WO2010068758A1 - Cellules pluripotentes dérivées de cellules stomatiques et leurs méthodes d'utilisation - Google Patents

Cellules pluripotentes dérivées de cellules stomatiques et leurs méthodes d'utilisation Download PDF

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WO2010068758A1
WO2010068758A1 PCT/US2009/067503 US2009067503W WO2010068758A1 WO 2010068758 A1 WO2010068758 A1 WO 2010068758A1 US 2009067503 W US2009067503 W US 2009067503W WO 2010068758 A1 WO2010068758 A1 WO 2010068758A1
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cells
spheres
cell
reprogrammed
culture
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PCT/US2009/067503
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Douglas Dean
Yongqing Liu
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University Of Louisville Research Foundation, Inc.
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Priority to EP09832539A priority Critical patent/EP2376625A4/fr
Priority to AU2009324641A priority patent/AU2009324641A1/en
Publication of WO2010068758A1 publication Critical patent/WO2010068758A1/fr
Priority to US12/951,678 priority patent/US20110243898A1/en
Priority to US13/976,879 priority patent/US20130312130A1/en

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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/405—Cell cycle regulated proteins, e.g. cyclins, cyclin-dependant kinases
    • C—CHEMISTRY; METALLURGY
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C—CHEMISTRY; METALLURGY
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/27—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from lung cells, from cells of the respiratory tract
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells

Definitions

  • the presently disclosed subject matter relates to reprogrammed somatic cells.
  • the presently disclosed subject matter provides reprogrammed somatic cells, methods for generating reprogrammed somatic cells, and uses for reprogrammed somatic cells.
  • iPSCs induced pluripotent stem cells
  • Retroviral expression vectors have been shown to be characterized by significant safety issues, most notably increased incidences of cancer resulting from the introduction and subsequent integration of the vectors into the cells of subjects to whom the retroviral vectors had been administered. What are needed, then, are methods for reprogramming somatic cells to reintroduce some degree of pluripotency desirably without the need to resort to the use of recombinant expression constructs, particularly in the form of retroviral constructs. This need, among others, is addressed by the presently disclosed subject matter.
  • the methods comprise (a) growing a plurality of fibroblasts in monolayer culture to confluency; and (b) disrupting the monolayer culture to place at least a fraction of the plurality of fibroblasts into suspension culture under conditions sufficient to form one or more embryoid body-like spheres, wherein the one or more embryoid body-like spheres comprise a reprogrammed cell (e.g., a reprogrammed fibroblast) comprising expressing one or more markers not expressed by a cell growing in a monolayer culture prior to the disrupting step.
  • the fibroblast is a mammalian fibroblast, optionally a human fibroblast.
  • the fibroblast is a non-recombinant fibroblast.
  • the disrupting comprises scraping the confluent monolayer off of a substrate upon which the confluent monolayer is being cultured.
  • the methods further comprise maintaining the one or more embryoid body-like spheres in suspension culture for at least one month.
  • the one or more embryoid body-like spheres are maintained in a medium comprising Dulbecco's Modified Eagle Medium (DMEM) and 10% fetal bovine serum (FBS).
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • the reprogrammed fibroblast expresses a stem cell marker selected from the group consisting of Oct4, Nanog, fibroblast growth factor-4 (FGF4), Sox2, Klf4, SSEA1 , and Stat3.
  • the reprogrammed fibroblast is non- tumorigenic in nude mice.
  • the presently disclosed subject matter also provides reprogrammed fibroblasts produced by the disclosed methods.
  • the presently disclosed subject matter also provides reprogrammed fibroblast cells non-recombinantly induced to express one or more endogenous stem cell markers.
  • the presently disclosed subject matter also provides formulations comprising the disclosed reprogrammed fibroblast cells in a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutically acceptable carrier or excipient is acceptable for use in humans.
  • the presently disclosed subject matter also provides embryoid body-like spheres comprising a plurality of reprogrammed fibroblasts.
  • the presently disclosed subject matter also provides cell cultures comprising the disclosed embryoid body-like spheres in a medium sufficient to maintain the embryoid body-like spheres in suspension culture for at least one month.
  • the presently disclosed subject matter also provides methods for inducing expression of one or more stem cell markers in a fibroblast.
  • the methods comprise (a) growing a plurality of fibroblasts in monolayer culture to confluency; and (b) disrupting the monolayer culture to place at least a fraction of the plurality of fibroblasts into suspension culture under conditions sufficient to form one or more spheres, wherein the one or more spheres comprise a fibroblast expressing one or more stem cell markers.
  • the presently disclosed subject matter also provides methods for differentiating a reprogrammed fibroblast cell into a cell type of interest.
  • the methods comprise (a) providing an embryoid body-like sphere comprising reprogrammed fibroblast cells; and (b) culturing the embryoid body-like sphere in a culture medium comprising a differentiation- inducing amount of one or more factors that induce differentiation of the reprogrammed fibroblast cells or derivatives thereof into the cell type of interest until the cell type of interest appears in the culture.
  • the cell type of interest is selected from the group consisting of a neuronal cell, an endodermal cell, and a cardiomyocyte, and derivatives thereof.
  • the cell type of interest is a neuronal cell or a derivative thereof.
  • the neuronal cell or derivative thereof is selected from the group consisting of an oligodendrocyte, an astrocyte, a glial cell, and a neuron.
  • the neuronal cell or derivative thereof expresses a marker selected from the group consisting of glial fibrillary acidic protein (GFAP), nestin, ⁇ III tubulin, oligodendrocyte transcription factor (Olig) 1 , and Olig2.
  • GFAP glial fibrillary acidic protein
  • Olig oligodendrocyte transcription factor 1
  • Olig2 oligodendrocyte transcription factor
  • the culture medium comprises about 10 ng/ml recombinant human epidermal growth factor (rhEGF), about 20 ng/ml fibroblast growth factor-2 (FGF2), and about 20 ng/ml nerve growth factor (NGF).
  • the cell type of interest is an endodermal cell or derivative thereof.
  • the culturing comprises culturing the embryoid body-like sphere in a first culture medium comprising Activin A; and thereafter culturing the embryoid body-like sphere in a second culture medium comprising N2 supplement-A, B27 supplement, and about 10 mM nicotinamide.
  • the culturing in the first culture medium is for about 48 hours.
  • the culturing in the second culture medium is for at least about 12 days.
  • the endodermal cell or derivative thereof expresses a marker selected from the group consisting of Nkx6-1 , Pdx 1 , and C-peptide.
  • the cell type of interest is a cardiomyocyte or a derivative thereof.
  • the culturing is for at least about 15 days.
  • the culture medium comprises a combination of basic fibroblast growth factor, vascular endothelial growth factor, and transforming growth factor ⁇ 1 in an amount sufficient to cause a subset of the embryoid body-like sphere cells to differentiate into cardiomyocytes.
  • the cardiomyocyte or derivative thereof expresses a marker selected from the group consisting of Nkx2-5/Csx and GATA4.
  • the embryoid body-like sphere is prepared by (a) growing a plurality of fibroblasts in monolayer culture on a tissue culture plate to confluency; and (b) disrupting the monolayer culture to place at least a fraction of the plurality of fibroblasts into suspension culture under conditions sufficient to form one or more embryoid body-like spheres, wherein the one or more embryoid body-like spheres comprise a reprogrammed fibroblast.
  • the presently disclosed subject matter also provides methods for treating a disease, disorder, or injury to a tissue in a subject comprising administering to the subject a composition comprising a plurality of reprogrammed fibroblast cells in a pharmaceutically acceptable carrier, in an amount and via a route sufficient to allow at least a fraction of the reprogrammed fibroblast cells to engraft the tissue and differentiate therein, whereby the disease, disorder, or injury is treated.
  • the disease, disorder, or injury is selected from the group consisting of an ischemic injury, a myocardial infarction, and stroke.
  • the subject is a mammal.
  • the mammal is selected from the group consisting of a human and a mouse.
  • the methods further comprise differentiating the reprogrammed fibroblast cells to produce a pre-determined cell type prior to administering the composition to the subject.
  • the pre-determined cell type is selected from the group consisting of a neural cell, an endoderm cell, a cardiomyocyte, and derivatives thereof.
  • FIGS 1 A-11 are a series of photographs showing that sphere formation triggered stable changes in triple knockout cells (TKOs; i.e., cells with disruptions in all three RB1 family genes: RB1, RBL1 , and RBL2) morphology.
  • TKOs triple knockout cells
  • Figure 1A shows TKOs at passage 4 in monolayer culture.
  • Figure 1 B shows TKOs lacked contact inhibition and formed mounds after reaching confluence in culture.
  • Figure 1C shows outgrowth of mounds, such as those shown in Figure 1 B, subsequently led to detachment from the plate and sphere formation.
  • Figure 1 D shows TKO spheres two weeks after transfer to a nonadherent plate.
  • Figure 1 E shows central cavity formation (arrow) evident in TKO spheres after several weeks in suspension culture.
  • Figure 1 F shows TKO spheres formed in suspension culture reattached when transferred back to tissue culture plates, and all cells in the spheres migrated back onto the plate to reform a monolayer.
  • Figure 1 G shows a higher power view of the boxed region in Figure 1F.
  • Figure 1H shows monolayers of sphere-derived cells two days after spheres were transferred back to a tissue culture plate.
  • Figure 11 shows cells in Figure 1 H after one week in culture. Note that cells in Figures 1 H and 11 had diverse morphologies, and further that they were smaller than TKO cells prior to sphere formation (Figure 1A).
  • FIG. 2A is a photograph of TKO cells placed in suspension following trypsinization. These cells did not form spheres in suspension. The cells died after 24 hours. Similar results were seen with RBT /- murine embryonic fibroblasts (MEFs).
  • Figure 2B is a photograph of TKO-Ras cells (TKO MEFs that were infected with a retrovirus expressing oncogenic V12 Ras; Sage et ai, 2000) showing that they also did not form spheres in suspension culture. Like TKO cells, TKO-Ras were not contact inhibited, but they detached from culture dishes as they became confluent and formed small groups or clusters of cells that survived in suspension and proliferated.
  • FIG. 3 is a set of bar graphs and photographs depicting the results of soft agar assays of TKOs, TKO cells derived from spheres (TKO Sphere), and TKO cells that overexpress Ras (TKO-Ras). Two independent assays are shown. Equal numbers of cells were plated, and visible colonies were counted after 3 weeks. Colony size was similar with TKO cells derived from spheres and TKO-Ras. Colonies formed with TKO cells were very small. The bar graphs below each photograph show the number of colonies per plate in each independent assay.
  • FIGS 4A and 4B show Western blot analyses of Ras expression and activity in MEFs, TKOs, and TKO-Ras cells.
  • TKOs were infected with a V12 Ha Ras-expressing retrovirus as described in Telang ef a/., 2006.
  • Figure 4A is a digital image of a Western blot showing total Ras expression in TKOs and in TKO-Ras cells.
  • the bottom panel of Figure 4A shows ⁇ -actin expression, which was included as a loading control.
  • Figure 4B is a digital image of a Western blot showing activated Ras that was detected by binding to GST-Raf.
  • the bottom panel of Figure 4B shows a Western blot of input total Ras protein used for each assay. Note that not only did TKO-Ras cells have an increased level of Ras relative to TKOs (Figure 4A), there was also an increased percentage of Ras that was activated in TKO-Ras cells ( Figure 4B).
  • Figures 5A-5D are a series of photographs showing sphere formation in RBT /- MEFs led to stable morphological changes.
  • Figure 5A shows RBT 1' MEFs in monolayer culture.
  • Figure 5B shows spheres formed when cells were scraped from dishes and placed in suspension culture.
  • Figure 5C shows re-adhesion of an RBT 7" MEF sphere to a tissue culture plate. Note that cells migrated from the sphere to reform a monolayer.
  • Figure 5D shows a higher power view of the cells in the box in Figure 5C.
  • Cells in Figures 5A and 5D are similar magnification. Note the difference in size and morphology in Figures 5A and 5D.
  • Figures 6A-6D provide the results of experiments showing that sphere formation led to expression of mRNAs for several stem cell markers in TKO and RBT 7" MEF spheres, and to downregulation of RB1 family members (RB1 , RBL1 , and RBL2) in RBT 7" MEFs.
  • Figure 6A is a bar graph depicting the results of Real Time PCR assays showing induction of mRNAs for stem cell markers in TKO and RBT 7" spheres after two weeks in suspension culture. Similar mRNA induction was maintained in monolayers derived from the spheres.
  • Figure 6B is a bar graph depicting the results of assays showing that Oct4 and Nanog mRNA increased in RBT 7" spheres with the number of days (d) in culture. Real Time PCR was used to analyze mRNA levels.
  • Figure 6C is a series of photomicrographs showing the results of immunostaining for Oct4 in sections of RBT 7" MEFs after 4 and 24 days in culture. The right hand panels depict a higher power view.
  • FIG. 6D is a bar graph providing the results of Real Time PCR demonstrating changes in expression of other mRNAs associated with stem cells and cancer stem cells after two weeks in suspension culture (see also Figure 7). The comparison with respect to relative abundance is to expression of the listed genes cells in subconfluent monolayers.
  • Figure 7 is a bar graph showing the results of Real Time PCR analysis of mRNA levels of the listed genes in RBT 7" cells after 8 days as spheres in suspension culture compared to RBT 7" cells maintained as monolayers.
  • Figures 8A-8D shows sphere formation in TKOs or RBT 7" MEFs generated cells with characteristics of a tumor side population (SP). lmmunostaining for Abcg2 and CD133 is shown on the left, and Hoechst dye staining is shown on the right.
  • Figure 8A is a set of fluorescence micrographs showing TKOs in subconfluent monolayer culture.
  • Figure 8B is a set of fluorescence micrographs showing cells derived from TKO spheres after two weeks in suspension culture. Similar results were seen with cells derived from RBT ;" MEF spheres.
