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Jan 31, 2012

Tripotent Differentiating Neural Precursors Generated Directly from Fibroblasts

  • Scientists report on a method that allows the direct conversion of mouse embryonic fibroblasts into neural precursor cells (NPCs) that are capable of differentiating into functional neurons, astrocytes, and oligodendrocytes. The Stanford University School of Medicine researchers built on previous work in which they used a defined set of transcription factors to convert mouse and human fibroblasts directly into cells that appeared to act as functional neurons. They claim their latest research using NPC-expressed transcription factors to trigger fibroblast conversion into NPCs could pave the way to the development of autologous cell transplantation-based therapies in the brain or spinal cord. Marius Wernig, Ph.D., and colleagues report their achievement in PNAS, in a paper titled “Direct conversion of mouse fibroblasts to self-renewing, tripotent neural precursor cells.”

    The ability to generate cardiomyocytes and NPC populations from fibroblasts by the administration of four iPS cell-reprogramming factors has already been demonstrated. However, NPC production was inefficient, and the cells couldn’t self-renew, and apparently lacked the ability to differentiate into oligodendrocytes, Dr. Wernig and colleagues report. The Stanford team thus aimed to see whether NPCs could be induced directly from fibroblasts using neural progenitor-specific transcription factors, and bypass the pluripotent state. Based on their previous work demonstrating that three transcription factors could directly convert mouse fibroblasts into functional induced neuronal (iN) cells, they hypothesized that it might be possible to trigger mouse embryonic fibroblast (MEF) conversion into an intermediate NPC population under appropriate conditions.

    The experiments used MEFs derived from Sox2-internal ribosome entry site (IRES)-EGFP knock-in mice expressing the reverse tetracycline transactivator (rtTA) under control of the Rosa26 locus. These MEFs were infected with a pool of 11 lineage-specific transcription factors (11F) under a tetO promoter. After infection, cells were grown in EGF- and FGF2-containing media in the presence of doxycycline. The 11 transcription factors were chosen because they had previously demonstrated functions in neural development and were expressed at high levels in NPCs.

    Twenty-four days after transgene induction, Sox2-EGFP+ cells were observed when MEFs were infected with the 11F pool, and some of these formed colonies, the researchers report. Transcription profiling of the Sox2-EGFP+ cells confirmed they expressed NPC-associated genes at similar levels to those expressed by embryonic stem cell-derived NPCs. On the subsequent withdrawal of the growth factors and doxycycline from cultures of these induced NPCs (iNPCs), the researchers were able to isolate NPCs that displayed functional properties of neural precursors, such as the capacity to differentiate into neurons, glia, and astrocytes under defined conditions.

    The next stage was to identify which of the 11 factors were critical to MEF reprogramming, by allowing the expression of only subsets of the transcription factors in transfected MEFs. They first manage to hone 11 factors down to five—Rfx4, ID4, FoxG1, Lhx2, and Sox2—that were sufficient for inducing Sox2-EGFP+ cells. By systematically omitting one factor at a time, the team found that FoxG1 and Sox2 were absolutely necessary for Sox2-EGFP+ colony formation and NPC-specific gene induction, whichever of the other factors were also included. In fact, they found that these two factors were sufficient to induce MEFs to generate colonies of Tuj1+ MAP2+ neurons that demonstrated electrophysiological characteristics of neurons, inclduing the capacity to generate action potentials.

    Importantly, Sox2-EGFP+ cells could be expanded in adherent conditions for at least 12 passages, and retained the ability to differentiate into both neuronal and astroglial cells. However, no oligodendrocytes were observed. The FoxG1/Sox2 iNPCs also demonstrated self-renewal potential and consisted mostly of neuron-restricted progenitor cells and some neuron/astroglial-restricted bipotent precursor cells, but again, no astroglial-restricted progenitors and no cells with oligodendroglial differentiation potential were observed.

    This apparent inability of the FoxG1/Sox2 mix to generate cells that could differentiate into oligodendrocytes led the team to investigate whether an additional factor might be able to induce a tripotent NPC population that could differentiate into neurons, glial cells, and oligodendrocytes. They thus screened the other nine of the transcription factor candidates in combination with FoxG1 and Sox2. “Strikingly, O4+ cells with a typical oligodendrocytic morphology could be produced only from the EGFP+ population generated with the addition of Brn2,” they write. And encouragingly, the resulting population of FoxG1/Sox2/Bm2 Sox2-EGFP+-sorted cells also had the potential to differentiate into GFP+ astrocytes and Tuji1+/MAP2+ neurons, “suggesting that all three cell types of the central nervous system could be derived from this three-factor population.”

    Interestingly, further experimentation showed that cells infected with FoxG1 and Brn2 alone could generate a Sox2-EGFP+ population that was capable of differentiating into oligodendrocyte-like cells in vitro and also in vivo in experimental Shiverer mice. In these animals, the iNPC-derived oligodendrocytes behaved similarly to oligodendrocytes derived from endogenous neural progenitor cells, and appropriately myelinating axons of the developing brain. Transduction of FoxG1 and Bm2 alone was also enough to produce iNPCs capable of differentiating into astrocytes. However, further in vitro studies and electrophysiological tests demonstrated that Sox2 was an absolute requirement for the differentiation of iNPCs into mature neurons under standard conditions.

    The ability to trigger fibroblasts to generate iNPCs that subsequently differentiate into neurons could have advantages over the direct conversion of fibroblasts into post-mitotic neuronal or glial cells, the Stanford team states. The ability of iNPCs to self-renew and expand over many passages will facilitate applications requiring large numbers of cells, such as drug screening or cell-transplantation therapies. This route also allows the generation of clonal populations that should lead to homogeneous populations of cells that can be more easily characterized than those from conversion of mixed fibroblasts. And, they remark, “unlike iPS cell or transient dedifferentiation, no overt oncogenes, such as c-Myc, or any other specific pluripotency factors are required to produce iNPCs.”

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