Direct Conversion of Skin Cells into Neural Precursor Cells

Cell reprogramming involves the use of genetic engineering techniques to push cells into a new cell type WITHOUT passing those cells through the embryonic stage. Several different studies have shown that transferring particular genes into specific cell types or removing distinct genes from them can drive them to become other cell types. There are several published examples of transdifferentiation:
1) In 1989, Weintraub and colleagues overexpressed a gene called MyoD in cultured fibroblasts to convert them into muscle cells. Unfortunately, this conversion was incomplete and required continuous expression of MyoD (Weintraub H et al., Proc. Natl. Acad. Sci. USA 1989;86:5434-8).
2) Tachibana and colleagues overexpressed a gene called MITF to transdifferentiate fibroblasts into pigment-synthesizing melanocytes (Tachibana et al., Nature Genetics 1996;14:50-4).
3) Xie and others overexpressed genes that encode two transcription factors (C/EBP and PU.1) in B cells, T cells, and fibroblasts into transdifferentiated them into cells that looked like macrophages (Xie et al., Cell 2004;117:663-76).
4) Deletion of a gene called Pax5 can transdifferentiate antibody-secreting B lymphocytes into common lymphoid progenitors, macrophages and antigen-presenting T cells (Cobaleda C, Jochum W, and Busslinger M. Nature 2007;449:473-7).
5) Doug Melton’s laboratory at Harvard University transferred a specific combination of three transcription factor genes (Ngn3, which is also known as Neurog3, Pdx1 and Mafa), into pancreatic exocrine cells (those cells that produce and secrete digestive enzymes).  This reprogrammed the cells into insulin-secreting beta cells (Qiao Zhou et a., In vivo reprogramming of adult pancreatic exocrine cells to β-cells. Nature 2008;455, 627-632).
6) Deletion of a gene that encodes a transcription factor called Foxl2 converts granulosa and thecal cells (found in the ovary) into Sertoli and Leydig cells, which are found in the testes (Uhlenhaut et al., Cell 2009;139:1130-42).
7) Thomas Vierbuchen and colleagues in the laboratory of Marius Wernig at Sanford University School of Medicine used a combination of three genes (Asc1, Brn2 and Myt1l) to convert fibroblasts into functional neurons (Vierbuchen et al., Nature 2010;463:1035-42).
8) In 2010, Ieda and co-workers in the laboratory of Deepak Srivastava have used ectopic expression of three genes (GATA4, MEF2C, and TBX5) to directly convert heart-based fibroblasts into heart muscle cells. These reprogrammed cells did not require expression of the introduced transgenes (Ieda et al., Cell 2010;142:375-86).

A recent study has extended these results even further. In an earlier study, Marius Wernig’s lab at Stanford University School of Medicine showed that skin fibroblasts can be transdifferentiated into functional neurons. Wernig’s lab has followed up in a paper that was published online on Jan. 30, 2012 in the Proceedings of the National Academy of Sciences.

In this study, Wernig’s lab used mouse skin cells and directly transdifferentiated them into the three main parts of the nervous system. These transdifferentiation experiments show that pluripotency (a term that describes the ability of stem cells to become nearly any cell in the body) is NOT necessary for a cell to transform from one cell type to another. Together, these results raise the possibility that embryonic stem cell research and induced pluripotency could be superseded by a more direct way of generating specific types of cells for therapy or research.

In the new study, Wernig and his colleagues converted fibroblasts in to neural precursor cells (NPCs). NPCs have the capacity to differentiate into neurons, but they can also become the two other main cell types in the nervous system: astrocytes and oligodendrocytes. In addition to their greater versatility, newly derived NPCs offer another advantage over neurons because they can be cultivated to large numbers in the laboratory — a feature critical for their long-term usefulness in transplantation or drug screening..