  • Figure 8C is a bar graph showing quantification of SP (Hoechst7Abcg2 + /CD133 + ) cells.
  • Figure 8D is a bar graph showing TKO and RBT 7" MEF sphere-derived cells separated into SP (Hoechst7Abcg2 + /CD133 + ) and main population (MP; Hoechst + /Abcg27CD133 " ) and placed in culture (day O). At the indicated times, the cells were again examined to quantify the appearance of MP cells within the SP population, and SP cells within the MP population.
  • Figure 9 is a series of fluorescence micrographs of wild type MEFs and TKO cells maintained as subconfluent monolayers showing that these cells do not express CD133 or Abcg2 (left panels) or exclude Hoechst dye (right panels).
  • Figure 10 is a FACS plot of TKO cells derived from spheres stained with Hoechst 33342 and propidium iodide (Pl) dyes followed by analysis and sorting using a MOFLOTM cell sorter. Living cells were visualized on dot-plots according to their Hoechst red (Ho Red) and Hoechst blue (Ho Blue) fluorescence. SP cells excluding Hoechst 33342 were sorted from region R2 and the region enclosing only living cells identified based on Pl staining (region R1 , not shown). The percentage represented the content of SP in total sorted cells. Gates were set stringently to ensure no contamination with MP cells. Assessment of sorted cells revealed 100% Hoechst7Abcg2 + /CD133 + cells.
  • Figure 11 is a bar graph showing about 50,000 sorted MP (Hoechst7Abcg27CD133 ⁇ ) and SP (Hoechst7Abcg27CD133 + ) cells derived from spheres after two weeks in suspension culture placed in culture at day 0. SP and MP cells were then counted in the two populations after 3 days in culture. Note that SP cell number remained constant in the sorted SP cells, while this population gave rise to MP cells. Also note that sorted MP cells gave rise to a small population of SP cells ( ⁇ 1%) by day three in culture.
  • Figures 12A-12E are a series of bar graphs showing that SP cells expressed mRNAs for stem cell markers, overexpressed the epithelial- mesenchymal transition (EMT) transcription factor Zeb1, and they had a CD44 high /CD24 l0W mRNA pattern.
  • Figures 12A shows TKO sphere-derived cells separated into SP
  • Figures 13A-13E show the results of lentiviral vector expression of green fluorescent protein (GFP) and shRNAs for Zebi or Zeb2 used to infect MEFs. Infection efficiency was >80%.
  • Figure 13A is a set of photomicrographs showing an example of GFP expression in MEFs infected with a GFP-expressing lentiviral vector.
  • Figures 13B and 13C are bar graphs showing RNA levels determined by Real Time PCR.
  • Figures 13D and 13E are digital images of Western blots. shRNA sequences for mouse Zeb1 and Zeb2 knockdown are described in Nishimura et a/., 2006 and in the Method and Materials for the EXAMPLES section hereinbelow.
  • Figures 14A-14D are a series of photomicrographs showing TKO cells formed spheres when cultured in non-adherent plates.
  • Figure 14A shows that after 2 weeks, spheres began to form central cavities (denoted by the arrow).
  • Figures 14B-14D show that the spheres aggregated into larger structures. Such structures are shown after 2 months in culture.
  • Figures 14C and 14D are hematoxylin and eosin (H&E)-stained sections of the boxed region in Figures 14B and 14C, respectively.
  • H&E hematoxylin and eosin
  • Figures 15A-15I are a series of photomicrographs of H&E-stained sections of TKO spheres and aggregates after 3 weeks in non-adherent plates. Diverse cell morphologies are shown in the photomicrographs.
  • Figure 15A shows a low power view of spheres containing cells of varying morphologies merging to form a large spherical structure.
  • Figures 15B and 15C show cells with morphologies of hematopoietic cells.
  • Figures 15D-15I show cells with neural tissue morphologies.
  • Figure 15D shows H&E staining demonstrating cells with elongated projects resembling neurons.
  • Figures 15E and 15F show cells with neuronal morphology and tissue resembling brain.
  • Figures 15G-15I show additional cells with elongated morphology of neurons.
  • Figures 16A-16F are a series of bar graphs showing sphere formation triggered induction of mRNAs representative of the three embryonic layers as well as mRNAs in important developmental signaling pathways.
  • Figures 16A- 16F show the results of Real Time PCR used to analyze the effect of sphere formation on expression of mRNAs representative of different embryonic layers (endoderm: Figure 16A; ectoderm: Figure 16B; and mesoderm: Figure 16C), and the Wnt ( Figure 16D), Notch (Figure 16E), and various growth factor (Figure 16F) developmental signaling pathways.
  • Relative m RNA expression in TKO subconfluent monolayers was compared to cells derived from TKO spheres which had been in suspension culture for three weeks. Similar results were seen with the spheres themselves. See Figure 7 for similar analyses of RB1 " ' " MEF spheres.
  • Figures 17A-17L are a series of photomicrographs of the results of immunostaining RB1 "A spheres showing expression of markers representative of the three embryonic layers.
  • Figure 17A is an H&E stained section of an RBT 7" MEF sphere after two weeks in suspension culture. An arrow denotes the edge of the sphere.
  • Figure 17B is a higher power view of the perimeter of the sphere in Figure 17A. Note the band of cells with endodermal-like morphology and eosinophilic cytoplasm.
  • Figure 17C is a higher power view of the region immediately interior to the band of cells at the perimeter of the sphere. Note cells with epithelial-like morphology.
  • Figures 17D-17L show the results of immunostaining sections of RBT A MEF spheres with antibodies directed against AFP (Figures 17D and 17E) 1 globin ( Figures 17F-17H), CD31 ( Figures 171 and 17J), Cdh1 ( Figure 17K), and ⁇ -lll tubulin ( Figure 17L).
  • AFP ⁇ -fetoprotein
  • HB hemoglobin
  • Tubb3 ⁇ -lll tubulin
  • Cdh1 E-cadherin.
  • Figure 18 is a series of photomicrographs of the results of immunostaining of 3 week old TKO spheres for representative markers of differentiation, ⁇ -fetoprotein (AFP); GATA4 (GATA); vimentin (Vim); ⁇ -tyrosine hydroxylase ( ⁇ TH); ⁇ -lll tubulin; myelin basic protein (MBP); IsM ; tyrosine hydroxylase (TH); and glial acidic fibrillary protein (GFAP). Wild type MEFs and TKOs prior to sphere formation did not immunostain for AFP, GATA4, TH, Isl1 , MBP, GFAP, or Tubb3. Wild type MEFs did express vimentin.
  • Figures 19A-19T are a series of photomicrographs of RBY 1' MEF spheres after 24 days in suspension.
  • Figures 19A-19P show autofluorescence in conjunction with H&E staining to allow assessment of cellular morphology. Note that most of the autofluorescent cells are nucleated, however, a subset of the cells lack nuclei ( Figures 19N-19P). Cells in the perimeter of the spheres immunostained for globin ( Figures 19N-19R). Little green autofluorescence was seen in the absence of the primary globin antibody ( Figures 19Q-19R). However, autofluorescence of the globin* cells was seen with a red filter. This autofluorescence completely overlapped with globin immunostaining. In addition to globin + cells, H&E staining showed cells with characteristics of other hematopoietic cells ( Figures 19S and 19T). Note the large multinucleated cell in the center resembling a megakaryocyte.
  • Figures 20A-20L are a series of photographs and photomicrographs showing that SP cells are the primary tumorigenic population in the spheres, and tumors derived from these cells consists of cancer cells and neuronal whorls.
  • Figure 2OA is a photograph showing tumors formed in nude mice three weeks after injection of 100 SP cells subcutaneously into the hind leg.
  • Figure 2OB is a photograph showing that tumors failed to form when 20,000 MP cells were injected.
  • Figures 20C and 2OD show that both TKO-Ras cells (Figure 20C) and MP cells (Figure 20D) formed tumors when 50,000 cells were injected. These tumors were indistinguishable histologically, and appeared to be spindle cell sarcomas. Multiple tumors from the two cell types showed the same histology. H&E-stained sections are shown.
  • Figure 2OE shows an H&E- stained section of a tumor formed three weeks after injection of 100 SP cells.
  • Figure 2OF is a higher power view of a whorl in the tumor from Figure 2OE.
  • Figure 2OG shows a Nomarksi image of a section of the tumor in Figure 2OE.
  • Figure 2OH shows immunostaining of the section in Figure 2OG for ⁇ -lll tubulin. Arrows indicate the same position in Figure 2OG and Figure 2OH. Only the whorls immunostained, and tumors derived form MP and TKO-Ras cells lacked these whorls and did not immunostain.
  • Figures 2Ol and Figure 2OJ show nuclear immunostaining for Oct4 in a section of an SP cell tumor. The boxed region in Figure 2Ol is shown at higher power in Figure 2OJ.
  • Figure 2OL show nuclear immunostaining for Nanog in a section of the SP tumor.
  • Figure 2OL is a higher power view of the section shown in Figure 2OK.
  • Figures 21A-21 D are a series of photomicrographs of tumors formed in nude mice.
  • Figure 21A is an H&E-stained section of a tumor formed following injection of spheres of small TKO after two weeks in suspension culture into nude mice.
  • Figure 21 B is an H&E section of a tumor formed following injection of two week old RBT /- MEF spheres into nude mice. Note spheres/whorls with eosinophilic centers.
  • Figure 21 C shows a Nomarski image of the tumor in Figure 21 B. Arrows indicate spheres/whorls.
  • Figure 21 D depicts immunostaining of Figure 21 C for ⁇ -lll tubulin (Tubb3).
  • Figures 22A-22D depict analysis of spheres formed from wild type (i.e., RB1 +/+ , RBL1 +/+ , and RBL2 +/+ ) murine embryonic fibroblasts (MEFs).
  • Figure 22A is a photomicrograph of spheres formed by wild type MEFs after one week in suspension culture, demonstrating that wild type fibroblasts can form spheres and survive in suspension culture.
  • Figure 22B is a bar graph showing the results of Real Time PCR assays of the induction of mRNAs for genes associated with embryonic stem (ES) cells.
  • ES embryonic stem
  • FIG. 22C is a series of photomicrographs of immunostaining of the spheres shown in Figure 22A showing regions of cells expressing the stem cell markers Oct4, Nanog, and SSEA1.
  • Figure 22D is a bar graph of Real Time PCR showing expression of mRNAs for a variety of transcription factors that drive differentiation as well as markers of differentiation of cell types from all three embryonic layers. mRNA expression was examined in spheres of wild type MEFs after one week in suspension culture.
  • Figures 23A-23P are photomicrographs of spheres formed from human foreskin fibroblasts ( Figures 23A-23G) or wild type MEFs (Figures 23H-23P) after 2 weeks in culture.
  • Figure 23A is a photomicrograph of endodermal-like cells at the border of the sphere after H&E staining.
  • Figures 23B and 23C show H&E staining of cells resembling nucleated blood cells.
  • Figure 23D shows benzidine staining which demonstrated the presence of hemoglobin.
  • Figures 23E-23G show the results of immunostaining the field shows in Figure 23A for the endodermal marker ⁇ -fetoprotein (AFP; see Figure 23E), the endothelial marker CD31 (see Figure 23F), and ⁇ -globin (see Figure 23G).
  • Each of Figures 23E-23G includes five panels: Nomarski images (panel 1), DAPI staining (panel 2), immunostaining for the indicated genes (panel 3), merges of panels 2 and 3 (panel 4), and merges of panels 1-3 (panel 5).
  • Figures 23H and 23I show low and high power views of H&E stained sections showing endothelial cells (gray arrow in Figure 23I) surrounding a blood vessel.
  • FIG. 23I A ductal structure is shown by the white arrow in Figure 23I.
  • Figure 23 J shows benzidine staining of wild type MEF spheres and demonstrates the presence of hemoglobin in the cells of these spheres.
  • Figure 23K panel 1 shows an H&E stain of an erythrocyte
  • Figure 23K panel 2 shows immunostaining of an adjacent section of the sphere for globin, demonstrating that this erythrocyte expressed hemoglobin.
  • Figure 23L shows immunostaining of another erythrocyte for globin.
  • FIG. 23M shows DAPI staining (panel 1 ); immunostaining for CD31 , which is a marker of endothelial cells (panel 2); and a merge of panels 1 and 2 (panel 3); and demonstrates that endothelial cells are formed in the wild type MEF spheres.
  • Figures 23N and 230 are photomicrographs showing a region of cartilage stained with alcian blue.
  • Figure 23P is a photomicrograph showing pearls of keratin (dark staining) in an keratinized cyst.
  • Figures 24A-24F are photomicrographs of wild type MEFs allowed to form spheres in suspension culture for 3 weeks, demonstrating that these cells gave rise to differentiated structures and tissues.
  • Figure 24A is a photomicrograph showing showing a secretory epithelial ascinar like structure with a central duct (arrow).
  • Figure 24B is a photomicrograph showing secretory ducts (gray arrows) and red blood cells (white arrow).
  • Figures 24C and 24D are photomicrographs showing immunostaining for the epithelial marker E cadherin (Cdh1) and the neuronal marker ⁇ -lll tubulin. Nuclear staining with DAPI is shown.
  • Figures 24E and 24F (the latter an enlargement of the field in the box in Figure 24E) show hair fibers at the border of the spheres (the border is identified by black arrows).
  • Figures 25A-25Q are a series of photomicrographs of spheres produced by Hoechst7Abcg2 + /CD133 + cells derived from wild type MEFs after 2 weeks in culture.
  • the Hoechst7Abcg2 + /CD133 + cells were isolated by cell sorting and cultured on a feeder layer of irradiated fibroblasts.
