNase I digestion. About 1 ug total RNA from each sample was reverse transcribed using the High Capacity cDNA Reverse Transcription kit (ABI) according to the manufacturer's protocol. Quantitative PCR reactions were performed with Power SYBR Green Master Mix (ABI) on an ABI Prism 7500 Real Time PCR System. Multiple transcripts were analyzed simultaneously for 40 cycles using an optimized qRT-PCR thermal profile. Data Analysis was performed using Real-Times SDS software (ABI). For each set of primers, a no template control and a no reverse amplification control were included. Post- amplification dissociation curves were performed to verify the presence of a single amplification product in the absence of DNA contamination. Fold changes in gene expression were determined using the Ct method with normalization to GAPDH endogenous control.

TRAPEZE® RT Telomerase Detection with Amplifluor® Primers

Telomerase activity was detected with a TRAPEZE® telomerase detection kit according to the manufacturer's instructions (Chemicon) with Platinum Taq Polymerase (Invitrogen). Briefly, protein extracts were prepared from each sample and 200ng of total protein was added to each reaction. If telomerase was active in the extract, it elongated the added primer, and the reaction product (templates) was amplified by PCR. This technique is highly sensitive and provided both qualitative (presence/absence) and quantitative evaluation.

Results

The present findings are focused on amniotic fluid cells that are abundantly present in the amniotic fluid and originate from the skin of the fetus. The invention disclosed herein is based on the unexpected observation that the normal amniotic fluid cells behave differently than other somatic cells upon induction to pluripotency. The amniotic fluid cells formed approximately 10-fold more colonies as compared to skin cells obtained from foreskin fibroblasts (Figure 1), and the time for colony formation was significantly shorter. Amniotic fluid cells typically needed approximately half the time to form a pluripotent colony compared to newborn foreskin fibroblasts. As disclosed herein, human AFC have been successfully reprogrammed into iPS by introducing factors Oct3/4, Sox-2, c-Myc and Klf-4, which have a typical ES cell-like morphology similar to those generated from foreskin fibroblasts (Figure 2). Also as disclosed herein, AFC expressed lower endogenous levels of 'sternness' genes oct3/4 and Sox-2 than embryonic stem cells, while c-myc and Klf-4 were expressed in comparative levels (Figure 3). The endogenous gene expression levels of the four factors by quantitative RT-PCR during the reprogramming process in AFC (Figure 4) and the foreskin fibroblasts (BJ) (Figure 5) were also analyzed. It has been found that in AFC the expression of Oct3/4, Sox-2, c-Myc and Klf-4 from the endogenous gene loci were reactivated and reached ES-like levels at an early iPS stage (AFC-iPS, clone #6, passage 1) (Figure 4). In contrast, foreskin fibroblasts showed a slower activation of their endogenous gene expression during reprogramming (BJ iPS) (Figure 5). Also, the AFC iPS colonies showed a higher telomerase activity than human ES cells (hES) (Figure 6).

Example 2 Production and Characterization of Induced Pluripotent Cells

MATERIALS AND METHODS

Cultured AFC as well as cultured neonatal and adult skin cells were induced to pluripotency with the transcription factor quartet (OCT3/4, SOX2, KLF4 and c-MYC) as described by Takahashi, K. et al. 2007. iPS colonies were characterized for morphological and growth characteristics, antigenic stem cell markers, stem cell gene expression, telomerase activity and differentiation using previously reported methods. Immunohistochemistry and gene expression profiling used to compare AF-iPS cells, neonatal and adult skin-derived iPS cells to undifferentiated hES cells was performed by standard methods. RT-PCR analysis was undertaken to compare a panel of key hES cell -specific markers and for the analysis of retroviral expression of the four exogenous transcription factors. Quantitative RT-PCR was used for detecting OCT3/4, SOX2, KLF4, c-MYC, TERT, NANOG, GDF3 and DPPA5 expression between the pairs of cultured parental AF and AF-iPS cells, cultured adult skin cells and adult skin-iPS, neonatal skin cells and neonatal skin-iPS cells. Telomerase activity was detected with a TRAPEZE telomerase detection kit (Chemicon). In vitro differentiation was evaluated by embryoid body formation and immunohistochemistry for each germ layer differentiation as described.