The switch from skin cells to NPCs occurred with high efficiency and only took about three weeks after the addition of just three transcription factors. Wernig’s research group used a different combination of three transcription factors than those used to generate mature neurons (Brn2, Sox2 and FoxG1) than was used to generate mature neurons. This combination of transcription factors drove the fibroblasts to transdifferentiate into “tripotent” NPCs that have the ability to form neurons and astrocytes but also into oligodendrocyte. The finding implies that it may one day be possible to generate a variety of neural-system cells for transplantation that would perfectly match a human patient.

The lab’s previous success with transdifferentiation experiments led Wernig to wonder if his lab could convert skin-based fibroblasts into the more-versatile NPCs. To do so, Wernig’s research group infected embryonic mouse skin cells — a commonly used laboratory cell line — with a virus that encoded 11 transcription factors known to be expressed at high levels in NPCs. Just over three weeks later, about 10 percent of the cells began to look and act like NPCs.

They then winnowed down the original panel of 11 transcription factors to just three that still converted fibroblasts to NPCs. Three of these genes (Brn2, Sox2 and FoxG1; in contrast, the conversion of skin cells directly to functional neurons requires the transcription factors Brn2, Ascl1 and Myt1l.) drove fibroblasts to differentiate into NPCs that were “tripotential” – that is, the NPCs could differentiate into not just neurons and astrocytes, but also oligodendrocytes, which make myelin that insulates nerve fibers and allows them to effectively transmit nerve impulses. Wernig’s lab workers dubbed the newly converted population “induced neural precursor cells,” or iNPCs.

In vitro experiments showed that the astrocytes, neurons and oligodendrocytes made from iNPCs expressed the same genes and morphologically resembled that they resembled astrocytes, neurons and oligodendrocytes found in living organisms. However, Wernig’s lab wanted to know how iNPCs would react when transplanted into an animal. Therefore, they injected them into the brains of newborn laboratory mice that were bred to lack the ability to myelinate neurons. After 10 weeks, they found that the injected cells had differentiated into oligodendroytes and had begun to coat the animals’ neurons with myelin.

Marius Wernig, MD, assistant professor of pathology and a member of Stanford’s Institute for Stem Cell Biology and Regenerative Medicine, said: “We are thrilled about the prospects for potential medical use of these cells. We’ve shown the cells can integrate into a mouse brain and produce a missing protein important for the conduction of electrical signal by the neurons. This is important because the mouse model we used mimics that of a human genetic brain disease. However, more work needs to be done to generate similar cells from human skin cells and assess their safety and efficacy.”

Pediatric cardiologist Deepak Srivastava, MD, who was not involved in these studies noted, “Dr. Wernig’s demonstration that fibroblasts can be converted into functional nerve cells opens the door to consider new ways to regenerate damaged neurons using cells surrounding the area of injury. It also suggests that we may be able to transdifferentiate cells into other cell types.” Srivastava is the director of cardiovascular research at the Gladstone Institutes at the University of California-San Francisco. In 2010, Srivastava’s lab transdifferentiated mouse heart fibroblasts into beating heart muscle cells.

The first author of this article, Ernesto Lujan, added: “Direct conversion has a number of advantages. It occurs with relatively high efficiency and it generates a fairly homogenous population of cells. In contrast, cells derived from iPS cells must be carefully screened to eliminate any remaining pluripotent cells or cells that can differentiate into different lineages.” Pluripotent cells can cause cancers when transplanted into animals or humans.

“Not only do these cells appear functional in the laboratory, they also seem to be able to integrate appropriately in an in vivo animal model,” said Lujan.

Wernig’s group is now working to replicate the work with skin-based fibroblasts from adult mice and humans, but Lujan emphasized that more research is needed before any human transplantation experiments could be conducted. Until that time, the ability to quickly and efficiently generate NPCs that can be grown in the laboratory to mass quantities and maintained over time will be valuable in disease and drug-targeting studies.

“In addition to direct therapeutic application, these cells may be very useful to study human diseases in a laboratory dish or even following transplantation into a developing rodent brain,” said Wernig.