  • Hoechst7Abcg27CD133 + cells are shown on feeder layers after one day ( Figures 25A and 25B) and after one week in culture ( Figure 25C). Immunostaining for the indicated markers is shown after one week in monolayer culture in Figures 25D-25Q.
  • Figures 25D-25Q includes three panels: the left panels show Nomarski images, the center panels show immunostaining for the indicated markers of the same fields as shown in the Nomarski images as well as nuclear localization with DAPI, and the right panels show merges of the left and center panels for each Figure.
  • Figures 26A-26E are a series of photomicrographs of teratoma formation by Hoechst7Abcg2 + /CD133 + cells derived from wild type MEF spheres after 2 weeks in suspension culture. Four independent preparations of 50,000 cells were injected into both hindlimbs of nude mice. Tumors were observed in all 8 injections, and were tumors were Collected after three weeks.
  • Figure 26A is a Normarski image of a teratoma.
  • Figure 26B is a higher power view of an adjacent section of the tumor stained with H&E. Note the variety of structures characteristic of a teratoma.
  • Figure 26C shows DAPI nuclear staining of the section presented in Figure 26A. The MEFs were isolated from Actin-GFP mice and immunostaining for GFP in Figure 26D, which shows that the tumor is GFP + whereas surrounding host tissue is GFP " .
  • Figure 26E is a merge of Figures 26C and 26D.
  • Figures 27A-27H are a series of photomicrographs of teratomas formed with Hoechst7Abcg2 + /CD133 + cells derived from wild type MEF spheres showing cobblestone epithelial morphology and expressing the epithelial specification protein E-cadherin.
  • Figures 27A-27D are a series of low power views.
  • a Nomarski image of the section is shown in Figure 27A.
  • DAPI nuclear Staining is shown in Figure 27B and E-cadherin immunostaining on the surface of the cells is shown in Figure 27C.
  • a merge of Figures 27Band 27C is shown in Figure 27D.
  • Figures 27E-27H are a series of higher power images.
  • a Nomarski image is shown in Figure 27E.
  • DAPI nuclear staining is shown in Figure 27F and E-cadherin immunostaining on the surface of the cells is shown in Figure 27G.
  • a merge of Figures 27F and 27G is shown in Figure 27H.
  • Figures 28A-28P are a series of photomicrographs showing the formation of differentiated tissues in teratomas produced from Hoechsf " /Abcg2 + /CD133 + cells isolated from wild type MEF spheres. Tumors were isolated 3 weeks after injection of 50,000 cells and sectioned for immunostaining.
  • Figure 28A is a Nomarski image of adipose tissue present in a teratoma.
  • Figure 28B shows DAPI staining showing cell nuclei.
  • Figure 28C shows immunostaining for GFP showing that the adipose tissue is derived from the injected Hoechst7Abcg2 + /CD133 + cells.
  • Figure 28D is a merge of Figures 28B and 28C.
  • Figure 28E is a Nomarski image of a neuronal structure in a teratoma.
  • Figure 28F shows DAPI nuclear staining of the section in Figure 28D.
  • Figure 28G shows immunostaining of the section of Figure 28E for ⁇ -lll tubulin, showing a cluster of neurons within a neuronal structure in the teratoma.
  • Figure 28H is a merge of Figures 28F and 28G.
  • Figure 28I is a Nomarski image of a region of intestinal-like epithelium in a teratoma.
  • Figure 28J shows DAPI nuclear staining of the section of Figure 28I.
  • Figure 28K shows immunostaining for GFP, and shows that this intestinal- like structure is derived from injected Hoechst7Abcg2 + /CD133 + cells.
  • Figure 28L is a merge of Figures 28J and 28K.
  • Figure 28M is a Nomarski image of a secretory epithelial structure in a teratoma.
  • Figure 28N shows DAPI nuclear staining in the structure of Figure 28M.
  • Figure 280 shows GFP immunostaining and demonstrates that the structure in Figure 28M is derived from the injected Hoechst7Abcg2 + /CD133 + cells.
  • Figure 28P shows the results of immunostaining for CDH 1 , which demonstrates that the structure shown is epithelial. These results demonstrates multiple differentiated tissues in the teratoma formed with Hoechst " /Abcg27CD133 + cells derived from wild type MEF cells following sphere formation.
  • Figures 29A-29I are a series of photomicrographs showing formation of skeletal muscle in a teratoma arising from injection of wild type MEF Hoechst " /Abcg27CD133 + cells derived from spheres into nude mice.
  • Figure 29A is a photomicrograph of an H&E stained section showing skeletal muscle fibers in the teratoma.
  • a Nomarski image of an adjacent section is shown as Figure 29B.
  • DAPI nuclear staining is shown in Figure 29C
  • GFP staining is shown in Figure 29D, demonstrating that the muscle cells ware tumor-derived.
  • a merge is shown in Figure 29E.
  • Figures 29F-29I are a series of control photomicrographs.
  • a Nomarski image of host skeletal muscle is shown in Figure 29F.
  • DAPI staining is shown in Figure 29G and GFP is shown in Figure 29H. There was a lack of GFP staining in Figure 29H, which is host muscle that does not express GFP
  • Figures 30A-30K are a series of micrographs of MEF spheres after two weeks in suspension culture. Spheres attached to the plates and cells began to migrate out onto the plate as with TKO and RBY 1' MEF spheres. However, in contrast to the TKO and RBT' " MEFS, only a portion of the cells from the wild type MEF spheres migrated back onto the plate. These cells were highly pigmented (see Figures 30A-30C). Initially, most of the cells were rounded or epithelial in appearance. However after several days on the plate, the cells remained pigmented but they began to elongate (see Figures 30D-30F). Figures 3OG and 3OH show lower power views of the cells. Figures 30I-30K each consist of five panels.
  • Figures 3Ol and 3OJ show immunostaining of these cells for the melanocyte marker Mitf
  • Figure 3OK shows immunostaining of the cells for a second melanocyte marker Mel5.
  • Figure 31 is a bar graph showing gene expression analysis of the cells shown in Figure 30.
  • the Real Time PCR results for mRNA levels were compared to monolayers of control wild type MEFs prior to sphere formation.
  • Figures 32A-32J are a series of photomicrographs showing primary cultures of human lung bronchial epithelial cells grown to confluence, scraped from tissue culture dishes, and placed in suspension culture in non-adherent plates as described herein for fibroblasts. Spheres were allowed to form for 5 days, and then the spheres were fixed and sectioned into 5 micron sections.
  • Figures 32A-32C show sections of the sphere stained with H&E ( Figure 32A) and globin (Figure 32B) to demonstrate erythrocyte differentiation in the sphere.
  • Figures 32D-32I show higher power views of the spheres showing erythrocytes immunostaining for globin.
  • Figure 32J shows benzidine staining of a section of the sphere, further demonstrating the presence of hemoglobin.
  • SEQ ID NOs: 1-70 are the nucleotide sequences of oligonucleotide primers that can be employed in pairwise combination (e.g., SEQ ID NOs: 1 and 2; SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, etc.) to detect the expression of the 25 genes listed in Table 1 below.
  • SEQ ID NO: 71 is the nucleotide sequence of an oligonucleotide that specifically binds to an SP6 promoter fragment.
  • SEQ ID NO: 72 is a nucleotide sequence of an exemplary shRNA sense strand that can be used to knockdown expression of Zeb1.
  • SEQ ID NO: 73 is a nucleotide sequence of an exemplary shRNA sense strand that can be used to knockdown expression of Zeb2.
  • SEQ ID NO: 74 is a nucleotide sequence of a control shRNA sense strand that can be used to test the specificity of the shRNAs comprising SEQ ID NO: 72 or SEQ ID NO: 73 used to knockdown expression of Zeb1 or Zeb2, respectively.
  • fibroblasts in which all three retinoblastoma (RB1) family members have been mutated led to stable reprogramming of the cells to a cancer stem cell phenotype. While fibroblasts containing only an RB1 mutation retained cell contact inhibition, bypassing this inhibition by forcing the cells to form spheres in suspension led to downregulation of RBL1 and RBL2, and to similar reprogramming of the RB1 "7" cells to a cancer stem cell phenotype.
  • TKOs retinoblastoma
  • results presented herein provide evidence of a potential pathway for generation of cancer stem cells from differentiated somatic cells. Based at least in part on these findings, disclosed herein is a new tumor suppressor function for the RB1 pathway that imposes contact inhibition to prevent outgrowth of differentiated somatic cells into spherical structures where reprogramming to cancer stem cells can occur.
  • a stem cell refers to one or more stem cells, unless the context clearly indicates otherwise. Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about”.
  • the phrase “A, B, C, and/or D” includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D.
  • the term “comprising”, which is synonymous with “including” “containing”, or “characterized by”, is inclusive or open-ended and does not exclude additional, unrecited elements and/or method steps. "Comprising” is a term of art that means that the named elements and/or steps are present, but that other elements and/or steps can be added and still fall within the scope of the relevant subject matter.
  • a pharmaceutical composition can "consist essentially of a pharmaceutically active agent or a plurality of pharmaceutically active agents, which means that the recited pharmaceutically active agent(s) is/are the only pharmaceutically active agent(s) present in the pharmaceutical composition. It is noted, however, that carriers, excipients, and other inactive agents can and likely would be present in the pharmaceutical composition.
  • compositions that comprise reprogrammed cells relates in some embodiments to compositions that comprise reprogrammed cells. It is understood that the presently disclosed subject matter thus also encompasses compositions that in some embodiments consist essentially of reprogrammed cells, as well as compositions that in some embodiments consist of reprogrammed cells.
  • the methods of the presently disclosed subject matter comprise the steps the steps that are disclosed herein and/or that are recited in the claims, in some embodiments the methods of the presently disclosed subject matter consist essentially of the steps that are disclosed herein and/or that are recited in the claims, and in some embodiments the methods of the presently disclosed subject matter consist of the steps that are disclosed herein and/or that are recited in the claim.
  • subject refers to a member of any invertebrate or vertebrate species.
  • the term "subject” is intended to encompass any member of the Kingdom Animalia including, but not limited to the phylum Chordata (i.e., members of Classes Osteichythyes (bony fish), Amphibia (amphibians), Reptilia (reptiles), Aves (birds), and Mammalia (mammals)), and all Orders and Families encompassed therein.
  • phylum Chordata i.e., members of Classes Osteichythyes (bony fish), Amphibia (amphibians), Reptilia (reptiles), Aves (birds), and Mammalia (mammals)
  • genes, gene names, and gene products disclosed herein are intended to correspond to homologs and/or orthologs from any species for which the compositions and methods disclosed herein are applicable.
  • the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates.
  • a given nucleic acid or amino acid sequence is intended to encompass homologous and/or orthologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds.
  • the methods of the presently disclosed subject matter are particularly useful for warm-blooded vertebrates.
  • the presently disclosed subject matter concerns mammals and birds. More particularly provided is the isolation, manipulation, and use of reprogrammed somatic cells from mammals such as humans and other primates, as well as those mammals of importance due to being endangered (such as Siberian tigers), of economic importance (animals raised on farms for consumption by humans) and/or social importance (animals kept as pets or in zoos) to humans, for instance, carnivores other than humans (such as cats and dogs), swine (pigs, hogs, and wild boars), ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), rodents (such as mice, rats, and rabbits), marsupials, and horses.
  • carnivores other than humans such as cats and dogs
  • swine pigs, hogs, and wild boars
  • domesticated fowl e.g., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economic importance to humans.
  • livestock including but not limited to domesticated swine (pigs and hogs), ruminants, horses, poultry, and the like.
  • isolated indicates that the nucleic acid or polypeptide exists apart from its native environment.
  • An isolated nucleic acid or polypeptide can exist in a purified form or can exist in a non-native environment.
  • nucleic acid molecule and “nucleic acid” refer to deoxyribonucleotides, ribonucleotides, and polymers thereof, in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar properties as the reference natural nucleic acid.
  • nucleic acid molecule and “nucleic acid” can also be used in place of "gene”, “cDNA”, and “mRNA”. Nucleic acids can be synthesized, or can be derived from any biological source, including any organism. Several genes are disclosed herein. Representative sequences of nucleic acid and amino acid products from these genes are set forth in Table 2.
  • Table 2 discloses Accession Numbers for certain of these genes that can be found in the GENBANK® database as they relate to humans and mice, other sequences from humans, mice, and other species are also included within the scope of the present disclosure and would be known and/or identifiable by one of ordinary skill in the art after consideration of the instant disclosure.
  • NM_000207 encodes the human C-peptide, which corresponds to amino acids 57-87 of NP_000198.
  • b NM_008386 is a nucleotide sequence of murine insulin. Nucleotides 351- 438 of NM_008386 encode the murine C-peptide, which corresponds to amino acids 57-85 of NP_032412.
  • isolated indicates that the cell exists apart from its native environment.
  • An isolated cell can also exist in a purified form or can exist in a non-native environment.
  • a cell exists in a "purified form" when it has been isolated away from all other cells that exist in its native environment, but also when the proportion of that cell in a mixture of cells is greater than would be found in its native environment. Stated another way, a cell is considered to be in "purified form" when the population of cells in question represents an enriched population of the cell of interest, even if other cells and cell types are also present in the enriched population.
  • a cell can be considered in purified form when it comprises in some embodiments at least about 10% of a mixed population of cells, in some embodiments at least about 20% of a mixed population of cells, in some embodiments at least about 25% of a mixed population of cells, in some embodiments at least about 30% of a mixed population of cells, in some embodiments at least about 40% of a mixed population of cells, in some embodiments at least about 50% of a mixed population of cells, in some embodiments at least about 60% of a mixed population of cells, in some embodiments at least about 70% of a mixed population of cells, in some embodiments at least about 75% of a mixed population of cells, in some embodiments at least about 80% of a mixed population of cells, in some embodiments at least about 90% of a mixed population of cells, in some embodiments at least about 95% of a mixed population of cells, in some embodiments at least about 99% of a mixed population of cells, and in some embodiments about 100% of a mixed population of cells, with the proviso that the cell comprises a greater percentage of the
  • the presently disclosed subject matter provides in some embodiments methods for producing a reprogrammed cell (e.g., a reprogrammed fibroblast).
