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Turning Human Fibroblasts Into Neurons; Making Safer Stem Cells

Adapted from a story that originally appeared on the Alzheimer Research Forum.

5 June 2011. Scientists are making slow but steady progress in stem cell technology. This week in Cell Stem Cell, researchers from Osaka University in Japan report a microRNA method to turn adult cells into induced pluripotent stem (iPS) cells without using a virus or leaving any scar in the DNA. This technique would likely be safer for clinical use. In the May 26 Nature online, scientists at Stanford University in Palo Alto, California, describe how to go directly from human fibroblasts to neurons, skipping the iPS stage altogether. Both methodologies could help researchers model neurodegenerative diseases and develop cell-based therapies.

The Stanford team was led by senior author Marius Wernig, who previously managed to turn mouse fibroblasts into what he calls induced neuron (iN) cells (see Vierbuchen et al., 2010). In mice, the changeover required viral expression of three transcription factors: ASCL1, BRN2, and MYT1Ll. Joint first authors Zhiping Pang and Nan Yang discovered that a fourth factor, NeuroD1, is required for the process to work in human embryonic stem cells. The transition took three to four weeks with human cells, compared to seven days for mouse—no surprise, Pang said, given that humans normally take much longer to develop than rodents.

The researchers found that the same protocol worked with embryonic fibroblasts. They used fibroblasts from neonatal foreskin, and from an 11-year-old child, Pang told ARF. The efficiency rate for making iN cells was 2 to 4 percent. The iN cells expressed neuronal markers, formed synapses, and carried action potentials. However, at this point they appear to be of a generic neural type. More than half expressed markers for glutamatergic neurons, but others had expression patterns closer to inhibitory neurons or catecholaminergic neurons.

“It is not clear whether any of them are bona fide neurons of a specific kind that you would find in a developing brain,” said Arnold Kriegstein of the University of California in San Francisco, who was not involved in the study. Pang and colleagues are now addressing the question of which specific neurons—say, a motor neuron or dopaminergic neuron—they can create with their technique. However, skipping straight to iN cells may not yield cells that fully mimic natural neurodegenerative disease, wrote Kristen Brennand and Fred Gage (see comment below). “We worry that bypassing neuronal differentiation and maturation will shortcut the cellular phenotype of these neurodevelopmental disorders,” wrote the scientists, both at the Salk Institute in La Jolla, California.

Another important consideration for stem cell-based therapies is the safety of the transplants. Many techniques use viral infection to deliver the necessary genes (Oct4, Sox2, Klf4, and c-Myc) to induce pluripotency, but viruses drop their genetic cargo randomly into the genome and it may land in or near oncogenes, causing cancer. Even with virus-free techniques that excise the transgenes once their work is done (see Kaji et al., 2009 and Woltjen et al., 2009), there remains the possibility that some integrated DNA would stay put, write the Japanese authors of the Cell Stem Cell paper.

To avoid these risks, the Osaka team—led by first author Norikatsu Miyoshi and joint senior authors Hideshi Ishii and Masaki Mori—designed an iPS protocol using double-stranded microRNAs to regulate the cells' pluripotency. They analyzed microRNAs in mouse embryonic stem and iPS cells, as well as adult fat cells, and selected those with twofold higher expression in the pluripotent lines: mir-200c, mir-302 s, and mir369 s. Oct4, one of the four transcription factors that can promote pluripotency, drives mir-302 s expression. The other two microRNAs, the researchers found, are involved in repressing epithelial-specific signaling.

The researchers transfected these microRNAs into the adult fat cells from mice, and within eight days the transfected cells turned on the pluripotency-associated gene Nanog. By 15 days, one in 10,000 colonies expressed Nanog—a rather low efficiency the researchers hope to improve. They named these colonies miRNA-induced pluripotent stem cells, mi-iPSCs.

Over the ensuing weeks, the clones expressed several more pluripotency markers. In suspension culture, they differentiated into cells found in all three germ layers; in mice, they formed teratomas. The technique also worked with human fat and skin cells as starting material. The “mi-iPSCs are subject to a reduced risk of mutations and tumorogenesis relative to most other protocols because mature miRNAs function without vectors or genomic integration,” the authors write. “We hope that mi-iPSC generation will eventually prove to be of significant benefit for both biochemical research and clinical regenerative medicine.”—Amber Dance.