Cell source

AF. Cultured AF cells were used for this study. Seventeen samples were obtained as discarded, anonymous, cultured amniotic fluid cells from the Cytogenetic Laboratory at the Mount Sinai School of Medicine in New York. All cells were chromosomally normal.

Adult and neonatal skin cells. Primary skin fibroblasts were cultured from skin biopsies of eight healthy adult volunteers (ages 45-77 years) with informed consent. Neonatal BJl fibroblasts were purchased from the ATTC. hES. An established HES-2 line obtained by material transfer agreement from WiCeIl was used for these studies.

Cell culture AF cells were cultured in AmnioMax medium (Invitrogen). Adult skin (Skin) and neonatal skin (BJ) fibroblasts were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 15% fetal calf serum (FCS). The hES cell line, HES-2, was cultured in serum-free medium DMEM/F12, supplemented with 20% knock out serum replacement (KSR Invitrogen) and 10 ng/ml bFGF. iPS cells were maintained on irradiated mouse embryonic fibroblasts (MEF) in Dulbecco's Modified Eagle Medium (DMEM) culture medium supplemented with 15% Fetal Calf Serum, 0.1 mM non-essential amino acids 1 mM L-glutamine, 0.1 mM β-mercaptoethanol and 10 ng/ml human recombinant fibroblast growth factor (hbFGF) (all from Invitrogen). AF cells were cultured at 37 ⁰C in a humified 5% CO₂ atmosphere and the media was changed twice a week. Cells were passaged at a ratio of 1 :4 every 5 days until they reached 80% confluence, when they were infected. For subsequent passages the media was aspirated, the cells were washed once with PBS and trypsinised with 0.05% trypsine for 5 min at 37 °C.

Retroviral Infection and iPS Cell Generation

PLAT-A packaging cells were plated at 8 x 10⁶ cells per 100 mm dish and incubated overnight. The next day they were transfected with a mixture containing equal amounts of OCT4/SOX2/KLF4/C-MYC¹ pMXs vectors with the Fugene-6 transfection protocol. Forty-eight hours after transfection, the medium was collected as the first virus- containing supernatant and replaced with fresh medium. Twenty- four hours later, the second virus-containing supernatant was collected. The supernatants were filtered through a 0.45 μm pore-size filter and supplemented with 8 μg/ml polybrene. The human AF cells were infected with the first virus-containing supernatant for 4 hours and the medium was replaced. Twenty-four hours later the cells were re-infected with the second supernatant. Six days after transduction the fibroblasts were harvested by trypsinization and re-plated on a feeder layer at 5 x 10⁴ cells per 100 mm dish. The medium was replaced with DMEM with 15% FCS supplemented with 10 ng/ml bFGF and was changed every other day. Twenty days later, colonies with hES cell morphology (iPS colonies) were picked, mechanically dissociated to small clumps and plated on feeder cells (passage 1) for expansion.

qRT-PCR Analysis

Relative gene expression was determined using a two-step quantitative real- time PCR protocol. Total RNA was isolated and purified as described in the RNeasy Isolation kit (Qiagen GmbH) with on column DNase I digestion. About 1 ug of total RNA from each sample was reverse transcribed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to the manufacturer's protocol. Quantitative PCR reactions were performed with Power SYBR Green Master Mix (Applied Biosystems) on an ABI Prism 7500 Real Time PCR System. Multiple transcripts were analyzed simultaneously for 40 cycles using an optimized qRT-PCR thermal profile. Data analysis was performed using Real-Times SDS software (Applied Biosystems). For each set of primers, a no template control and a no reverse amplification control were included. Post- amplification dissociation curves were generated to verify the presence of a single amplification product in the absence of DNA contamination. Fold changes in gene expression were determined using the ΔΔCt method with normalization to GAPDH endogenous control.

