NIH Public Access Author Manuscript Science. Author manuscript; available in PMC 2014 November 03.
NIH-PA Author Manuscript
Published in final edited form as: Science. 2011 December 23; 334(6063 Suppl): 53–54.
Reprogrammed Astrocytes as a Potential Therapy for Neurodegenerative Disorders Changhai Tian1,2,4 and Jialin C. Zheng1,2,3,4,* 1Laboratory
of Neuroimmunology and Regenerative Therapy, Omaha, NE, U.S.A
2Department
of Pharmacology/Experimental Neuroscience, Omaha, NE, U.S.A
3Department
of Pathology/Microbiology, Omaha, NE, U.S.A
4University
of Nebraska Medical Center, Omaha, NE, U.S.A
NIH-PA Author Manuscript
Summary
NIH-PA Author Manuscript
We sought to determine which parental cell type was best suited for creating reprogrammed induced pluripotent stem cells (iPSCs) and what differences might exist between iPSCs generated from various tissues. To do this, we reprogrammed primary mouse astrocytes, a central nervous system derived cell, into astrocyte-derived iPSCs (mAsiPSCs) using an ectopic expression of Oct3/4, Sox2, Klf4 and c-Myc. These mAsiPSCs expressed stem cell markers and formed teratomas. Importantly, mAsiPSCs grew more slowly and were more likely to differentiate into neuronal-type cells compared to mouse embryonic fibroblastderived iPSCs (MEFsiPSCs), a non-central nervous system-derived cell type. These results suggest that mAsiPSCs retain a “memory” of their central nervous system origin and may have a higher incidence of neuronal differentiation. Moreover, direct conversion of somatic cells into lineage-specific progenitor cells in one step, bypassing the intermediated pluripotent stage, provides an alternative strategy for generating promising cell types. We demonstrated the conversion of adult dermal fibroblasts into induced neural progenitor cells (iNPCs) using a novel combination of genes different from those previously reported. These iNPCs exhibit typical NPC morphology and marker gene expression, and a normal capacity for proliferation, self-renewal, and differentiation. Our findings suggest new cellreplacement strategies that may be beneficial for studies involving neurodegenerative diseases, disease modeling, and neural development. Somatic cells have been successfully reprogrammed to form induced pluripotent stem cells (iPSCs), altering their pattern of gene expression, and hence their fate, by ectopic expression of a defined set of factors including Oct3/4, Sox2, and either c-Myc and Klf4 or Nanog and LIN28 (1–4). It was recently reported that reprogrammed cells may retain a “memory” of their original cell type, potentially biasing their differentiation towards the cell’s original lineage and suggesting that iPSCs from different origins may exhibit distinct differentiation preferences (5–7). To elucidate the differences between cells originating from reprogramming ectoderm vs. mesoderm origins, we used mouse astrocytes as the parental
*
Corresponding author: [email protected].
Tian and Zheng
Page 2
NIH-PA Author Manuscript
cell type to generate iPSCs, named mAsiPSCs. Astrocytes were chosen because they are the most abundant neural cell type in the brain and play a critical role in neurodegenerative disorders. Our results showed that mAsiPSCs exhibit morphologies, differentiative potential, and teratoma formation in severe combined immune deficiency (SCID) mice, with derivatives of all three germ layers. Expression profiling of stem cell markers in mAsiPSCs and mouse embryonic fibroblast-derived iPSCs (MEFsiPSCs) showed significant similarities. However, this comparison also suggested that the central nervous systemderived mAsiPSCs were more likely to become neuronal cell types than were the mesodermderived MEFsiPSCs. Further, mAsiPSCs were especially prone to dopaminergic neuron differentiation (see Figure 1). This is consistent with the concept that the differentiation potential for iPSCs is influenced by the epigenetic “memory” of their tissue of origin. Such information is useful when reprogramming cell types for different desired endpoints.
NIH-PA Author Manuscript
Returning a somatic cell to its pluripotent state through reprogramming and then pushing it toward a particular differentiation pathway is demanding, and may increase the risk of obtaining undesired cell types or even neoplasia formation. However, the direct conversion of somatic cells into another cell type has recently been achieved by ectopic expression of defined transcription factors (8–13). Using gene expression profiling and parental cells from E/Nestin:EGFP transgenic mouse as a monitor system, we screened nine candidate transcription factors for the ability to directly convert fibroblasts into neural progenitor cells (NPCs). We found that five of the nine transcription factors can directly convert adult dermal fibroblasts into NPC-like cells, or induced NPCs (iNPCs), and the resulting iNPCs possessed the same properties as wild-type NPCs, including proliferation, self-renewal, and differentiation (Figure 2). These findings may provide an alternative strategy to generate NPCs for cell replacement therapy of neurodegenerative diseases.