  • a reprogrammed cell e.g., a reprogrammed fibroblast
  • reprogrammed refers to a cell that has be manipulated in culture in order to acquire a degree of pluripotency that it would not have acquired had the manipulation in culture not taken place.
  • exemplary reprogrammed cells include, but are not limited to fibroblasts that as a result of the manipulations disclosed herein are induced to express markers associated with stem cells or with differentiated cells other than fibroblasts that the fibroblasts in culture do not and/or would not have expressed if maintained in monolayer culture.
  • the phrases “markers associated with stem cells”, “stem cell markers”, and “mRNA for stem cell markers” refer to genes the expression of which is generally associated with stem cells and other pluripotent and/or totipotent cells including, but not limited to embryonic stem (ES) cells and induced plurippotent cells (iPSCs), but that that is not generally associated with reprogrammed cells in culture prior to the in vitro manipulation(s) that caused the cells to become reprogrammed.
  • ES embryonic stem
  • iPSCs induced plurippotent cells
  • the genes Oct4, Nanog, fibroblast growth factor-4 (FGF4), Sox2, Klf4, SSEA1 , and Stat3 are all expressed by ES cells and other pluripotent cells, but are not expressed or expressed to a much lower level in fibroblasts.
  • stem cell genes As such, they are referred to herein as “stem cell genes” or “stem cell markers”. Upon reprogramming, fibroblasts upregulate one or more of these genes, and the upregulation of the one or more of these stem cell markers is indicative of reprogramming.
  • the methods comprise (a) growing a plurality of cells (e.g., fibroblasts) in monolayer culture to confluency; and (b) disrupting the monolayer culture to place at least a fraction of the plurality of cells into suspension culture under conditions sufficient to form one or more embryoid body-like spheres, wherein the one or more embryoid body-like spheres comprise a reprogrammed cell induced to express at least one endogenous gene not expressed by the cell growing in the monolayer culture prior to the disrupting step.
  • a plurality of cells e.g., fibroblasts
  • condition sufficient to form one or more embryoid body-like spheres refers to any culture conditions wherein cells growing in monolayers that are disrupted initiate sphere formation while growing in suspension. Such conditions include various tissue culture media as well as different disruption techniques.
  • the monolayers and/or the spheres that are generated therefrom are grown in a tissue culture medium.
  • Tissue culture media that can be employed in the growth and maintenance of the cells and spheres of the presently disclosed subject matter include, but are not limited to any tissue culture medium that is generally used for growing and maintaining mammalian cells, particularly stem cells such as embryonic stem cells.
  • Non-limiting examples of such media are DMEM, F12, RPMI-1640, and combinations thereof, which can be augmented with mammalian serum (e.g., 5-20% fetal bovine or fetal calf serum) and/or serum substitutes (e.g., OPTI- MEM® Reduced Serum Medium available from INVITROGENTM), glutamine and/or other essential amino acids, antibiotics and/or antimycotics, etc. as would be understood by one of ordinary skill in the art.
  • mammalian serum e.g., 5-20% fetal bovine or fetal calf serum
  • serum substitutes e.g., OPTI- MEM® Reduced Serum Medium available from INVITROGENTM
  • glutamine and/or other essential amino acids e.g., antibiotics and/or antimycotics, etc.
  • Exemplary media that can be employed in the practice of the presently disclosed subject matter are disclosed in Nagy et al., 2003 and in U.S. Patent Nos
  • disruption and grammatical variants thereof refer to a manipulation of a monolayer of cells in culture that results in at least a subset of the monolayer detaching from the substrate on which it is growing
  • Non-limiting examples of other disruption strategies include using light trypsinization and/or collagenase treatment to remove sheets of cells and scraping of monolayer cells followed by moderate pipetting with a pipetting device to generate the spheres.
  • a hanging drop method wherein lightly trypsinized cells in suspension are allowed to adhere to underside of tissue culture plate top can also be employed.
  • drops can be removed and placed in suspension culture. This procedure has been employed with ES cells to produced uniformly sized spheres or embryoid bodies, and can also be employed with the methods and compositions of the presently disclosed subject matter.
  • a reprogrammed cell of the presently disclosed subject matter has the property of long term self renewal.
  • long term self renewal refers to an ability to self renew in culture over a period of at least one month, two month, three month, four month, five month, six months, or longer.
  • a cell of the presently disclosed subject matter is a fibroblast.
  • Fibroblasts can come from many sources from various species.
  • the fibroblast is a mammalian fibroblast, optionally a human fibroblast. Methods for isolating fibroblasts from various species are also known.
  • a fibroblast is isolated from a source and grown in culture without any genetic manipulation (i.e., without the introduction of any exogenous coding and/or regulatory sequences using recombinant DNA technology).
  • the cell is selected from the group including adult human skin fibroblasts, adult peripheral blood mononuclear cells, adult human bone marrow-derived mononuclear cells, neonatal human skin fibroblasts, human umbilical vein endothelial cells, human umbilical artery smooth muscle cells, human postnatal skeletal muscle cells, human postnatal adipose cells, human postnatal peripheral blood mononuclear cells, or human cord blood mononuclear cells.
  • the cell i.e., the fibroblast
  • a cell can be genetically manipulated by introducing into the cell one or more exogenous nucleic acid sequences.
  • the exogenous nucleic acid sequences can include coding sequences.
  • the exogenous nucleic acid sequence can include one or more regulatory sequences designed to regulate the expression of the exogenous coding sequences, endogenous coding sequences present in the cell, or both.
  • the monolayers are disrupted to place at least a fraction of the fibroblasts into suspension culture.
  • the term "disrupted” refers to a physical manipulation of the monolayer such that a plurality of cells becomes detached from the rest of the monolayer and from the growth surface and grows in suspension.
  • the disruption can be anything that causes pluralities of cells as a unit to detach from the growth surface and grow in suspension.
  • the disrupting comprises scraping at least a fraction of the confluent monolayer off of a substrate upon which the confluent monolayer is being cultured.
  • the disrupted cells grow in culture, they can form one or more embryoid body-like spheres.
  • the phrase "embryoid body-like sphere” refers to an aggregate of disrupted cells that appears morphologically similar to an embryoid body formed by embryonic stem (ES) cells under appropriate in vitro culturing conditions (see e.g., Nagy etal., 2003; U.S. Patent No. 5,914,268).
  • ES embryonic stem
  • These embryoid body-like spheres are stable in culture; in some embodiments, they can be maintained in suspension culture for at least one month, and in some embodiments, they can be maintained in suspension culture for at least two months.
  • the one or more embryoid body-like spheres are maintained in a medium comprising Dulbecco's Modified Eagle Medium (DMEM) and 10% fetal bovine serum (FBS).
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • some of the cells present therein are reprogrammed cells (in some embodiments, reprogrammed fibroblasts).
  • the reprogrammed cells can be characterized by the expression of one or more stem cell markers that are not expressed (or are expressed to a much lower degree) by the cells (e.g., fibroblasts) in monolayer culture prior to formation of the embryoid body-like sphere.
  • the reprogrammed fibroblasts express a stem cell marker selected from the group including, but not limited to Oct4, Nanog, FGF4, Sox2, Klf4, Sseal , and Stat3.
  • Reagents that can be employed to assay for the expression of these stem cell markers and others include oligonucleotide primers comprising the sequences set forth in Table 1 hereinabove (e.g., for use in expression assays such as the RT-PCR assay).
  • the reprogrammed fibroblasts of the presently disclosed subject matter are in some embodiments non-tumorigenic in nude mice.
  • the presently disclosed subject matter also provides methods for inducing expression of one or more stem cell markers in a cell (in some embodiments, a fibroblast).
  • the methods comprise (a) growing a plurality of cells in monolayer culture to confluency; and (b) disrupting the monolayer culture to place at least a fraction of the plurality of cells into suspension culture under conditions sufficient to form one or more spheres, wherein the one or more spheres comprise a cell with upregulated expression of one or more stem cell markers.
  • the presently disclosed subject matter also provides reprogrammed cells produced by the presently disclosed methods, reprogrammed cells non- recombinantly induced to express one or more endogenous stem cell markers, embryoid body-like spheres comprising a plurality of reprogrammed cells, and cell cultures comprising the presently disclosed embryoid body-like spheres in a medium sufficient to maintain the embryoid body-like spheres in suspension culture for at least one month.
  • the cells are fibroblasts.
  • reprogrammed cells e.g., fibroblasts
  • the presently disclosed subject matter also provides methods for differentiating a reprogrammed cell into a cell type of interest.
  • the methods comprise (a) providing an embryoid body-like sphere comprising reprogrammed cells; and (b) culturing the embryoid body-like sphere in a culture medium comprising a differentiation-inducing amount of one or more factors that induce differentiation of the reprogrammed cells or derivatives thereof into the cell type of interest until the cell type of interest appears in the culture.
  • the reprogrammed cells of the presently disclosed subject matter can thus be differentiated into cell-types of various lineages, if desired.
  • differentiated cells include any differentiated cells from ectodermal (e.g., neurons and fibroblasts), mesodermal (e.g., cardiomyocytes), or endodermal (e.g., pancreatic cells) lineages.
  • the differentiated cells can be one or more: pancreatic beta cells, neural stem cells, neurons (e.g., dopaminergic neurons), oligodendrocytes, oligodendrocyte progenitor cells, hepatocytes, hepatic stem cells, astrocytes, myocytes, hematopoietic cells, or cardiomyocytes.
  • neurons e.g., dopaminergic neurons
  • oligodendrocytes oligodendrocyte progenitor cells
  • hepatocytes e.g., hepatic stem cells
  • astrocytes e.g., myocytes, hematopoietic cells
  • cardiomyocytes e.g., cardiomyocytes.
  • the differentiated cells derived from the reprogrammed cells of the presently disclosed subject matter can in some embodiments be terminally differentiated cells, or they can be capable of giving rise to cells of a specific lineage.
  • reprogrammed cells of the presently disclosed subject matter can be differentiated into a variety of multipotent cell types, e.g., neural stem cells, cardiac stem cells, or hepatic stem cells. These stem cells can then be further differentiated into new cell types, e.g., neural stem cells can be differentiated into neurons; cardiac stem cells can be differentiated into cardiomyocytes; and hepatic stem cells can be differentiated into hepatocytes.
  • Methods of differentiating reprogrammed cells can be similar to and based on those methods used to differentiate stem cells, particularly ES cells, MSCs, MAPCs, MIAMI, and hematopoietic stem cells (HSCs).
  • the differentiation occurs ex vivo; in some embodiments the differentiation occurs in vivo.
  • neural stem cells can be generated by culturing the reprogrammed cells of the presently disclosed subject matter in the presence of noggin, or other bone morphogenetic protein antagonists (see e.g., ltsykson et a/., 2005).
  • neural stem cells can be generated by culturing the reprogrammed cells of the presently disclosed subject matter in the presence of growth factors including, but not limited to FGF-2 (see Zhang et al., 2001).
  • the cells are cultured in serum-free medium containing FGF-2.
  • the reprogrammed cells of the presently disclosed subject matter are co-cultured with a mouse stromal cell line, e.g., PA6 in the presence of serum-free medium comprising FGF-2.
  • the reprogrammed cells of the presently disclosed subject matter are directly transferred to serum-free medium containing FGF-2 to directly induce differentiation.
  • Neural stems derived from the reprogrammed cells of the presently disclosed subject matter can be differentiated into neurons, oligodendrocytes, and/or astrocytes. Often, the conditions used to generate neural stem cells can also be used to generate neurons, oligodendrocytes, and/or astrocytes.
  • reprogrammed cells of the presently disclosed subject matter can be co-cultured with a PA6 mouse stromal cell line under serum-free conditions (see e.g., Kawasaki etal., 2000).
  • Other methods have also been described in, for example, Pomp et a/., 2005; U.S. Patent No. 6,395,546; Lee et al., 2000.
  • Oligodendrocytes can also be generated from the reprogrammed cells of the presently disclosed subject matter. Differentiation of the reprogrammed cells of the presently disclosed subject matter into oligodendrocytes can be accomplished by known methods for differentiating ES cells or neural stem cells into oligodendrocytes. For example, oligodendrocytes can be generated by co-culturing reprogrammed cells of the presently disclosed subject matter or neural stem cells derived therefrom with stromal cells (see e.g., Hermann etal., 2004).
  • oligodendrocytes can be generated by culturing the reprogrammed cells of the presently disclosed subject matter or neural stem cells in the presence of a fusion protein, in which the lnterleukin (IL)-6 receptor, or derivative, is linked to the IL-6 cytokine, or derivative thereof. Oligodendrocytes can also be generated from the reprogrammed cells of the presently disclosed subject matter by other methods known in the art (see e.g. Kang ef a/,, 2007).
  • Astrocytes can also be produced from the reprogrammed cells of the presently disclosed subject matter. Astrocytes can be generated by culturing reprogrammed cells of the presently disclosed subject matter or neural stem cells derived therefrom in the presence of neurogenic medium with bFGF and EGF (see e.g., Housele et al., 1999).
  • Reprogrammed cells of the presently disclosed subject matter can be differentiated into pancreatic beta cells by methods known in the art (see e.g., Assady et al., 2001 ; Lumelsky et al., 2001 ; D'Amour et a/., 2005; D'Amour et a/., 2006).
  • the method can comprise culturing the reprogrammed cells of the presently disclosed subject matter in serum-free medium supplemented with Activin A, followed by culturing in the presence of serum-free medium supplemented with all-trans retinoic acid, followed by culturing in the presence of serum-free medium supplemented with bFGF and nicotinamide (see e.g., Jiang etal., 2007).