References:
Pang ZP, Yang N, Vierbuchen T, Ostermeier A, Fuentes DR, Yang TQ, Citri A, Sebastiano V, Marro S, Südhof TC, Wernig M. Induction of human neuronal cells by defined transcription factors. Nature. 2011 May 26. Abstract

Miyoshi N, Ishii H, Nagano H, Haraguchi N, Dewi DL, Kano Y, Nishikawa S, Tanemura M, Mimori K, Tanaka F, Saito T, Nishimura J, Takemasa I, Mizushima T, Ikeda M, Yamamoto H, Sekimoto M, Doki Y, Mori M. Reprogramming of mouse and human cells to pluripotency using mature microRNAs. Cell Stem Cell. 2011 Jun 3;8(6):633-8. Abstract

Comments on News and Primary Papers


Primary Papers: Induction of human neuronal cells by defined transcription factors.

Comment by:  Kristen BrennandFred Gage
Submitted 3 June 2011
Posted 3 June 2011

In part due to the lack of patient material for study, the molecular mechanisms responsible for many central nervous system (CNS) diseases are not well understood. Fibroblasts from patients and controls can be directly reprogrammed into human induced pluripotent stem cells (hiPSC) and subsequently differentiated into disorder-specific neurons. hiPSCs represent a new strategy for studying disease; a number of recent publications have demonstrated that CNS diseases ranging from type I spinal muscular atrophy (Ebert et al., 2009), familial dystonia (Lee et al., 2009), Parkinson’s disease (PD) (Park et al., 2008; Soldner et al., 2009), amyotrophic lateral sclerosis (ALS) (Dimos et al., 2008), Rett's syndrome (Hotta et al., 2009; Marchetto et al., 2010), and schizophrenia (Brennand et al., 2011) can be modeled using hiPSC neurons.

A new method to generate patient neurons, obviating the time-consuming step of generating and validating hiPSCs has now been developed. Last year, it was demonstrated that viral expression of just three factors—ASCL1, BRN2, and MYT1Ll—is sufficient to convert adult mouse fibroblasts into functional induced neurons (iNs) in vitro (Vierbuchen et al., 2010). With the addition of a fourth factor—NeuroD—direct reprogramming of human cells to iNs has recently been demonstrated (Pang et al., 2011). From both mouse and human cells, the conversion is incredibly rapid, generating iNs capable of producing action potentials within 14 days, and permanent, with stable neuronal fate maintained up to three weeks following the repression of viral genes. Compared to mouse iNs, however, human iNs seems relatively immature: they have slightly depolarized membrane potentials, lower amplitude synaptic responses, and spontaneous synaptic activity appears more slowly, being first detectable at five to six weeks.

The ability to generate iNs from healthy and diseased patients means that this may be a new tool with which to study neurological disorders. The rapid experimental timeframe of iN generation and the theoretical potential to reprogram to specific neuronal subtypes make this an appealing experimental strategy for in vitro models of neurological disease. Current technical limitations concern efficiency and neuronal identity. While the conversion is relatively efficient, occurring at an estimated rate of 2-4 percent, whether this is sufficient for disease modeling remains to be determined. Furthermore, the ability to generate specific neuronal subtypes remains undemonstrated; the current method generates a heterogeneous mix of neurons, predominantly glutamatergic and dopaminergic, expressing markers of either forebrain or peripheral neuron patterning.

Two important issues must be considered as one contemplates modeling psychiatric disorders using iNs. We worry that bypassing neuronal differentiation and maturation will shortcut the cellular phenotype of these neurodevelopmental disorders. For example, if psychiatric disease results from abnormal synaptic maturation, iN generation may bypass the developmental window in which the disease phenotype can be observed in vitro. Second, if ASCL1, BRN2, or MYT1L contribute to the disease state, persistent overexpression might be sufficient to mask or rescue cellular phenotypes in vitro. It is not unreasonable to predict that overexpression of one or more of these key neuronal genes might affect disease initiation or progression: mutations disrupting MYT1L expression and binding have been linked to schizophrenia (Vrijenhoek et al., 2008; Riley et al., 2010), BRN2 regulates expression of a conductance calcium-activated potassium channel (KCNN3) implicated in schizophrenia (Sun et al., 2001), and ASCL1 has been linked to Parkinson’s disease (Ide et al., 2005).