References
NIH-PA Author Manuscript
1. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007; 131:861. [PubMed: 18035408] 2. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006; 126:663. [PubMed: 16904174] 3. Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature. 2007; 448:318. [PubMed: 17554336] 4. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007; 318:1917. [PubMed: 18029452] 5. Hu Q, Friedrich AM, Johnson LV, Clegg DO. Memory in induced pluripotent stem cells: reprogrammed human retinal-pigmented epithelial cells show tendency for spontaneous redifferentiation. Stem Cells. 2010; 28:1981. [PubMed: 20882530] 6. Kim K, Doi A, Wen B, Ng K, Zhao R, et al. Epigenetic memory in induced pluripotent stem cells. Nature. 2010; 467:285. [PubMed: 20644535] 7. Polo JM, Liu S, Figueroa ME, Kulalert W, et al. Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells. Nat Biotechnol. 2010; 28:848. [PubMed: 20644536] 8. Pfisterer U, Kirkeby A, Torper O, Wood J, Nelander J, et al. Direct conversion of human fibroblasts to dopaminergic neurons. Proc Natl Acad Sci USA. 2011; 108:10343. [PubMed: 21646515] 9. Sekiya S, Suzuki A. Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors. Nature. 2011; 475:390. [PubMed: 21716291]
Science. Author manuscript; available in PMC 2014 November 03.
Tian and Zheng
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10. Caiazzo M, Dell’Anno MT, Dvoretskova E, Lazarevic Dejan, Taverna Stefano, et al. Direct generation of functional dopaminergic neurons from mouse and human fibroblasts. Nature. 2011; 476:224. [PubMed: 21725324] 11. Ambasudhan R, Talantova M, Coleman R, Yuan X, Zhu S, et al. Direct reprogramming of adult human fibroblasts to functional neurons under defined conditions. Cell Stem Cell. 2011; 9:113. [PubMed: 21802386] 12. Kim J, Efe JA, Zhu S, Talantova M, Yuan X, et al. Direct reprogramming of mouse fibroblasts to neural progenitors. Proc Natl Acad Sci USA. 2011; 108:7838. [PubMed: 21521790] 13. Pang ZP, Yang N, Vierbuchen T, Ostermeier A, Fuentes DR, et al. Induction of human neuronal cells by defined transcription factors. Nature. 2011; 476:220. [PubMed: 21617644]
NIH-PA Author Manuscript NIH-PA Author Manuscript Science. Author manuscript; available in PMC 2014 November 03.
Tian and Zheng
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NIH-PA Author Manuscript NIH-PA Author Manuscript Fig. 1. Comparison of neuronal differentiation between MEFsiPSCs and mAsiPSCs
NIH-PA Author Manuscript
The embryonic bodies (EBs) from MEFsiPSCs and mAsiPSCs were cultured with serumfree Insulin/Transferrin/Selenium/Fibronectin (ITSFn) medium to screen nestin-positive cells. After 6–10 days in culture, the cells were plated on collagen IV-coated coverslips and incubated under neuronal differentiation conditions for 7 days, and then subjected to immunostaining with polyclonal anti-β-tubulin III antibody (Green) and nuclear staining with DAPI (Blue) (A). Cells cultured in dopaminergic (DA) neuron differentiation medium were collected at 0 day, day 7, and day 9, and then subjected to total mRNA extraction and real-time PCR analysis with mouse tyrosine hydroxylase 1-specific primers (B).
Science. Author manuscript; available in PMC 2014 November 03.
Tian and Zheng
Page 5
NIH-PA Author Manuscript NIH-PA Author Manuscript
Fig. 2. Direct conversion of adult mouse skin fibroblasts into induced neural progenitor cells (iNPCs) and their identification
NIH-PA Author Manuscript
Skin fibroblasts from adult Nestin-Enhanced Green Fluorecent Protein transgenic mice (kindly provided by Richard Miller, Northwestern University) were infected with different combinations of pMXs-based transcription factors (TF7; TF5; TF4; TF3). After 12 days in culture with NPC medium, neurospheres were observed in cultures with TF7 or TF5 infection, but not in the TF4 and TF3 cultures (A). Green fluorescent protein-positive single cells from TF7 and TF5 cultures were passaged and neurospheres were reformed (B). iNPCs were cultured on collagen IV-coated coverslips and subjected to conditions resulting in differentiation down neuron or astrocyte pathways, followed by immunostaining with anti-βtubulin III, anti-Glial fibrillary acidic protein GFAP and anti-tyrosine hydroxylase1 antibodies (C, D).