  • the method comprises culturing the reprogrammed cells of the presently disclosed subject matter in the presence of serum-free medium, activin A, and Wnt protein from about 0.5 to about 6 days, e.g., about 0.5, 1 , 2, 3, 4, 5, 6, days; followed by culturing in the presence of from about 0.1 % to about 2%, e.g., 0.2%, FBS and activin A from about 1 to about 4 days, e.g., about 1 , 2, 3, or 4 days; followed by culturing in the presence of 2% FBS, FGF-10, and KAAD-cyclopamine (keto-N- aminoethylaminocaproyl dihydro cinnamoylcyclopamine) and retinoic acid from about 1 to about 5 days, e.g.
  • Hepatic cells or hepatic stem cells can be differentiated from the reprogrammed cells of the presently disclosed subject matter.
  • culturing the reprogrammed cells of the presently disclosed subject matter in the presence of sodium butyrate can generate hepatocytes (see e.g., Rambhatla etal., 2003).
  • hepatocytes can be produced by culturing the reprogrammed cells of the presently disclosed subject matter in serum-free medium in the presence of Activin A, followed by culturing the cells in fibroblast growth factor-4 and bone morphogenetic protein-2 (see e.g., Cai et al., 2007).
  • the reprogrammed cells of the presently disclosed subject matter are differentiated into hepatic cells or hepatic stem cells by culturing the reprogrammed cells of the presently disclosed subject matter in the presence of Activin A from about 2 to about 6 days, e.g., about 2, about 3, about 4, about 5, or about 6 days, and then culturing the reprogrammed cells of the presently disclosed subject matter in the presence of hepatocyte growth factor (HGF) for from about 5 days to about 10 days, e.g., about 5, about 6, about 7, about 8, about 9, or about 10 days.
  • HGF hepatocyte growth factor
  • the reprogrammed cells of the presently disclosed subject matter can also be differentiated into cardiac muscle cells. Inhibition of bone morphogenetic protein (BMP) signaling can result in the generation of cardiac muscle cells or cardiomyocytes (see e.g., Yuasa et al., 2005).
  • BMP bone morphogenetic protein
  • the reprogrammed cells of the presently disclosed subject matter are cultured in the presence of noggin for from about two to about six days, e.g., about 2, about 3, about 4, about 5, or about 6 days, prior to allowing formation of an embryoid body, and culturing the embryoid body for from about
  • 1 week to about 4 weeks e.g., about 1 , about 2, about 3, or about 4 weeks.
  • cardiomyocytes can be generated by culturing the reprogrammed cells of the presently disclosed subject matter in the presence of leukemia inhibitory factor (LIF), or by subjecting them to other methods known in the art to generate cardiomyocytes from ES cells (see e.g. , Bader et al., 2000; Kehat et al., 2001 ; Mummery et al., 2003).
  • LIF leukemia inhibitory factor
  • Examples of methods to generate other cell-types from reprogrammed cells of the presently disclosed subject matter include:
  • the cell type of interest is selected from the group including, but not limited to a neuronal cell, an endodermal cell, a cardiomyocyte, and derivatives thereof. In some embodiments, the cell type of interest is a neuronal cell or a derivative thereof.
  • the neuronal cell or derivative thereof is selected from the group including, but not limited to an oligodendrocyte, an astrocyte, a glial cell, and a neuron.
  • the neuronal cell or derivative thereof expresses a marker selected from the group including, but not limited to GFAP, nestin, ⁇ III tubulin, Oligi, and Oiig2.
  • the culture medium comprises about 10 ng/ml rhEGF, about 20 ng/ml FGF2, and about 20 ng/ml NGF, optionally wherein the culturing is for at least about 10 days.
  • Neuronal cells and/or derivatives thereof can be identified using techniques known in the art including, but not limited to the use of antibodies that bind to GFAP, nestin, ⁇ III tubulin, Olig1 , and Olig2, and/or other neuronal cell markers, or Reverse Transcription PCR using oligonucleotides are specific for GFAP, nestin, ⁇ III tubulin, Oligi , and Olig2 and/or other genes expressed in neuronal cells or their derivatives. Exemplary oligonucleotides are set forth in Table 1 hereinabove.
  • the cell type of interest is an endodermal cell or derivative thereof.
  • Culture conditions that can give rise to endodermal cells and/or derivatives thereof from reprogrammed fibroblasts include, but are not limited to culturing an embryoid body-like sphere in a first culture medium comprising Activin A; and thereafter culturing the embryoid body-like sphere in a second culture medium comprising N2 supplement-A, B27 supplement, and about 10 mM nicotinamide.
  • the culturing in the first culture medium is for about 48 hours.
  • the culturing in the second culture medium is for at least about 12 days.
  • Culturing under one or more of these conditions can be sufficient to cause a differentiated derivative of a reprogrammed fibroblast to express a marker selected from the group including, but not limited to Nkx6-1 , Pdx 1 , and C-peptide.
  • Endodermal cells and/or derivatives thereof can be identified using techniques known in the art including, but not limited to the use of antibodies that bind to Nkx6-1, Pdx 1, and C-peptide, and/or other endodermal cell markers, or Reverse Transcription PCR using oligonucleotides are specific for Nkx6-1 , Pdx 1 , C-peptide, and/or other genes expressed in endodermal cells or their derivatives.
  • the cell type of interest is a cardiomyocyte or a derivative thereof.
  • the culturing is in some embodiments for at least about 15 days, optionally, in a culture medium comprising a combination of basic fibroblast growth factor, vascular endothelial growth factor, and transforming growth factor ⁇ 1 in an amount sufficient to cause a subset of the embryoid body-like sphere cells to differentiate into cardiomyocytes. Culturing under these conditions can lead to the cardiomyocyte or the derivative thereof expressing a marker selected from the group including, but not limited to Nkx2-5/Csx and GATA4.
  • Cardiomyocytes and/or derivatives thereof can be identified using techniques known in the art including, but not limited to the use of antibodies that bind to Nkx2-5/Csx and GATA4, and/or other cardiomyocyte markers, or Reverse Transcription PCR using oligonucleotides are specific for Nkx2-5/Csx, GATA4, and/or other genes expressed in cardiomyocytes and/or their derivatives. Exemplary oligonucleotides are set forth in Table 1 hereinabove. J]L Applications
  • somatic cells e.g., human somatic cells
  • the methods include obtaining a cellular sample (e.g., by a biopsy such as, but not limited to a skin biopsy), blood draw, or alveolar or other pulmonary lavage. It is to be understood that initial plating densities of cells prepared from a tissue can be varied based on such variables as expected viability or adherence of cells from the particular tissue.
  • Methods for obtaining various types of somatic cells include, but are not limited to, the following exemplary methods.
  • Skin tissue containing the dermis is harvested, for example, from the back of a knee or buttock.
  • the skin tissue is then incubated for 30 minutes at 37°C in 0.6% trypsin/Dulbecco's Modified Eagle's Medium (DMEM)/F-12 with 1% antibiotics/antimycotics, with the inner side of the skin facing downward.
  • DMEM trypsin/Dulbecco's Modified Eagle's Medium
  • tweezers are used to lightly scrub the inner side of the skin.
  • the skin tissue is finely cut into 1 mm 2 sections and is then centrifuged at 1200 rpm for 10 minutes at room temperature. The supernatant is removed, and 25 ml of 0.1% trypsin/DMEM/F-12/1% antibiotics, antimycotics, is added to the tissue precipitate.
  • the mixture is stirred at 200- 300 rpm using a stirrer at 37°C. for 40 minutes.
  • 3 ml fetal bovine serum (FBS) is added, and filtered sequentially with gauze, a 100 ⁇ m nylon filter, and a 40 ⁇ m nylon filter.
  • FBS fetal bovine serum
  • DMEM/F-12/1% antibiotics, antimycotics is added to wash the precipitate, and then centrifuged at 1200 rpm at room temperature for 10 minutes.
  • the cell fraction thus obtained is then cultured as described herein.
  • Dermal cells can be enriched by isolating dermal papilla from scalp tissue.
  • Human scalp tissue 0.5 - 2 cm 2 or less
  • DMEM fetal mesenchymal calf serum
  • the epidermis is peeled off from the dermis; and hair follicles are pulled out from the dermis. Hair follicles are washed with phosphate-buffered saline (PBS); and the epidermis and dermis are removed.
  • PBS phosphate-buffered saline
  • Single dermal-papilla derived cells are generated by culturing the explanted papilla on a plastic tissue culture dish in the medium containing DMEM and 10% fetal calf serum (FCS) for 1 week.
  • FCS fetal calf serum
  • FGM-2 SINGLEQUOTS® Longza Inc., Allendale, New Jersey, United States of America
  • Epidermal cells can be also enriched from human scalp tissues (0.5 - 2 cm 2 or less). Human scalp issues is rinsed, trimmed to remove excess adipose tissues, and cut into small pieces. These tissue pieces are enzymatically digested in 12.5 mg/ml dispase (INVITROGENTM) in Dulbecco's modified Eagle's medium (DMEM) for 24 hours at 4°C. After the enzymatic treatment, the epidermis is peeled off from the dermis; and hair follicles are pulled out from the dermis. The bulb and intact outer root sheath (ORS) are dissected under a microscope. After the wash, the follicles are transferred into a plastic dish.
  • human scalp tissues 0.5 - 2 cm 2 or less. Human scalp issues is rinsed, trimmed to remove excess adipose tissues, and cut into small pieces. These tissue pieces are enzymatically digested in 12.5 mg/ml dispase (INVITROGENTM) in D
  • the bulge region is dissected from the upper follicle using a fine needle. After the wash, the bulge is transferred into a new dish and cultured in medium containing DM EIWF 12 and 10% FBS. After the cells are identified, culture medium is changed to the EPILIFETM Extended-Lifespan Serum-Free Medium (Sigma-Adrich Corp., St. Louis, Missouri, United States of America).
  • the presently disclosed subject matter provides in some embodiments methods for treating a disease, disorder, or injury to a tissue in a subject.
  • the methods comprise administering to the subject a composition comprising a plurality of reprogrammed cells (e.g. , fibroblasts) in a pharmaceutically acceptable carrier in an amount and via a route sufficient to allow at least a fraction of the reprogrammed cells to engraft the target tissue and differentiate therein, whereby the disease, disorder, or injury is treated.
  • the disease, disorder, or injury can be any disease, disorder, or injury in which cell replacement therapy might be expected to be beneficial.
  • the disease, disorder, or injury is selected from the group including, but not limited to an ischemic injury, a myocardial infarction, and stroke.
  • target tissue and "target organ” as used herein refer to an intended site for accumulation of a reprogrammed cell of the presently disclosed subject matter and/or a differentiated derivative thereof (e.g., an in vitro differentiated derivative thereof) following administration to a subject.
  • a differentiated derivative thereof e.g., an in vitro differentiated derivative thereof
  • the methods of the presently disclosed subject matter involve a target tissue or a target organ that has been damaged, for example by ischemia or other injury.
  • control tissue refers to a site suspected to substantially lack accumulation of an administered cell.
  • a tissue or organ that has not been injured or damaged is a representative control tissue, as is a tissue or organ other than the intended target tissue.
  • selective targeting and “selective homing” as used herein refer to a preferential localization of a cell (for example, a reprogrammed cell of the presently disclosed subject matter and/or an in vitro differentiated derivative thereof) that results in an accumulation of the administered reprogrammed cell of the presently disclosed subject matter and/or an in vitro differentiated derivative thereof in a target tissue that is in some embodiments about 2-fold greater than accumulation of the administered reprogrammed cell of the presently disclosed subject matter and/or an in vitro differentiated derivative thereof in a control tissue, in some embodiments accumulation of the administered reprogrammed cell of the presently disclosed subject matter and/or an in vitro differentiated derivative thereof that is about 5-fold or greater, and in some embodiments an accumulation of the administered reprogrammed cell of the presently disclosed subject matter and/or an in vitro differentiated derivative thereof that is about 10-fold or greater than in an control tissue.
  • a cell for example, a reprogrammed cell of the presently disclosed subject matter and/or
  • selective targeting and “selective homing” also refer to accumulation of a reprogrammed cell of the presently disclosed subject matter and/or an in vitro differentiated derivative thereof in a target tissue concomitant with an absence of accumulation in a control tissue, in some embodiments the absence of accumulation in all control tissues.
  • Techniques that can be employed for targeting reprogrammed cells of the presently disclosed subject matter are disclosed in PCT International Patent Application Publication Nos. WO 2007/067280 and WO 2009/059032, the disclosure of each of which is incorporated by reference herein in its entirety.
  • absence of targeting is used herein to describe substantially no binding or accumulation of a reprogrammed cell of the presently disclosed subject matter and/or an in vitro differentiated derivative thereof in one or more control tissues under conditions wherein accumulation would be detectable if present.
  • the phrase also is intended to include minimal, background accumulation of a reprogrammed cell of the presently disclosed subject matter and/or an in vitro differentiated derivative thereof in one or more control tissues under such conditions.
  • the administering is of a reprogrammed cell, or a differentiated derivative thereof, which is from a donor.
  • the donor is the same individual as the recipient, but in some embodiments the donor is a different individual.
  • the donor can be immunocompatible with the recipient.
  • the donor is identified as immunocompatible if the HLA genotype matches the HLA genotype of the recipient.
  • the immunocompatible donor is identified by genotyping a blood sample from the immunocompatible donor.
  • the methods can further comprise differentiating the reprogrammed cells (e.g., fibroblasts) to produce a pre-determined cell type prior to administering the composition to the subject.
  • the pre-determined cell type can be selected from the group including, but not limited to a neural cell, an endoderm cell, a cardiomyocyte, and derivatives thereof, although the presently disclosed subject matter is not limited to just these cell types of interest. II1.B.1.