TRAPEZE® RT Telomerase Detection with Amplifluor® Primers

Telomerase activity was detected with a TRAPEZE telomerase detection kit according to the manufacturer's instructions (Chemicon) with Platinum Taq Polymerase (Invitrogen). Briefly, protein extracts were prepared from each sample and 200 ng of total protein was added to each reaction. If telomerase activity was present in the extract, it elongated the added primer, and the reaction product (templates) was amplified by PCR. This technique was highly sensitive and provided both qualitative (presence/absence) and quantitative analyses.

Immunofluoresence analysis iPs cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Cells were stained with primary antibodies against SOX2 (1 :200, Abeam), NANOG (1:2000, Santa Cruz), nestin (1:200, Chemicon), smooth-muscle actin (1:2000, Chemicon), troponin (1:2000, Chemicon), AFP (1:100, R&D Systems) followed by staining with secondary AlexaFluor-conjugated antibodies (Invitrogen). Teratoma formation

Teratoma formation was induced by injecting 2-5 x 10⁶ cells from AF-iPS, skin-iPS, neonatal BJ-iPS, and hES cells into the subcutaneous tissue above the rear haunch of 6 week old athymic, immunocompromised SCID mice. Six to eight weeks post-injection, teratomas were harvested and fixed overnight at 4°C in 4% paraformaldehyde. Samples were immersed in 30% sucrose followed by embedding the tissue in O.C.T freezing compound (Tissue-Tek). lOμm cryosections were incubated with the appropriate antibodies and analyzed by confocal microscopy for the presence of differentiation markers: Tujl (ectoderm: Covance); alpha-actinin (mesoderm; DAKO), and alpha-fetoprotein (endoderm; DAKO). Nuclei were stained blue with 4',6-diamidine-2-phenylidole dihydrochloride (DAPI). Teratomas were sectioned and hematoxylin-eosin (H&E) stained to visualize the general morphology and derivatives of the three germ layers.

Results

To confirm that human AF-iPS cells were more efficiently established than iPS cells from other human somatic cells, iPS clone formation was characterized. By induction of pluripotency with the transcription factor quartet (OCT3/4, SOX2, KLF4 and c- MYC) as described by Takahashi et al. 2007, the cultured AF-derived skin cells formed approximately 10-times more iPS colonies than cultured adult skin cells (Fig. 7a). Of note, cultured AF skin cells formed iPS colonies twice as fast as adult cultured skin cells (Fig. 7b). In addition, the AF-iPS colonies were characterized for morphological and growth characteristics, antigenic stem cell markers, stem cell gene expression, telomerase activity and differentiation. Morphologically, the AF-iPS colonies were similar to hES cells and iPS colonies derived from adult skin. AF-iPS colonies formed tightly packaged, sharp-edged, flat colonies, as did hES cells, and aggregated colonies as adult iPS cells from adult skin (Fig. 7). Immunohistochemistry revealed that AF-iPS cells expressed the same markers as hES and human iPS cells from adult skin (Fig. 7).

Using a panel of markers to characterize the "sternness" and the undifferentiated state of AF-iPS cells, it was determined that key developmentally regulated markers were highly expressed at several time points (Fig. 8). RT-PCR analysis was performed to compare the key hES cell-specific markers, including OCT3/4, SOX2, KLF4, C-MYC, TERT, NANOG, FGF4, REXl, GDF3, DPP A5 in cultured AF cells, AF- iPS clones (AF-iPS-2, AF-iPS-3) and an hES cell line, HES-2 (Fig. 8a). Four of the retrovirally expressed transcription factors demonstrated the presence of the viral-encoded OCT3/4, SOX2, KLF4, C-MYC transcripts (Fig. 8b). Telomerase activity in AF- iPS cells was compared with that in iPS cells from adult skin fibroblasts and hES cells (Fig. 8c). Each of the iPS clones (AF-iPS-2, AF-iPS-3) expressed high telomerase activity (Fig. 8c), which was comparable to that in hES cell line, HES-2. In contrast, the cultured AF cells prior to reprogramming had negligible telomerase activity.