Science. Author manuscript; available in PMC 2014 November 03.
NIH-PA Author Manuscript
Published in final edited form as: Science. 2011 December 23; 334(6063 Suppl): 53–54.
Reprogrammed Astrocytes as a Potential Therapy for Neurodegenerative Disorders Changhai Tian1,2,4 and Jialin C. Zheng1,2,3,4,* 1Laboratory
of Neuroimmunology and Regenerative Therapy, Omaha, NE, U.S.A
2Department
of Pharmacology/Experimental Neuroscience, Omaha, NE, U.S.A
3Department
of Pathology/Microbiology, Omaha, NE, U.S.A
4University
of Nebraska Medical Center, Omaha, NE, U.S.A
NIH-PA Author Manuscript
Summary
NIH-PA Author Manuscript
We sought to determine which parental cell type was best suited for creating reprogrammed induced pluripotent stem cells (iPSCs) and what differences might exist between iPSCs generated from various tissues. To do this, we reprogrammed primary mouse astrocytes, a central nervous system derived cell, into astrocyte-derived iPSCs (mAsiPSCs) using an ectopic expression of Oct3/4, Sox2, Klf4 and c-Myc. These mAsiPSCs expressed stem cell markers and formed teratomas. Importantly, mAsiPSCs grew more slowly and were more likely to differentiate into neuronal-type cells compared to mouse embryonic fibroblastderived iPSCs (MEFsiPSCs), a non-central nervous system-derived cell type. These results suggest that mAsiPSCs retain a “memory” of their central nervous system origin and may have a higher incidence of neuronal differentiation. Moreover, direct conversion of somatic cells into lineage-specific progenitor cells in one step, bypassing the intermediated pluripotent stage, provides an alternative strategy for generating promising cell types. We demonstrated the conversion of adult dermal fibroblasts into induced neural progenitor cells (iNPCs) using a novel combination of genes different from those previously reported. These iNPCs exhibit typical NPC morphology and marker gene expression, and a normal capacity for proliferation, self-renewal, and differentiation. Our findings suggest new cellreplacement strategies that may be beneficial for studies involving neurodegenerative diseases, disease modeling, and neural development. Somatic cells have been successfully reprogrammed to form induced pluripotent stem cells (iPSCs), altering their pattern of gene expression, and hence their fate, by ectopic expression of a defined set of factors including Oct3/4, Sox2, and either c-Myc and Klf4 or Nanog and LIN28 (1–4). It was recently reported that reprogrammed cells may retain a “memory” of their original cell type, potentially biasing their differentiation towards the cell’s original lineage and suggesting that iPSCs from different origins may exhibit distinct differentiation preferences (5–7). To elucidate the differences between cells originating from reprogramming ectoderm vs. mesoderm origins, we used mouse astrocytes as the parental
*
Corresponding author: [email protected].
Tian and Zheng
Page 2
NIH-PA Author Manuscript
cell type to generate iPSCs, named mAsiPSCs. Astrocytes were chosen because they are the most abundant neural cell type in the brain and play a critical role in neurodegenerative disorders. Our results showed that mAsiPSCs exhibit morphologies, differentiative potential, and teratoma formation in severe combined immune deficiency (SCID) mice, with derivatives of all three germ layers. Expression profiling of stem cell markers in mAsiPSCs and mouse embryonic fibroblast-derived iPSCs (MEFsiPSCs) showed significant similarities. However, this comparison also suggested that the central nervous systemderived mAsiPSCs were more likely to become neuronal cell types than were the mesodermderived MEFsiPSCs. Further, mAsiPSCs were especially prone to dopaminergic neuron differentiation (see Figure 1). This is consistent with the concept that the differentiation potential for iPSCs is influenced by the epigenetic “memory” of their tissue of origin. Such information is useful when reprogramming cell types for different desired endpoints.
NIH-PA Author Manuscript
Returning a somatic cell to its pluripotent state through reprogramming and then pushing it toward a particular differentiation pathway is demanding, and may increase the risk of obtaining undesired cell types or even neoplasia formation. However, the direct conversion of somatic cells into another cell type has recently been achieved by ectopic expression of defined transcription factors (8–13). Using gene expression profiling and parental cells from E/Nestin:EGFP transgenic mouse as a monitor system, we screened nine candidate transcription factors for the ability to directly convert fibroblasts into neural progenitor cells (NPCs). We found that five of the nine transcription factors can directly convert adult dermal fibroblasts into NPC-like cells, or induced NPCs (iNPCs), and the resulting iNPCs possessed the same properties as wild-type NPCs, including proliferation, self-renewal, and differentiation (Figure 2). These findings may provide an alternative strategy to generate NPCs for cell replacement therapy of neurodegenerative diseases.