  • compositions of the presently disclosed subject matter comprise in some embodiments a composition that includes an active agent (e.g., a reprogrammed cell and/or a derivative thereof, as well as pluralities thereof) and a carrier, particularly a pharmaceutically acceptable carrier, such as but not limited to a carrier pharmaceutically acceptable for use in humans.
  • an active agent e.g., a reprogrammed cell and/or a derivative thereof, as well as pluralities thereof
  • a carrier particularly a pharmaceutically acceptable carrier, such as but not limited to a carrier pharmaceutically acceptable for use in humans.
  • Any suitable pharmaceutical formulation can be used to prepare the compositions for administration to a subject.
  • suitable formulations can include aqueous and nonaqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostatics, bactericidal antibiotics, and solutes that render the formulation isotonic with the bodily fluids of the intended recipient.
  • formulations of the presently disclosed subject matter can include other agents conventional in the art with regard to the type of formulation in question.
  • sterile pyrogen-free aqueous and nonaqueous solutions can be used.
  • adjuvants and/or biological response modifiers include, but are not limited to monoclonal antibodies, interferons (IFNs, including but not limited to IFN- ⁇ and IFN- ⁇ ), interleukins (ILs, including but not limited to IL2, IL4, IL6, and IL10), cytokines (including, but not limited to tumor necrosis factors), and colony-stimulating factors (CSFs, including by not limited to GM-CSF and GCSF).
  • IFNs interferons
  • ILs interleukins
  • CSFs colony-stimulating factors
  • Suitable methods for administration of the compositions of the presently disclosed subject matter include, but are not limited to intravenous administration and delivery directly to the target tissue or organ.
  • the method of administration encompasses features for regionalized delivery or accumulation of the compositions of the presently disclosed subject matter at the site in need of treatment.
  • the compositions of the presently disclosed subject matter are delivered directly into the tissue or organ to be treated.
  • selective delivery of the cells present in the compositions of the presently disclosed subject matter is accomplished by intravenous injection of the presently disclosed compositions, where the cells present therein can home to the target tissue and/or organ and engraft therein. III.B.3. Dose
  • a “treatment effective amount” or a “therapeutic amount” is an amount of a therapeutic composition sufficient to produce a measurable response (e.g., a biologically or clinically relevant response in a subject being treated).
  • a measurable response e.g., a biologically or clinically relevant response in a subject being treated.
  • Actual dosage levels of an active agent or agents (e.g., a reprogrammed cell and/ or a differentiated derivative thereof) in the compositions of the presently disclosed subject matter can be varied so as to administer an amount of the active agent(s) that is effective to achieve the desired therapeutic response for a particular subject.
  • the selected dosage level will depend upon the activity of the therapeutic composition, the route of administration, combination with other drugs or treatments, the severity of the condition being treated, and the condition and prior medical history of the subject being treated. However, it is within the skill of the art to start doses of the compositions of the presently disclosed subject matter at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
  • the potency of a composition can vary, and therefore a "treatment effective amount" can vary. However, one skilled in the art can readily assess the potency and efficacy of a therapeutic composition of the presently disclosed subject matter and adjust the therapeutic regimen accordingly.
  • the presently disclosed subject matter also provides methods for analyzing differentiation of different cell lineages.
  • the reprogramming strategies disclosed herein, and the cells produced therewith can be employed to study the differentiation of cells representative of all three embryonic layers.
  • the results disclosed herein with respect to erythrocytes and the Real Time PCR results demonstrating expression of early and late stage markers of differentiation demonstrated that reprogrammed cells progressed along pathways of differentiation under the disclosed conditions.
  • Molecular events including sequential gene expression patterns as well as epigenetic changes in each of the cell types can be investigated using the compositions and methods of the presently disclosed subject matter.
  • the presently disclosed subject matter also provides methods for analyzing the transition of differentiated somatic cells to cancer stem cells during tumor formation and/or progression.
  • the present disclosure includes a large amount of data that demonstrates that mutations of the members of the RB1 family can lead to the generation of cells with properties of cancer stem cells. Mutations in RB family members are known to be important events in cancer, as most if not all cancers appear to inactivate one or more RB1 family members as a step toward transformation.
  • compositions and methods of the presently disclosed subject matter can be employed as a model for RB1 family-dependent transition of cells (e.g., ES cells, iPSCs, or other cells) to cancer stem cells.
  • cells e.g., ES cells, iPSCs, or other cells
  • EMT epithelial-mesenchymal transcription
  • the presently disclosed subject matter can be employed in investigations of other events that might be responsible for transition of cells to cancer stem cells.
  • cancers can be initiated by an outgrowth of fully differentiated somatic cells into sphere-like structures with concomitant loss of cell-cell contact inhibition.
  • Cells within these growing spheres undergo dedifferentiation to form cells with properties of cancer stem cells.
  • the methods and compositions of the presently disclosed subject matter could be employed as a model in culture and also in vivo in tumor formation models to define the steps in cancer formation that are initiated by outgrowth of differentiated somatic cells lacking cell-cell contact inhibition. In some embodiments, this could involve investigation of gene expression changes as well as epigenetic changes responsible for such alterations in gene expression.
  • Wild type mouse embryo fibroblasts were isolated from E13.5 mouse embryos, and Rb family mutant MEFs were kind gifts from Tyler Jacks (Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America), Julien Sage (Stanford University, Palo Alto, California, United States of America), and Gustavo Leone (The Ohio State University, Columbus, Ohio, United States of America).
  • Fibroblasts in which all three RB1 family members have been mutated referred to herein as "triple knockouts"; TKOs) derived from four separate embryos were used in the experiments described herein with similar results. Cells were analyzed beginning at passage 4, but similar results were also seen at passage 11.
  • the cells were cultured in DMEM with 10% heat-inactivated fetal bovine serum.
  • CHEMICON® Chemicon Inc., a division of Millipore Corp., Billerica,
  • Millipore Millipore Corp., Billerica, Massachusetts, United States of America; Santa Cruz: Santa Cruz Biotechnology Inc., Santa Cruz, California, United
  • Tumor formation in nude mice Either spheres (after two weeks in suspension culture) or trypsinized monolayers of cells derived from spheres were injected subcutaneously into the right hind limb of Balb/cAnNCr-nu/n ⁇ nude mice (available from the National Cancer Institute at Fredrick, Frederick, Maryland, United States of America). Tumors were fixed in 10% buffered formalin, embedded in paraffin, sectioned at 5 ⁇ m, and stained with hematoxylin and eosin (H&E) or used for immunostaining.
  • H&E hematoxylin and eosin
  • SP cells were identified and isolated using a MOFLOTM cell sorter (Dako North America, Inc., Carpinteria, California, United States of America) after excitation of the Hoechst dye with a 350 nm UV laser (100 mW power was used). Fluorescence light emitted by cells was directed toward a 510 nm DCLP dichroic mirror and collected simultaneously by two independent detectors following a 450/65 nm and a 670/30 nm band pass filters, respectively. Cells were analyzed on a linearly amplified fluorescence scale.
  • RNA extraction and Real Time PCR RNA was extracted using TRIZOL® reagent (INVITROGENTM Corp.), and cDNA was synthesized using the INVITROGENTM RT kit (INVITROGENTM Corp.), and SYBR® Green Real Time PCR was performed using a Stratagene Mx3000P Real Time PCR system (Stratagene, La JoIIa, California, United States of America). PCR primers are described in Table 1 hereinabove.
  • a mouse stem cell Real Time PCR Array was also analyzed (Catalogue No. APMM-405, SABIOSCIENCESTM Corporation, Frederick, Maryland, United States of America). Three independent samples, each in triplicate, were analyzed for each Real Time PCR condition.
  • shRNA oligomers used for and Zeb2 silencing were described previously (Nishimura et al. , 2006).
  • the shRNAs were first cloned into a CMV-GFP lentiviral vector where its expression was driven by the mouse U6 promoter.
  • each shRNA construct was generated by synthesizing an 83-mer oligonucleotide containing: (i) a 19-nucleotide sense strand and a 19-nucleotide antisense strand separated by a nine-nucleotide loop (5'-TTCAAGAGA-3'); (ii) a stretch of five adenines as a template for the PoIIII promoter termination signal; (iii) 21 nucleotides complimentary to the 3 1 end of the PoIIII U6 promoter; and (iv) a 5' end containing a unique Xba ⁇ restriction site.
  • the long oligonucleotide was used together with a SP6 oligonucleotide (5'- ATTTAGGTGACACTATAGAAT-3'; SEQ ID NO: 71) to PCR-amplify a fragment containing the entire U6 promoter plus shRNA sequences.
  • the resulting product was digested with Xba ⁇ and Spel, ligated into the Nhel site of the lentivirus vector, and the insert was sequenced to ensure that no errors had occurred during the PCR or cloning steps.
  • sequences of the 19-nucleotide sense strands were ⁇ '-AAGACAACGTGAAAGACAA-S' (SEQ ID NO: 72) for Zeb1 and ⁇ '-GGAAAAACGTGGTGAACTA-S' (SEQ ID NO: 73) for Zeb2.
  • a negative control shRNA was also tested that had a sense strand of 5'- AACAAGATGAAGAGCACCA-3' (SEQ ID NO: 74).
  • RB1 Family Mutation Allows Outgrowth of Cells into Spheres Leading to Survival in Suspension and Stable Changes in Cell Morphology Consistent with their lack of cell-cell contact inhibition, once mouse embryo fibroblasts (MEFs) in which all three RB1 family members had been mutated (referred to herein as "TKOs") became confluent in culture, they began to stack up on one another leading to the generation of mounds of cells on the plates. See Figures 1A and 1 B. Similar results were seen with cells at passages 4, 11 , and 40, and with TKOs isolated from four different litters of mice.
  • the TKO spheres visually resembled embryoid bodies seen when embryonic stem cells are placed in suspension culture (see Figures 1C and 1 D; Desbaillets et al., 2000), and when transferred to non-adherent plates, these spheres could be maintained for at least two months in suspension. During this period, they increased in size and formed a central cavity (see Figure 1E). When the spheres were transferred back to a tissue culture plate, they adhered to the plate and all of the cells within the spheres migrated back onto the plate, reforming a monolayer (see Figures 1 F and 1G).
  • TKO-Ras TKO-Ras
  • Figure 4 expression of activated V12Ras in TKOs
  • TKO-Ras cells could form colonies in soft agar.
  • Sage et al. reported that TKO-Ras cells could indeed form colonies in soft agar and tumors in nude mice (Sage et al., 2000), but Peeper et al. reported that V12Ras expression did not allow for growth of TKOs in soft agar (Peeper et al., 2001).
  • TKO-Ras cells did form colonies in soft agar and tumors in nude mice when 50,000 cells were injected (Figure 3; see below).
  • the differential effects of V12Ras in the TKO-Ras cells could be due to the levels of Ras expression in different cells, since three different V12Ras-expressing cells were used in the studies.
  • TKO-Ras cells did not form spheres in suspension that resembled those formed by TKOs (see Figure 2B). Instead, single cells and small clusters of TKO-Ras cells began to appear in suspension after the TKO- Ras cells achieved confluence in culture.
  • TKO-Ras cells in suspension were allowed to reattach to culture plates, they were visually indistinguishable from cells maintained in monolayer culture. Thus, the TKO-Ras cells in suspension did not undergo the morphological changes seen with TKO cells in spheres.
  • activated Ras allows for survival and proliferation of single TKO cells in suspension. Formation of spheres allowed the TKOs to survive and proliferate in suspension in the absence of activated Ras.
  • Nanog is a downstream target of Oct4 and thus its expression can be viewed as a functional readout of Oct4 activity.
  • the level of Nanog mRNA paralleled that of Oct4 during this time course of sphere culture ( Figure 6B).
  • upregulation of other genes associated with stem cells was observed in both TKO and RBT 7" MEF spheres ( Figure 6D; Figure 7).
  • Expression of CD44 and CD133 was induced, and CD24 expression was downregulated (see Figure 6D).
  • EXAMPLE 4 A Subset of Cells with Properties of a Side Population Generated in TKO and RB1 " ' " MEF spheres
  • Hoechst7Abcg27CD133 ⁇ cells that were typically greater than 10 microns in diameter. See Figure 10.
  • RB1 " ' " cells were then examined for SP properties including exclusion of Hoechst dye; cell surface expression of Abcg2 and CD133; small size (e.g., about 5-7 microns in diameter); and expression of the Klf4, Oct4, Sox2, and c- myc in levels similar to those seen in ES cells. Additional properties identified for these cells included an ability to divide asymmetrically to yield additional SP cells and MP cells (which lack these properties), and ability of a low number (as few as 100 cells) to generate tumors in nude mice. As opposed to MP cells, the tumors formed with SP cells contained cancer cells as well as differentiated cells expressing the neuronal marker beta3 tubulin. MP tumors did not contain differentiated cells (see below).
  • the sorted MP cells were analyzed. These cells were proliferative, but they did not divide asymmetrically to give rise to SP cells (Figure 8D). However, it is of note that while the sorted MP cells were originally devoid of SP cells, a small number of SP cells appeared in the dividing MP culture ( ⁇ 1%), and this number remained relatively constant in the proliferating MP population for at least one month ( Figure 11). Taken together, it appears that SP cells from both TKO and RB1 "A spheres can give rise to MP cells via asymmetric division, and that the MP cells in turn can divide symmetrically to increase their number in the population (although there was a low level of SP cell generation in the MP).
  • Zeb1 mRNA is Induced in SP cells and is Associated with a CD44 hiqh/CD24 low mRNA Expression Pattern
  • EMT E-cadherin and epithelial-mesenchymal transition
  • Snail repression of E-cadherin and EMT appears to be mediated at least in part through induction of Zeb1 and Zeb2 (Peinado et a/., 2007).