The expression of OCT3/4, SOX2, KLF4, C-MYC, TERT, NANOG, GDF3 and DPP A5 was assessed by quantitative real-time PCR in AF-iPS and cultured AF cells (Fig. 9a), adult skin fibroblasts and iPS cells from adult skin (Fig. 3b) and human foreskin fibroblasts (BJ line) and BJ-iPS cells (Fig. 9c). Relative levels of endogenous OCT4, SOX2, KLF4, MYC expression in AF-iPS cells and the levels in the hES cell line, HES-2 showed similar patterns over the course of reprogramming. hES cells spontaneously form ball-like embryo-like structures termed embryoid bodies (EB), which consist of a core of mitotically active and differentiating hES cells and a periphery of fully differentiated cells from all three germ layers. AF- iPS cells also developed into embryoid bodies (EB), which were similar to the EB formation of hES cells (Fig. 10a). The AF-iPS cells formed EB with peripheral differentiated cells similar to that of iPS cells from adult skin. After EB formation, aggregates were transferred to gelatin- coated plates and allowed to spontaneously differentiate over 10 days. Resulting cells were differentiated into neurons (Fig. 10b) and epithelial-like cells (Fig. 10c). After EB differentiation, cells derived from AF-iPS, iPS cells from adult skin and hES cells were stained with anti-nestin (an ectodermal marker), anti-α-smooth muscle actin and anti- troponin I (mesodermal markers) and anti-α-fetoprotein (AFP) (an endodermal marker). The hES, AF-iPS and iPS cells from adult skin all had differentiated into the three germ layers. Teratoma formation was induced by injecting 2-5 x 10⁶ cells into the subcutaneous tissue above the rear haunch of 6 week old athymic, immunocompromised SCID mice. Eight to twelve weeks postinjection, antibody reactivity was detected for markers of all three germ layers (Fig. 11 Panel A). H&E stained teratomas contained tissues representing all three germ layers ectoderm, epidermal and neural tissue; mesoderm, bone and cartilage; and endoderm, respiratory epithelium and intestinal-like epithelium (Fig. 11 Panel B). The teratoma formation experiments confirmed that AF-iPS cells have the same differentiation potential as skin-iPS cells, neonatal BJ-iPS cells and hES cells

In summary, cultured AF cells can be reprogrammed to an undifferentiated ES cell-like state. The demonstration that these terminally differentiated cells can be reprogrammed to pluripotency more efficiently than other cell types is notable, as AF cells are easily obtainable by amniocentesis, which can provide large numbers of cells from 5-10 ml of human AF. In the United States, AF cell prenatal screening has been recommended recently not only for advanced maternal age, but also for all pregnancies. Additionally, AF cells are easily obtainable from pregnancies close to term. Efficient generation of abundant AF-iPS permits investigation of prenatal cellular therapy for various recessive genetic diseases or for patient-specific personalized therapies. Fetal AF cells are more efficiently reprogrammed to pluripotency than adult cells and provide an abundant source for various basic studies.

Example 3 Cardiac Differentiation of Induced Pluripotent Cells

Developmental potency of patient-specific iPS cell lines

The developmental potential of the generated iPS cell lines is determined by teratoma formation. Briefly, iPS cells (1 x 10⁶) are subcutaneously transplanted into SCID mice and after 8 weeks teratomas are formed. These tumors are histologically analyzed for various tissues of all three germ layers.

Cardiac differentiation of Human iPS cells

Prior to differentiation, the human iPS cells are feeder-depleted by culturing on a thin layer of matrigel in hESC medium for 24 to 48 hours. To generate embryoid body (EB) like colonies, cells are dissociated to small clusters (~ 10-20 cells) using collagenase B for 20 minutes followed by trypsin-EDTA for approximately 2 minutes. The clusters are washed and then plated in 6-well low cluster plates in StemPro-34 supplemented with penicillin/streptomycin, 10 ng/mL BMP-4, 2 mM glutamine, 4 x 10^~4 M monothioglycerol, 50 μg/mL ascorbic acid and 0.5 ng/ml BMP4.

