We would like to thank all members of the Stem Cell and Regenerative Medicine Group of the University of W&#252;rzburg for helpful suggestions and Martina Gebhardt as well as Heike Arthen for excellent technical support. This work was supported by grants from the Deutsche Forschungsgemeinschaft DFG (ED79/1-2), the German Ministry of Education and Research BMBF (01 GN 0813), the Bavarian Research Network Induced Pluripotent Stem Cells &#8220;forIPS&#8221; and the &#8220;Stiftung Sibylle Assmus&#8221;. Figure 1 was produced using Servier Medical Art, available from www.servier.com.

The human fibroblasts used in this study were obtained from a skin punch biopsy after getting informed consent and ethical clearance by the ethics committee of the University of W&#252;rzburg, Germany (ethical report no: 96/11 dated 10.06.2011).
1. Punch Biopsy
<ol>
	<li>Disinfect skin of patient. Anesthetize skin part of which biopsy will be taken from (preferentially a less sun-exposed area) using 0.5 to 1 ml mepivacaine hydrochloride intracutaneously and prepare skin biopsy using a steril...

The authors declare that they have no competing financial interests.

Here we show the isolation and direct conversion of human fibroblasts into expandable transgene-free neural progenitor cells and their differentiated progeny as a putative basis for cell-replacement therapy or application in drug screening analyses. Direct lineage conversion of somatic cells into NPCs has been achieved by forced expression of lineage-specific transcription factors 26,33,34. Nevertheless, in many cases fetal fibroblasts were used for transdifferentiation experiments33,34. For biomedi...