  • EMT factors can also trigger a CD44 hl9h /CD24 l0W pattern on epithelial cells, which is associated with the somatic cells acquiring stem cell and cancer stem cell properties (Mani et al., 2008). Therefore, whether expression of these EMT transcription factors was induced in the sphere-derived SP cells was tested.
  • CD44 mRNA and downregulation of CD24 mRNA in SP cells was tested. Indeed, CD44 mRNA was induced in SP cells, whereas CD24 mRNA was diminished ( Figure 12D). In addition to this CD44 high /CD24 l0W mRNA pattern in the SP cells, it is of note that CD133 mRNA and protein was also induced in the SP cells.
  • the data in the AVG columns present fold changes of expression in MEFs as compared to TKOs (individual levels normalized based on ACTB expression levels, n.d., not determined as the gene product was not detected in one or the other sample.
  • Induction of mRNAs for markers of all three embryonic layers was seen in the sphere-derived cells (see also Figures 7 and 16A-16C). These markers included important developmental transcription factors such as GATA4, T, Msx1 , Foxa2, MyoD, Ascl2, PDX1 , PPAR ⁇ and isleti , and components of development signaling pathways including TGF- ⁇ /BMP, notch, wnt, and FGF ( Figures 7 and 16A-16P).
  • markers of terminal differentiation such as cardiac actin, myosin heavy chain, osteocalcin, aggrecan, E-cadherin, transferin, ⁇ -fetoprotein (AFP), myelin basic protein, GFAP, tyrosine hydroxylase, ⁇ -lll tubulin, NCAM, Neurog2, Col9a1 , CD19, CD3, CD4, and CD8.
  • markers of terminal differentiation such as cardiac actin, myosin heavy chain, osteocalcin, aggrecan, E-cadherin, transferin, ⁇ -fetoprotein (AFP), myelin basic protein, GFAP, tyrosine hydroxylase, ⁇ -lll tubulin, NCAM, Neurog2, Col9a1 , CD19, CD3, CD4, and CD8.
  • the perimeter of embryoid bodies formed from ES cells typically contain early endodermal cells characterized by expression of AFP and GATA4, and this region is a site of hematopoietic and endothelial differentiation resembling embryonic yolk sac blood islands (Burkert et al., 1991).
  • a band of cells was observed around the perimeter of RBT 7" MEF spheres which resembled endodermal cells ( Figures 17A-17C), and these cells immunostained for AFP ( Figures 17D and 17E).
  • This region also immunostained for GATA4 and mRNAs for GATA4 and the early endodermal transcription factors Foxa2, PDX1 , and IsH were also induced in spheres ( Figures 7, 17A, and 18).
  • This region of the spheres also contained a number of cells with eosinophilic cytoplasm, and these cells immunostained for globin, indicating that they were erythroid ( Figures 17F-17H and 19). While most of these globin + cells were nucleated, some of the cells lacked nuclei ( Figures 17H and 19), implying that they might have been progressing from erythroblast like progenitors toward erythrocytes in the spheres.
  • This perimeter region of the spheres also contained cells with elongated morphology resembling endothelial cells (Figures 17A-17C), and indeed these cells immunostained for the endothelial marker CD31 ( Figures 171 and 17J).
  • Figures 19A-19M show that the cells in spheres differentiated to form erythrocytes at various stages of differentiation, some of which have nuclei and some of which have lost their nuclei.
  • Figures 19N-19R show immunostaining for hemoglobin demonstrating that the forming erythrocytes expressed hemoglobin. Other cells of hematopoietic origin were also evident in the spheres.
  • Figures 19S and 19T show a megakaryocyte. Together, these results demonstrated that cells in the spheres differentiated into various hematopoietic lineages, which is also a characteristic of ES cells and iPSC cells.
  • TKO-SDC TKO sphere-derived cells containing approximately 10% SP and 90% MP cells (see Figure 8).
  • TKOs or RB1 " ' " MEFs form colonies in soft agar ( Figure 3).
  • the sphere-derived TKO and RBY 1' MEF cells also formed tumors (500 ⁇ 18 mg).
  • tumors formed from small spheres or sphere-derived cells were indistinguishable, and tumors from TKO or RB1 " ' " sphere-derived cells were also indistinguishable ( Figure 21 ). These tumors also appeared to be spindle cell sarcomas similar to those formed with TKO-Ras cell. However, tumors from sphere-derived cells also contained sphere-like whorls with eosinophilic centers (which were not evident in TKO-Ras tumors;
  • SP cells were originally identified as the subpopulation of tumors capable of efficiently regenerating the tumor when transplanted. Therefore, different numbers of sorted SP and MP cells were injected into nude mice to assess which population was tumorigenic. Two independent experiments were performed with two injections of each cell number in the following experiments. Initially, 50,000, 20,000, 5,000, or 1 ,000 MP cells were injected. While tumors formed with each injection of 50,000 MP cells (523 ⁇ 93 mg after 31 days), no tumors were observed in any injection with 20,000 or fewer MP cells, even after two months. However, when 5,000; 2,000; 500; or 100 SP cells were injected, tumors formed at each injection level and grew rapidly (e.g., 813 ⁇ 279 mg at three weeks with 100 SP cells injected).
  • SP cells were the primary initiators of tumor formation among the sphere derived cells. Even though the sorted MP population was initially devoid of SP cells, it is of note that a small percentage of SP cells ( ⁇ 1%) became evident with passage of the MP population in culture, and this number of SP cells remained relatively constant for at least one month in culture ( Figure 11). Therefore, the appearance of a small percentage of SP cells among the MP population might account for tumor formation seen when a large number of MP cells (50,000) was injected.
  • the tumors formed from SP and MP cells were histologically distinct (see Figures 20D-20F).
  • the MP tumors were indistinguishable histologically from those formed with TKO-Ras cells ( Figures 2OC and 20D), whereas SP tumors contained neuronal whorls ( Figures 2OE and 20F). These whorls were similar in appearance those seen in tumors derived from unsorted sphere-derived TKO or RBr' " cells ( Figure 21), but they were more numerous; they also immunostained for the neuronal marker ⁇ -lll tubulin ( Figures 2OG and 20H).
  • the SP tumors also contained clusters of cells expressing nuclear Oct4 and Nanog throughout the tumor ( Figures 20I-20L), suggesting that SP cells were maintained in the forming tumor.
  • wild type MEFs from E13.5 mouse embryos i.e., mouse embryos at embryonic day 13.5 post coitus (p.c.)
  • E13.5 mouse embryos i.e., mouse embryos at embryonic day 13.5 post coitus (p.c.)
  • MEFs were grown to confluency, scraped from tissue culture plates, and placed in suspension as described hereinabove.
  • Cells immediately formed spheres (see Figure 22A) and these spheres were viable in culture for at least two months.
  • RNA was isolated from the spheres and used in Real Time PCR assays. As described hereinabove, there was induction of mRNAs for several stem cell genes (see Figure 22B).
  • Histological sections of sphere after one month in culture showed the presence of both nucleated and anucleated red blood cells that immunostained for globin and reacted with benzadine, which demonstrated the presence of hemoglobin in the cells. Megakarocytes and neutrophils were also evident. Other bone marrow cells were also present, lmmunostaining for ⁇ -lll tubulin demonstrated the presence of neurons, and immunostaining for E-cadherin and ZO1 was evident on the surface of epithelial cells arranged in secretory ducts.
  • Hoechst7Abcg2 + /CD133 + SP cells have been isolated from wild type MEF spheres, and it was determined that the Hoechst " /Abcg2 + /CD133 + SP cells were the cells that expressed markers. Additionally, these cells had an additional property that distinguished them from other cells in the spheres; they were small in diameter, ranging from 5-7 microns. Taken together, these results demonstrated that cells with the size and expression pattern of stem cells could be generated from wild type MEFs after one week of culture as spheres in suspension culture.
  • ES cells When cultured under similar sphere-forming conditions, ES cells undergo differentiation into cells representative of all three embryonic layers. Indeed, the results demonstrated that mRNA indicative of each of the three embryonic layers were induced in the spheres. Thus, stem cell-like cells in the spheres had the same property as ES cells in that they were capable of generating differentiated cells representing each of the three embryonic layers in spheres.
  • Figure 23 Similar studies were performed with human fibroblasts (see Figure 23). These included primary cultures of human foreskin fibroblasts and primary cultures of fibroblasts from lung (IMR90 and WI38).
  • Figure 23A shows the presence of endodermal-like cells at the border of the sphere after H&E staining as evidenced by immunostaining with the endodermal marker ⁇ - fetoprotein (AFP; see Figure 23E). These same cells were positive for the endothelial marker CD31 (see Figure 23F) and ⁇ -globin (see Figure 23G). Cells resembling nucleated blood cells were also present (see Figures 23B and 23C), which was confirmed by benzidine staining, which demonstrated the presence of hemoglobin (see Figure 23D).
  • Figure 23J shows benzidine staining of wild type MEF spheres. Benzidine staining demonstrated the presence of hemoglobin in cells of MEF spheres.
  • Figure 23K1 shows H&E staining of an erythrocyte
  • Figure 23K2 shows immunostaining of an adjacent section of the sphere for hemoglobin, demonstrating that this erythrocyte expressed hemoglobin.
  • Figures 23L1-23L3 show immunostaining of another erythrocyte for hemoglobin, and this cell was nucleated as demonstrated by DAPI nuclear staining.
  • wild type MEF spheres contained both nucleated (i.e., immature) and mature erythrocytes.
  • Figures 23M1-23M3 show innumostaining for CD31 , which is a marker of endothelial cells.
  • DAPI staining was used to show the nucleus of the cell.
  • CD31 staining demonstrated that endothelial cells were formed in the wild type MEF spheres, which also occurs in ES cell and iPSC spheres.
  • Figures 23N and 230 are photomicrographs showing a region of cartilage stained with alcian blue.
  • Figure 23P is a photomicrograph showing pearls of keratin (dark staining) in an keratinized cyst.
  • Figure 24A is a photomicrograph showing a secretory epithelial ascinar like structure with a central duct (arrow), and Figure 24B shows evidence of the formation of secretory ducts (gray arrows) and red blood cells (white arrow).
  • Figure 24 The top middle and top right photomicrographs of Figure 24 show hair fibers at the border of the spheres (the border is identified by black arrows), and Figures 24C and 24D shows immunostaining for the epithelial marker E cadherin (Cdh1) and the neuronal marker ⁇ -lll tubulin.
  • Figures 24E and 24F (the latter an enlargement of the field in the box in Figure 24E) show hair fibers at the border of the spheres (the border is identified by black arrows).
  • Figures 25A-25Q are a series of photomicrographs of spheres produced by Hoechst7Abcg2 + /CD133 + cells derived from wild type MEFs after 2 weeks in culture.
  • the Hoechst7Abcg2 + /CD133 + cells were isolated by cell sorting and cultured on a feeder layer of irradiated fibroblasts.
  • the wild type MEFs were isolated from ⁇ -actin-GFP mice obtained from The Jackson Laboratory (Bar Harbor, Maine, United States of America). Cells in the center of the colonies maintained a Hoechsf " phenotype (characteristic of ES cells), whereas cells on the edges of the colonies became Hoechst* (which is characteristic of differentiating cells).
  • Hoechst + cells gave rise to a variety of differentiated cells that migrated away from the original colony. These differentiated cells expressed ⁇ -lll tubulin ( ⁇ 3Tub), GFAP, Troponin I, CD34, CD45, AFP, ZO1 , Ter119, or globin as shown in Figures 25D-25Q. These results demonstrated that Hoechst7Abcg2 + /CD133 + cells derived from the wild type MEF spheres could be maintained in an undifferentiated state in culture, and that these cells could give rise to lineages representative of all three embryonic layers.
  • Hoechst " /Abcg2 + /CD133 + cells could differentiate into a variety of different lineages in monolayer culture: ⁇ -lll tubulin indicative of neurons; GFAP indicative of glial cells; AFP indicative of endodermal cells; ZO1 indicative of epithelial cells; troponin I indicative of cardiomyocytes; CD34 and CD45 indicative of hematopoietic lineages; Ter119 indicative of erythrocyte progenitors; and globin indicative of erythrocytes.
  • This ability of Hoechst7Abcg27CD133 + cells from wild type MEF spheres to differentiate into a variety of lineages is shared by ES cells and inducible pluripotent stem cells (iPSC).
  • the cells behaved like ES cells and iPSC in monolayer culture as well as in the spheres.
  • sphere formation with both mouse and human fibroblasts led to expression of proteins indicative of all three embryonic layers.
  • the morphologies of the cells in these spheres were consistent with such differentiation.
  • Figures 26A-26E are a series of photomicrographs of teratoma formation by Hoechst7Abcg2 + /CD133 + cells derived from wild type MEF spheres after 2 weeks in suspension culture. Four independent preparations of 50,000 cells were injected into both hindlimbs of nude mice. Tumors were observed in all 8 injections, and were tumors were collected after three weeks.
  • Figure 26A is a Normarski image of a representative teratoma
  • Figure 26B is a higher power view of an adjacent section of the tumor stained with H&E.
  • the MEFs were isolated from Actin-GFP mice and immunostaining for GFP (see Figure 26D), which showed that the tumor was GFP + whereas surrounding host tissue was GFP " .
  • These results demonstrate Hoechst7Abcg2 + /CD133 + cells derived from wild type MEF spheres had another property of ES cells and iPSCs: they formed teratomas.
  • FIGS 27A-27H are a series of photomicrographs of teratomas formed with Hoechst7Abcg2 + /CD133 + cells derived from wild type MEF spheres showing cobblestone epithelial morphology and expressing the epithelial specification protein E-cadherin (see
  • Figures 27C and 27D low power and 27G and 27H (higher power), which present E-cadherin immunostaining on the surface of the cells).