In brief, for differentiation to the cardiac lineage, the following cytokines are used: days 0-1, BMP4 (0.5 ng/ml); days 1-4, BMP4 (10 ng/ml), bFGF (5 ng/ml) and activin A (3 ng/ml); days 4-8, DKKl (150 ng/ml) and VEGF (10 ng/ml); days 8-14, VEGF (10 ng/ml), DKKl (150 ng/ml) and bFGF (5 ng/ml).

Cultures are maintained in a 5% CO₂/5% O₂/90% N₂ environment for the first 10-12 days and are transferred into a 5% CO₂/air environment.

Harvesting and phenotvpic analysis of embrvoid bodies (EBs) iPS generating EB-like colonies are collected and dissociated to single cells by a 5-minute treatment with 0.25% trypsin-EDTA. Following trypsinization, 1 mL media with serum is added and the EBs are dissociated to single cells by passaging 6 times through a 20-gauge needle. Anti-KDR-PE and anti-CD 117- APC are used to test cardiac lineage commitment by flow cytometry analysis. For intracellular staining, the harvested and dissociated EBs are fixed and stained with primary and secondary antibodies in PBS with 0.5% saponin.

IPS cell growth and differentiation

ES cells are maintained on irradiated embryonic feeder cells in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 15% fetal calf serum (FCS), penicillin, streptomycin, LIF (1% conditioned medium) and 1.5xlO^"4 M monothioglycerol (MTG; Sigma). Two days prior to the onset of differentiation, cells are transferred on gelatinized plates in the same media. For the generation of EBs, ES cells are trypsinized and plated at various densities in differentiation cultures. Differentiation of EBs is carried out in 60 mm Petri grade dishes in IMDM supplemented with 15% FCS, 2 mM L-glutamine (Gibco/BRL), transferrin (200 μg/ml), 0.5 mM ascorbic acid (Sigma), and 4.5XlO^"4 M MTG. For reaggregation, sorted cells are cultured in the media used to differentiate EBs. Cells are cultured for 20 hours at a density of 4xlO⁵/ml in ultra low attachment 24-well plates (Costar). Cultures are maintained in a humidified chamber in a 5% CO₂/air mixture at 37⁰C.

For reaggregation, 5 x 10⁵ sorted cells are reaggregated in 500 ul StemPro-34 SF medium (GIBCO), 2 mM L-glutamine, transferrin (200 ug/ml), 0.5 mM ascorbic acid (Sigma), 4.5 x 10^"4 M MTG, human (h) VEGF (5 ng/ml) (R&D Systems), hbFGF (30 ng/ml) (R&D Systems) per well in 24-well low-cluster plates (Costar). For direct plating of populations, 6 x 10⁴ cells are seeded onto individual wells of a 96- well flat bottom plate (Becton Dickenson, Franklin Lakes, NJ) coated with gelatin in StemPro-34 SF medium, 2 mM L-glutamine, hVEGF (5 ng/ml), and hbFGF (30 ng/ml).

Flow cytometry and cell sorting

EBs are harvested, trypsinized and the single cell suspension analyzed on a Facscalibur flow cytometer (Becton Dickinson) or sorted on a Moflo cell sorter (Cytomation Systems). Staining with mAb Flkl bio, Kit-PE or CD31-bio (PharMingen) is performed.

Characterization of hAFC iPS induced cardiomyocytes

The cardiac phenotype of these cardiomyocytes is evaluated at the subcellular and cellular levels.

Subcellular studies:

Sarcoplasmic Reticulum (SR) Ca²⁺ uptake: SR is isolated by differential centrifugationin these myocytes, and SR Ca²⁺-transport rates over a wide-range of free Ca- concentrations (±Ryanodine; ±thapsigargin) is examined. These SR Ca ⁺-transport assays can also be performed in cardiac homogenates under conditions which restrict Ca -uptake to SR membrane vesicles.

