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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.

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...  Read more

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