The most prudent course of action would be to recapitulate the cellular and molecular phenotypes observed in patient hiPSC neurons with patient iNs. With this validation complete, we predict that many studies of psychiatric disorders using iNs will begin.

References:

Brennand, K. J., Simone, A., Jou, J., Gelboin-Burkhart, C., Tran, N., Sangar, S., Li, Y., Mu, Y., Chen, G., Yu, D. et al. (2011). Modelling schizophrenia using human induced pluripotent stem cells. Nature. Abstract

Dimos, J. T., Rodolfa, K. T., Niakan, K. K., Weisenthal, L. M., Mitsumoto, H., Chung, W., Croft, G. F., Saphier, G., Leibel, R., Goland, R. et al. (2008). Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science (New York, N.Y 321, 1218-1221. Abstract

Ebert, A. D., Yu, J., Rose, F. F., Jr., Mattis, V. B., Lorson, C. L., Thomson, J. A. and Svendsen, C. N. (2009). Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature 457, 277-280. Abstract

Hotta, A., Cheung, A. Y., Farra, N., Garcha, K., Chang, W. Y., Pasceri, P., Stanford, W. L. and Ellis, J. (2009). EOS lentiviral vector selection system for human induced pluripotent stem cells. Nature protocols 4, 1828-1844. Abstract

Ide, M., Yamada, K., Toyota, T., Iwayama, Y., Ishitsuka, Y., Minabe, Y., Nakamura, K., Hattori, N., Asada, T., Mizuno, Y. et al. (2005). Genetic association analyses of PHOX2B and ASCL1 in neuropsychiatric disorders: evidence for association of ASCL1 with Parkinson's disease. Hum Genet 117, 520-527. Abstract

Lee, G., Papapetrou, E. P., Kim, H., Chambers, S. M., Tomishima, M. J., Fasano, C. A., Ganat, Y. M., Menon, J., Shimizu, F., Viale, A. et al. (2009). Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs. Nature 461, 402-406. Abstract

Marchetto, M. C., Carromeu, C., Acab, A., Yu, D., Yeo, G. W., Mu, Y., Chen, G., Gage, F. H. and Muotri, A. R. (2010). A model for neural development and treatment of rett syndrome using human induced pluripotent stem cells. Cell 143, 527-539. Abstract

Pang, Z., Yang, N., Vierbuchen, T., Ostermeier, A., Fuentes, D., Yang, T., Citri, A., Sebastiano, V., Marro, S., Südhof, T. et al. (2011). Induction of human neuronal cells by defined transcription factors. Nature. 2011 May 26. Abstract

Park, I. H., Arora, N., Huo, H., Maherali, N., Ahfeldt, T., Shimamura, A., Lensch, M. W., Cowan, C., Hochedlinger, K. and Daley, G. Q. (2008). Disease-specific induced pluripotent stem cells. Cell 134, 877-886. Abstract

Riley, B., Thiselton, D., Maher, B. S., Bigdeli, T., Wormley, B., McMichael, G. O., Fanous, A. H., Vladimirov, V., O'Neill, F. A., Walsh, D. et al. (2010). Replication of association between schizophrenia and ZNF804A in the Irish Case-Control Study of Schizophrenia sample. Molecular psychiatry 15, 29-37. Abstract

Soldner, F., Hockemeyer, D., Beard, C., Gao, Q., Bell, G. W., Cook, E. G., Hargus, G., Blak, A., Cooper, O., Mitalipova, M. et al. (2009). Parkinson's disease patient-derived induced pluripotent stem cells free of viral reprogramming factors. Cell 136, 964-977. Abstract

Sun, G., Tomita, H., Shakkottai, V. G. and Gargus, J. J. (2001). Genomic organization and promoter analysis of human KCNN3 gene. J Hum Genet 46, 463-470. Abstract

Vierbuchen, T., Ostermeier, A., Pang, Z. P., Kokubu, Y., Sudhof, T. C. and Wernig, M. (2010). Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463, 1035-1041. Abstract

Vrijenhoek, T., Buizer-Voskamp, J. E., van der Stelt, I., Strengman, E., Sabatti, C., Geurts van Kessel, A., Brunner, H. G., Ophoff, R. A. and Veltman, J. A. (2008). Recurrent CNVs disrupt three candidate genes in schizophrenia patients. American journal of human genetics 83, 504-510. Abstract

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