Myofibrillar Function: The effects of Hsp20 overexpression on the myofibrillar organization, myofibrillar creatine kinase (CK) levels and TnI phosphorylation is investigated under basal conditions. Myofibrils are also prepared and myofibrillar ATPase activity is determined. Quantitative immunoblotting assesses the protein and phosphorylation levels of myofibrillar regulatory proteins (MHC, TnT, TnC, TnI, MLC and actin). Furthermore, the levels of the SR regulatory proteins (PLB, SERCA, RyR, CSQ, HRC, junctin, and triadin) and phosphoproteins are assessed.

Cardiomvocyte function: Myocytes offer an experimental system wherein contractility is not influenced by geometric constraints, extracellular matrix, loading conditions and neurohumoral factors. In addition, contractile parameters and Ca -kinetics may be measured in the same cells, allowing the use of various perturbations to elucidate the mechanisms involved in Ca²⁺-handling and excitation/contraction coupling. Contractile and Ca²⁺-kinetic parameters are recorded to assess SR Ca²⁺-load, Ca²⁺-current, excitation- contraction (E-C) coupling and Ca²⁺-transport rates of the SR Ca²⁺-pump, Na⁺/ Ca²⁺ exchange and slower systems (sarcolemmal Ca²⁺-ATPase and mitochondrial Ca²⁺-uptake). The rates of myocyte shortening (+dL/dt) and relengthening (-dL/dt) and the Ca²⁺-kinetics are obtained (twitch, caffeine-induced contracture, and caffeine-induced contracture in Na⁺-free, Ca²⁺-free solution). The SR Ca²⁺-load of genetically altered and wild type cells is matched and then the rate of decay of similar amplitude Ca²⁺-transients is estimated. The ryanodine receptor (RyR) and E-C coupling are likely to be sensitive to SR Ca²⁺-load, and the SR Carload is manipulated as an independent variable (by conditioning with various frequencies or voltage clamp protocols). A standard series of [Ca²⁺]i (and parallel contraction) measurements are made during: 1) steady state twitches; 2) caffeine-induced contractures by rapid application of 10 mM caffeine; and 3) caffeine induced contractions in Na⁺-free, Ca²⁺- free solution, to release the SR Ca²⁺ (while net SR Ca²⁺-reuptake and Na/ Ca²⁺ exchange are prevented) and to provide SR Ca²⁺-content measurements. The steady state twitch D[Ca²⁺],, compared to the D[Ca²⁺Ji in caffeine-induced contraction, provides indirect information about the fraction of SR Ca²⁺-content released during a twitch (related to E-C coupling gain).

While the invention has been described with reference to certain exemplary embodiments thereof, those skilled in the art may make various modifications to the described embodiments of the invention without departing from the scope of the invention. The terms and descriptions used herein are set forth by way of illustration only and are not meant as limitations, hi particular, although the present invention has been described by way of examples, a variety of compositions and methods would practice the inventive concepts described herein. Although the invention has been described and disclosed in various terms and certain embodiments, the scope of the invention is not intended to be, nor should it be deemed to be, limited thereby and such other modifications or embodiments as may be suggested by the teachings herein are particularly reserved, especially as they fall within the breadth and scope of the claims here appended. Those skilled in the art will recognize that these and other variations are possible within the scope of the invention as defined in the following claims and their equivalents. All references cited herein are incorporated herein in their entirety. References

Aasen, T. et al. (2008). Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat Biotechnol 26, 1276-1284. Aoi T. et al. (2008). Generation of pluripotent stem cells from adult mouse liver and stomach cells. Science 321, 699-702. Atala, A. (2008). Advances in tissue and organ replacement. Curr Stem Cell Res Ther 3(1):

21-31. Baker M. (2008). Embryonic-like stem cells from a single human hair. Nature Reports Stem

Cells. Published online: dol:10.1038/stemcells.2008.142 Brambrink T. et al. (2008). Sequential Expression of Pluripotency Markers during Direct

Reprogramming of Mouse Somatic Cells. Cell Stem Cell 2, 151-159. Carey, B. W. et al. (2009). Reprogramming of murine and human somatic cells using a single polycistronic vector. Proc Natl Acad Sd USA. 106, 157-162. De Coppi, P. et al. (2007a). Isolation of amniotic stem cell lines with potential for therapy.