  • These teratomas contained cells representative of all three embryonic layers as well as differentiated tissues, similar to teratoma formation by ES cells.
  • Hoechst7Abcg2 + /CD133 + isolated from MEF-derived spheres formed teratomas containing differentiated epithelial cells.
  • Figure 28A is a Nomarski image of adipose tissue present in a teratoma.
  • Figure 28C shows immunostaining for GFP showing that the adipose tissue was derived from the injected Hoechst " /Abcg2 + /CD133 + cells.
  • Figure 28E is a Nomarski image of a neuronal structure in a teratoma.
  • Figure 28G shows immunostaining of the section of Figure 28E for ⁇ -lll tubulin, showing a cluster of neurons within a neuronal structure in the teratoma.
  • Figure 28I is a Nomarski image of a region of intestinal-like epithelium in a teratoma.
  • Figure 28K shows immunostaining of the cells presented in Figure 28I for GFP, and shows that this intestinal-like structure was derived from injected Hoechst7Abcg2 + /CD133 + cells.
  • Figure 28M is a Nomarski image of a secretory epithelial structure in a teratoma.
  • Figure 280 shows GFP immunostaining and demonstrated that the structure in Figure 28M is derived from the injected Hoechst7Abcg27CD133 + cells.
  • Figure 28P shows the results of immunostaining the structure for CDH 1 expression, which demonstrated that the structure was epithelial.
  • Figures 29A-29I are a series of photomicrographs showing formation of skeletal muscle in a teratoma arising from injection of wild type MEF Hoechsf " /Abcg2 + /CD133 + cells derived from spheres into nude mice.
  • Figure 29A shows skeletal muscle fibers in the teratoma by H&E staining.
  • a Nomarski image of an adjacent section is shown as Figure 29B and GFP staining is shown in Figure 29D, demonstrating that the muscle cells ware tumor-derived.
  • Control photomicrographs are presented in Figures 29F-29I .
  • a Nomarski image of host skeletal muscle is shown in Figure 29F.
  • DAPI staining is shown in Figure 29G and GFP is shown in Figure 29H.
  • GFP staining There was a lack of GFP staining in Figure 29H 1 which is host muscle that does not express GFP, indicating that Hoechst7Abcg27CD133 + cells derived from wild type MEF spheres formed teratomas in nude mice containing skeletal muscle, which is also known to occur with teratomas derived from ES cells.
  • the presently disclosed subject matter demonstrated the presence of multiple differentiated tissues in the teratoma formed with Hoechst " /Abcg2 + /CD133 + cells derived from wild type MEF cells following Sphere formation.
  • Hoechst " /Abcg2 + /CD133 + cells derived from wild type MEF spheres had properties of ES cells or inducible pluripotent stem cells (iPSC).
  • iPSC inducible pluripotent stem cells
  • MEF spheres after two weeks in suspension culture were transferred to tissue culture dishes. Spheres attached to the plates and cells began to migrate out onto the plate as was observed with TKO and RB1 "A MEF spheres. However, in contrast to the TKO and RBT 7" MEF cells, only a portion of the cells from the wild type MEF spheres migrated back onto the plate. These cells were highly pigmented (see Figures 30A-30C). Initially, most of the cells were rounded or epithelial in appearance. However after several days on the plate, the cells remained pigmented but they began to elongate (see Figures 30D- 30F). After several more days, the cells were still pigmented but then began to send out multiple dendritic-like projections resembling melanocytes.
  • the cells were immunostained for two melanocyte-specific markers: Mitf and mel5. All of the pigmented cells immunostained for both markers, suggesting that the pigmented cells which migrated out of the MEF spheres were melanosome-like and that they took on the morphology and gene expression pattern of melanocytes after several days in culture. Similar results were seen with spheres formed from human foreskin fibroblasts and with the normal human lung fibroblast lines IMR90 and WI38 obtained from the American Type Culture Collection (ATCC®; Manassas, Virginia, United States of America). EXAMPLE 12
  • Tyr and Tyrpi are key genes in the pigment synthesis cascade. Pax3 and Sox10 cooperate with
  • MITF-M isoform in specification of melanocytes.
  • RPE65 is a marker of retinal pigment epithelial cells which is not expressed in melanocytes and thus was employed as a control.
  • MEFs human foreskin fibroblasts, or normal human lung fibroblast cell lines IMR90 and Wi38 were grown to confluence and then scraped from tissue culture plates and placed in suspension culture in non-adherent plates. After two weeks in culture, the resulting spheres were transferred to culture dishes and as with TKO and RB1 null MEFs, cells in the sphere migrated back onto the tissue culture dishes to reform a monolayer. However, in contrast to the mutant MEFs, not all of the cells in the wild type spheres migrated back out of the spheres.
  • Mitf and mel5 (tyrosinase related protein 75) antibodies were from Abeam Inc., Cambridge, Massachusetts, United States of America and were used at a dilution of 1 :50 as described by the manufacturer.
  • these epithelial spheres contained a number of nucleated and non-nucleated eosinophilic cells resembling erythrocytes and erythrocyte progenitors similar to those seen with spheres of fibroblasts. Sections of the epithelial spheres were then immunostained for the alpha globin chain of hemaglobin, and the sections were also stained with benzidine-peroxide, which produced a dark blue reaction in the presence of hemoglobin (see arrows in Figures 32A-32J).
  • human lung epithelial cells could also form spheres in suspension culture and underwent a similar differentiation into cells resembling erythrocytes as seen with fibroblast spheres. As such, it appeared that epithelial cells induced to form spheres in suspension also underwent reprogramming and differentiated into other cell types. Discussion of the EXAMPLES
  • Embryonic stem (ES) cells and induced pluripotent stem cells (iPSC) can classically differentiate into cells representing each of the three embryonic lineages (ectoderm, endoderm, and mesoderm) when placed in suspension culture, and this differentiation is accompanied by activation of signaling pathways including Wnt, Notch, and growth factors such as BMP and FGF.
  • the Real Time PCR results disclosed herein demonstrated that TKO cells placed in spheres can, like ES cells and iPSC, differentiate into cells expressing mRNAs for markers of all three embryonic layers.
  • TKO cells could also give rise to cancer cells, suggesting that mutation of the RB1 family might associated with cancer generation in these cells.
  • wild type MEFs without the RB1 family mutations i.e., that are RBI + , RBLI + , and RBL2 +
  • wild type MEFs without the RB1 family mutations also differentiated into cells expressing mRNAs for markers of all three embryonic layers, but did not give rise to cancer cells in the same fashion as did TKO MEFs.
  • the RB1 pathway was mutated, these reprogrammed cells gave rise to both differentiated cells as well as cancer stem cells, which in turn gave rise to cancer cells.
  • sphere formation using wild type mouse or human fibroblasts led to similar reprogramming, but cancer cells were not produced.
  • maintaining a functional RB1 pathway could prevent the production of cancer cells during reprogramming of fibroblast via sphere formation.
  • Sphere formation can provide reprogramming, but since the endogenous stem cell genes were reexpressed (i.e., without requiring ectopic expression from recombinant vectors), there was no need for viral infection and its associated cancer risk.
  • Undifferentiated ES cells form teratomas when injected into hosts, thus these cells must be partially differentiated in culture prior to injection. Nevertheless, a cancer risk remains from any remaining undifferentiated cells. Additionally, partial differentiation of ES cells seems to be required for their ability to facilitate repair of tissues in vivo. Sphere-derived cells from wild type mouse or human fibroblasts did not appear to pose a cancer risk. Therefore, progenitors representative of cells in all three embryonic layers can be sorted from spheres using specific cell surface markers and can be used in similar therapies as partially differentiated ES cells or induced pluripotent fibroblasts. Based on the discoveries described herein, cells in spheres can be directed toward specific differentiation pathways by using the various differentiation protocols that have been established for ES cells.
  • An exemplary approach is that skin fibroblasts from a patient following punch biopsy are placed in culture and used to form spheres.
  • the sphere derived cells can be exposed to appropriate growth factors and cytokines designed to enhance and/or facilitate formation of a specific cellular lineage. Cells surface markers specific for this lineage can be used to sort the differentiated cells, which can then in turn be used therapeutically in cell transfer back to the patient.
  • Exemplary advantages of employing the presently disclosed cells rather than ES cells include, but are not limited to the fact that the former are not characterized by the ethical concerns raised by use of the latter, apparently have greatly reduced or no risk of teratoma formation, and would not give rise to histocompatibility issues (or other genetic or infection issues) because the sphere-derived cells can be isolated from the subject into which they would thereafter be introduced (unlike ES cells).
  • induced pluripotent fibroblasts disclosed herein would be expected to have over ES cells is that endogenous "pluripotency markers" (e.g., Oct4, Sox2, and Klf4) are caused to be re- expressed in the sphere-derived cells without the need to resort to employing viral infection, which has been linked to cancer risk.
  • endogenous "pluripotency markers” e.g., Oct4, Sox2, and Klf4
  • sphere formation is a mechanism for reprogramming of fibroblasts to a multipotential phenotype. While the instant co-inventors do not wish to be bound by any particular theory of operation, a proposed model for a pathway for generation of cells with properties of cancer stem cells from differentiated somatic cells is presented in Figure 33.

Abstract

L'invention concerne des méthodes de production d'un fibroblaste reprogrammé. Les méthodes peuvent consister à faire croître une pluralité de fibroblastes en culture monocouche afin d'obtenir une confluence; et à rompre la culture monocouche pour placer au moins une fraction de la pluralité de fibroblastes en culture en suspension dans des conditions suffisantes pour former une ou plusieurs sphères de type corps embryoïde, la ou les sphères de type corps embryoïde comprenant un ou plusieurs fibroblastes reprogrammés qui expriment un ou plusieurs marqueurs non exprimés par un fibroblaste croissant dans la culture monocouche avant l'étape de rupture. L'invention concerne également des fibroblastes reprogrammés produits selon les méthodes susmentionnées, des préparations qui comprennent des fibroblastes reprogrammés, ainsi que des méthodes de traitement d'une lésion d'un tissu chez un sujet par administration au sujet en attente d'un tel traitement d'une composition de cellules fibroblastiques reprogrammées dans un support pharmaceutiquement acceptable.
PCT/US2009/067503 2008-12-10 2009-12-10 Cellules pluripotentes dérivées de cellules stomatiques et leurs méthodes d'utilisation WO2010068758A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012071108A1 (fr) * 2010-11-22 2012-05-31 University Of Louisville Research Foundation, Inc. Cellules pluripotentes dérivées de cellules somatiques et procédés d'utilisation de celles-ci
CN104087551A (zh) * 2014-07-17 2014-10-08 济南磐升生物技术有限公司 一种体外分离培养人的表皮细胞的新方法
US9730975B2 (en) 2014-11-25 2017-08-15 The Penn State Research Foundation Chemical reprogramming of human glial cells into neurons for brain and spinal cord repair
KR102224273B1 (ko) * 2019-10-10 2021-03-08 고려대학교 산학협력단 줄기세포 유래 성숙 심근세포 및 이를 이용한 심혈관 질환 모델

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040161419A1 (en) * 2002-04-19 2004-08-19 Strom Stephen C. Placental stem cells and uses thereof
US20080233649A1 (en) * 2004-03-05 2008-09-25 Raewyn Seaberg Pancreatic stem cells
US20080268054A1 (en) * 2000-12-04 2008-10-30 Eugene Bell Dermal derived human stem cells and compositions and methods thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080268054A1 (en) * 2000-12-04 2008-10-30 Eugene Bell Dermal derived human stem cells and compositions and methods thereof
US20040161419A1 (en) * 2002-04-19 2004-08-19 Strom Stephen C. Placental stem cells and uses thereof
US20080233649A1 (en) * 2004-03-05 2008-09-25 Raewyn Seaberg Pancreatic stem cells

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
KIM ET AL.: "Pegylated recombinant human epidermal growth factor (rhEGF) for sustained release from biodegradable PLGA microspheres.", BIOMATERIALS, vol. 23, no. 11, 2002, pages 2311 - 2317, XP004345260, Retrieved from the Internet <URL:http://www.ncbi.nlm.nih.gov/sites/entrez> [retrieved on 20100427] *
LIGON ET AL.: "Olig2-regulated lineage-restricted pathway controls replication competence in neural stem cells and malignant glioma.", NEURON, vol. 53, no. 4, 2007, pages 503 - 517, XP008167380 *
NAKAGAWA ET AL.: "Generation of induced pluripotent stem cells withoutMyc from mouse and human fibroblasts.", NAT BIOTECHNOL., vol. 26, no. 1, January 2008 (2008-01-01), pages 101 - 106, XP009098334 *
See also references of EP2376625A4 *
TAKAHASHI ET AL.: "Induction of Pluripotent Stem Cells from Adult Human Fibroblastsby Defined Factors.", CELL, vol. 131, 2007, pages 861 - 872, XP002530547 *
WERNIG ET AL.: "In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state.", NATURE, vol. 448, 2007, pages 318 - 324, XP002621304 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012071108A1 (fr) * 2010-11-22 2012-05-31 University Of Louisville Research Foundation, Inc. Cellules pluripotentes dérivées de cellules somatiques et procédés d'utilisation de celles-ci
CN104087551A (zh) * 2014-07-17 2014-10-08 济南磐升生物技术有限公司 一种体外分离培养人的表皮细胞的新方法
US9730975B2 (en) 2014-11-25 2017-08-15 The Penn State Research Foundation Chemical reprogramming of human glial cells into neurons for brain and spinal cord repair
US11850273B2 (en) 2014-11-25 2023-12-26 The Penn State Research Foundation Chemical reprogramming of human glial cells into neurons for brain and spinal cord repair
KR102224273B1 (ko) * 2019-10-10 2021-03-08 고려대학교 산학협력단 줄기세포 유래 성숙 심근세포 및 이를 이용한 심혈관 질환 모델

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