References
NIH-PA Author Manuscript
1. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007; 131:861. [PubMed: 18035408] 2. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006; 126:663. [PubMed: 16904174] 3. Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature. 2007; 448:318. [PubMed: 17554336] 4. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007; 318:1917. [PubMed: 18029452] 5. Hu Q, Friedrich AM, Johnson LV, Clegg DO. Memory in induced pluripotent stem cells: reprogrammed human retinal-pigmented epithelial cells show tendency for spontaneous redifferentiation. Stem Cells. 2010; 28:1981. [PubMed: 20882530] 6. Kim K, Doi A, Wen B, Ng K, Zhao R, et al. Epigenetic memory in induced pluripotent stem cells. Nature. 2010; 467:285. [PubMed: 20644535] 7. Polo JM, Liu S, Figueroa ME, Kulalert W, et al. Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells. Nat Biotechnol. 2010; 28:848. [PubMed: 20644536] 8. Pfisterer U, Kirkeby A, Torper O, Wood J, Nelander J, et al. Direct conversion of human fibroblasts to dopaminergic neurons. Proc Natl Acad Sci USA. 2011; 108:10343. [PubMed: 21646515] 9. Sekiya S, Suzuki A. Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors. Nature. 2011; 475:390. [PubMed: 21716291]
Science. Author manuscript; available in PMC 2014 November 03.
Tian and Zheng
Page 3
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10. Caiazzo M, Dell’Anno MT, Dvoretskova E, Lazarevic Dejan, Taverna Stefano, et al. Direct generation of functional dopaminergic neurons from mouse and human fibroblasts. Nature. 2011; 476:224. [PubMed: 21725324] 11. Ambasudhan R, Talantova M, Coleman R, Yuan X, Zhu S, et al. Direct reprogramming of adult human fibroblasts to functional neurons under defined conditions. Cell Stem Cell. 2011; 9:113. [PubMed: 21802386] 12. Kim J, Efe JA, Zhu S, Talantova M, Yuan X, et al. Direct reprogramming of mouse fibroblasts to neural progenitors. Proc Natl Acad Sci USA. 2011; 108:7838. [PubMed: 21521790] 13. Pang ZP, Yang N, Vierbuchen T, Ostermeier A, Fuentes DR, et al. Induction of human neuronal cells by defined transcription factors. Nature. 2011; 476:220. [PubMed: 21617644]
NIH-PA Author Manuscript NIH-PA Author Manuscript Science. Author manuscript; available in PMC 2014 November 03.
Tian and Zheng
Page 4
NIH-PA Author Manuscript NIH-PA Author Manuscript Fig. 1. Comparison of neuronal differentiation between MEFsiPSCs and mAsiPSCs
NIH-PA Author Manuscript
The embryonic bodies (EBs) from MEFsiPSCs and mAsiPSCs were cultured with serumfree Insulin/Transferrin/Selenium/Fibronectin (ITSFn) medium to screen nestin-positive cells. After 6–10 days in culture, the cells were plated on collagen IV-coated coverslips and incubated under neuronal differentiation conditions for 7 days, and then subjected to immunostaining with polyclonal anti-β-tubulin III antibody (Green) and nuclear staining with DAPI (Blue) (A). Cells cultured in dopaminergic (DA) neuron differentiation medium were collected at 0 day, day 7, and day 9, and then subjected to total mRNA extraction and real-time PCR analysis with mouse tyrosine hydroxylase 1-specific primers (B).
Science. Author manuscript; available in PMC 2014 November 03.
Tian and Zheng
Page 5
NIH-PA Author Manuscript NIH-PA Author Manuscript
Fig. 2. Direct conversion of adult mouse skin fibroblasts into induced neural progenitor cells (iNPCs) and their identification
NIH-PA Author Manuscript
Skin fibroblasts from adult Nestin-Enhanced Green Fluorecent Protein transgenic mice (kindly provided by Richard Miller, Northwestern University) were infected with different combinations of pMXs-based transcription factors (TF7; TF5; TF4; TF3). After 12 days in culture with NPC medium, neurospheres were observed in cultures with TF7 or TF5 infection, but not in the TF4 and TF3 cultures (A). Green fluorescent protein-positive single cells from TF7 and TF5 cultures were passaged and neurospheres were reformed (B). iNPCs were cultured on collagen IV-coated coverslips and subjected to conditions resulting in differentiation down neuron or astrocyte pathways, followed by immunostaining with anti-βtubulin III, anti-Glial fibrillary acidic protein GFAP and anti-tyrosine hydroxylase1 antibodies (C, D).
Science. Author manuscript; available in PMC 2014 November 03.