Nat Biotechnol 25(1): 100-106. De Coppi, P. et al. (2007b). Amniotic fluid and bone marrow derived mesenchymal stem cells can be converted to smooth muscle cells in the cryo-injured rat bladder and prevent compensatory hypertrophy of surviving smooth muscle cells. J Urol 177(1):

369-376. Ebert, AD. et al. (2009). Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature 457, 277-280 EgIi, D. et al. (2008). Mediators of reprogramming: transcription factors and transitions through mitosis. Nature Reviews MoI Cell Biology 9, 505-516. Fauza O. (2004). Amniotic fluid and placental stem cells. Best Paractice and Res Clin Obstet

Gynecol 18, 877-891. In't Anker, P. S. et al. (2003). Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantation, Blood 102, 1548-1549. Jaenisch, R. et al. (2008). Stem Cells, the Molecular Circuitry of Pluripotency and Nuclear

Reprogramming. Cell, 132, 567-582 Kaji, K. et al. (2009). Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature 458, 771-775. Kim, J.B et al. (2008). Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors. Nature 454(7204): 646-650. Lengner, CJ et al. (2008). The pluripotency regulator Oct4: a role in somatic stem cells? Cell

Cycle 7(6): 725-728. Lowry,WE. et al. (2008). Generation of human induced pluripotent stem cells from dermal fibroblasts. Proc Natl Acad Sci USA 105, 2883-2888 Maherali, N. et al. (2008). Induced pluripotency of mouse and human somatic cells. Cold

Spring Harbor Symp Quant Biol 73, 157-162. Mali, P. et al. (2008). Improved efficiency and pace of generating induced pluripotent stem cells from human adult and fetal fibroblasts. Stem Cells 26, 1998-2005. Meissner, A. et al. (2007). Direct reprogramming of geneticallyunmodified fibroblasts into pluripotent stem cells Nature Biotechnology 25, 1177 - 1181. Nakagawa, M et al. (2008). Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol 26(1): 101-106. Park, LH. et al. (2008a). Generation of human-induced pluripotent stem cells. Nat Protoc 3,

1180-1186 Park, LH. et al. (2008b). Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451(7175): 141-146. Polgar, K. et al. (1989). Characterization of rapidly adhering amniotic fluid cells by combined immunofluorescence and phagocytosis assays. Am J Hum Genet 45(5):

786-792. Polgar, K. et al. (1984). Neutral-red uptake by amniotic-fluid macrophages in neural-tube defects: a rapid test. N Engl J Med 310(22): 1463-1464. Priest, R.E. et al. (1978). Origin of cells in human amniotic fluid cultures: ultrastructural features. Lab Invest 39(2): 106-109. Prusa, A.R. et al. (2003). OCT4-expressing cells in human amniotic fluid: a new source for stem cell research? Hum Reprod 18, 1489-1493. Sieber-Blum, M. et al. (2008). Epidermal Neural Crest Stem Cells (EPI-NCSC) and

Pluripotency. Stem Cell Rev A, 256-260. Siegel, N. et al. (2008). Human amniotic fluid stem cells: a new perspective. Amino Acids

35(2): 291-293. Siegel, N. et al. (2007). Stem cells in amniotic fluid as new tools to study human genetic diseases. Stem Cell Rev 3(4): 256-264. Takahashi, K. et al. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5): 861-872. Wernig, M. et al. (2007). In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448(7151): 318-324. Woltjen, K. et al. (2009). piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature 458, 766-770. Yu, J. et al. (2007). Induced pluripotent stem cell lines derived from human somatic cells.

Science 318(5858): 1917-1920. Zsebo, K.M. et al. (1990). Stem cell factor is encoded at the Sl locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor. Cell 63(1): 213-224.

Claims

What is claimed is:

1. A method of producing cells having induced pluripotency comprising: isolating cells from a sample of amniotic fluid; delivering to the cells at least one transcription factor associated with pluripotency; and selecting cells having induced pluripotency.

2. The method of claim 1 , wherein the at least one transcription factor associated with pluripotency is selected from the group consisting of Klf4, Oct4, Sox2 and c-Myc.

3. The method of claim 1, wherein the transcription factor is delivered to the cells by transducing the cells with a vector encoding transcription factors Oct4 and Sox2.

4. The method of claim 1 , wherein the transcription factor is delivered to the cells by transducing the cells with a vector encoding transcription factors Klf4, Oct4 and Sox2.

5. The method of claim 1, wherein the transcription factor is delivered to the cells by transducing the cells with vectors encoding transcription factors Klf4, Oct4, Sox2 and c-Myc.

6. The method of Claim 1 wherein the transcription factor is delivered to the cells by treating the cells with at least one protein comprising a transcription factor.

7. The method of claim 1, wherein the cells are epithelial cells.

8. Pluripotent cells produced according to the method of claim 1.

9. A method of treating a disease in a patient comprising administering to the patient cells derived from a pluripotent cell according to claim 8.

10. A method of making a pluripotent, autologous, patient-specific cell bank comprising: obtaining a sample of amniotic fluid from a pregnant subject; isolating cells from the sample of amniotic fluid; delivering to the cells at least one transcription factor associated with pluripotency; selecting cells having induced pluripotency; and maintaining the cells having induced pluripotency.

11. The method of claim 10, wherein the at least one transcription factor associated with pluripotency is selected from Klf4, Oct4, Sox2 or c-Myc.

12. The method of claim 10, wherein the transcription factor is delivered to the cells by transducing the cells with a vector encoding transcription factors Klf4, Oct4 and Sox2.

13. The method of claim 10, wherein the transcription factor is delivered to the cells by transducing the cells with a vector encoding transcription factors Oct4 and Sox2.

14. The method of claim 10, wherein the transcription factor is delivered to the cells by transducing the cells with vectors encoding transcription factors Klf4, Oct4, Sox2 and c-Myc.

15. The method of Claim 10 wherein the transcription factor is delivered to the cells by treating the cells with at least one protein comprising a transcription factor.

16. The method of claim 10, wherein the cells are epithelial cells.

17. A pluripotent, autologous, patient-specific cell bank made according to the method of claim 10.

18. A method of treating a disease in a patient comprising administering to the patient cells derived from a pluripotent, autologous, patient-specific cell bank according to claim 17.

19. A method of increasing the relative number of cells in a cell population expressing one or more transcription factor associated with pluripotency comprising: isolating cells from a sample of amniotic fluid; delivering to the cells at least one transcription factor associated with pluripotency; selecting cells exhibiting embryonic stem cell morphology, wherein cells exhibiting embryonic stem cell morphology express one or more transcription factors associated with pluripotency.

20. The method of claim 19, wherein the at least one transcription factor associated with pluripotency is selected from Klf4, Oct4, Sox2 or c-Myc.

21. The method of claim 19, wherein the transcription factor is delivered to the cells by transducing the cells with a vector encoding transcription factors Oct4 and Sox2.

22. The method of claim 19, wherein the transcription factor is delivered to the cells by transducing the cells with a vector encoding transcription factors Klf4, Oct4 and Sox2.

23. The method of claim 19, wherein the transcription factor is delivered to the cells by transducing the cells with vectors encoding transcription factors Klf4, Oct4, Sox2 and c-Myc.

24. The method of Claim 19 wherein the transcription factor is delivered to the cells by treating the cells with at least one protein comprising a transcription factor.

25. A cell produced by the method of claim 19.

26. A method of providing therapeutic treatment for an individual comprising: obtaining a sample of amniotic fluid from the mother of a patient prior to the birth of the patient; isolating cells from the sample of amniotic fluid; delivering to the cells at least one transcription factor associated with pluripotency; selecting cells having induced pluripotency; causing the pluripotent cells to differentiate into a differentiated cell for therapeutic treatment; and administering the differentiated cells to the patient.