In 2006 Yamanaka and colleagues could show for the first time the possibility of reprogramming of somatic cells into a pluripotent state1. This dedifferentiation was achieved by overexpression of four transcription factors Oct4, Sox2, Klf4, and c-Myc in murine fibroblasts. The generated so-called induced pluripotent stem cells (iPSCs) show functional equivalence to embryonic stem cells (ESCs) and can thus be differentiated into all cell types of the adult organism. One year later reprogramming to iPSCs could also be achieved for human fibroblasts2. Experiments in animal models demonstrate that iPSC-derived cells can generally be used for cell replacement therapy, e.g., in Parkinson&#8217;s Disease (PD) 3-5. However, several limitations associated with the use of iPSCs represent roadblocks for the full realization of their therapeutic potential. First of all, reprogramming of cells into a pluripotent state and subsequent quality control is generally a time-consuming and inefficient process yielding in extensive and thus costly cell culture procedures. Second, iPSCs need to be re-differentiated into the desired cell type of interest before biomedical application and the probability of residual pluripotent cells in the differentiated population harbors a significant tumorigenic potential and thus displays a high risk after cell transplantation6. Third, the reprogramming process is usually achieved by inducing the reprogramming factors by lenti- or retroviral infection. The integration of these viruses into the host genome might lead to insertional mutagenesis and/or uncontrolled reactivation of the transgenes7,8. Non-integrative systems have been developed to deliver the reprogramming factors to target cells, which minimize the risk of insertional mutagenesis and transgene reactivation. Examples for these transgene-free approaches are the reprogramming of cells using non-integrating Adeno or Sendai virus9,10, DNA-based vectors11 or the application of DNA-free methods, like transfection of synthetic mRNA12 or transduction of recombinant proteins13,14. Another promising method for the derivation of transgene-free iPSC is the use of loxP-modified lentiviral reprogramming constructs and subsequent deletion of transgenes using the Cre-loxP DNA recombination system15,16.
A more straightforward approach to generate neural cells for cell replacement therapy represents direct conversion of fibroblasts into post-mitotic neurons17-20. Vierbuchen et al. reported that the overexpression of transcription factors Ascl1, Brn2 and Myt1l results in the generation of 20% neurons from murine fibroblasts17. In 2011 it was shown, that the same three transcription factors in combination with overexpression of NeuroD1 enable transdifferentiation of human fibroblasts into neurons19. Human induced neurons could also be generated by overexpression of Ascl1 and Ngn2 under dual SMAD- and GSK3&#946;- inhibition20. Notably, direct conversion of fibroblasts into neurons generates a non-proliferative, post-mitotic cell population that does not allow further expansion and biobanking.
Recently, the direct conversion of fibroblasts into a proliferating neural stem/progenitor cell population was reported21-26. For sake of clarity, all these cell types will be named as induced neural progenitor cells (iNPCs) in this report. Han et al. overexpressed Brn4, Sox2, c-Myc and Klf4 to generate iNPCs. Like their neural stem cell counterparts derived from either primary tissue or pluripotent cells these iNPCs are tripotential and could be differentiated into neurons, astrocytes and oligodendrocytes21. Our group reported a slightly different conversion protocol involving overexpression of Sox2, Klf4, and c-Myc and induced Oct4-expression for 5 days only. With this approach we could generate stably proliferating iNPCs from murine embryonic and adult fibroblasts that exhibit complete silencing of the reprogramming factors22. In contrast to iPSCs, iNPCs do not exhibit a tumorigenic potential after transplantation27. We used converted cells successfully in an animal model of demyelination, myelin deficient rats, demonstrating that iNPCs are clinically useful22. Until then, NPCs could be only generated from pluripotent stem cells or primary neural tissue28-32. iNPCs are stably expandable cells that can be cryopreserved and are able to differentiate into neurons, astrocytes and oligodendrocytes. Much effort has been made to adapt the direct conversion protocol from mouse to human cells23,26,33,34. In 2012 it was published that overexpression of the single factor Sox2 in fibroblasts is sufficient to generate murine and human iNPCs33. The authors reported generation of human iNPCs from fetal foreskin fibroblasts and characterized them by staining against Sox2 and Nestin. However, the target cells used for reprogramming represent a very particular cell type that won&#8217;t be available in clinical practice and there was no functional characterization of converted cells by transplantation in an animal model performed. A more recent publication describes the generation of neuronal restricted progenitors from human fetal fibroblasts by overexpression of Sox2, c-Myc and either Brn2 or Brn434. The generated cell lines showed self-renewal capacity and could be differentiated into various types of terminal neurons. However, the usage of fetal fibroblasts is unfavorable, as these cells are of heterogeneous origin and one cannot exclude the presence of residual e.g., neural crest stem cells in the preparations. In 2014, Zhu et al. reported the direct conversion of human adult and neonatal fibroblasts into tripotential neural progenitor cells by overexpression of Sox2 together with Oct4 or Oct4 alone and addition of small molecules to the cell culture media. Notably, based on their studies Sox2 alone was insufficient to induce direct conversion26. More recently, Lu et al. reported that the overexpression of the Yamanaka factors Oct4-, Sox2-, Klf4-, c-Myc by Sendai virus for 24 hr and subsequent inactivation of the virus by increased temperature results in the generation of expandable tripotential neural precursor cells23. In conclusion, although the conversion protocols published for human cells thus far have in common the overexpression of at least one or more of the Yamanaka factors, often in a timely restricted manner, there is no clear indication of the minimal molecular factors needed to drive direct conversion into iNPCs. The timely restricted overexpression of Oct4 by either genetic means, transfection with synthetic mRNA, or cell-permeant protein together with constitutive expression of Sox2, Klf-4, and c-Myc did not result in stable human iNPC lines yet. Thus, the application of Sendai virus to overexpress all Yamanaka factors and timely restrict their activity by heat inactivation of the virus23 together with optimized neural media induction conditions22,31 represents the preferred strategy thus far.
Several studies demonstrate the cellular functionality of NSCs or their differentiated counterparts in different animal disease models. Neural progenitors from human pluripotent stem cells have been transplanted in mouse models of the neuroinflammatory disorder multiple sclerosis35,36. The applicability of hESC-derived neural progenitor cells in EAE (experimental autoimmune encephalomyelitis)-mice was first shown in 2008 35. Multipotent neural precursor cells were injected into the ventricles of mouse brain, and the transplantation resulted in the reduction of clinical signs of EAE. Kim et al. generated oligodendroglial precursors from hESCs and transplanted those intracerebroventricularly into EAE-mice. Although transplants did not survive for more than 10 days, mice showed significant improvement of neurological function and reduction of proinflammatory immune cells in the white matter36. Stem cell therapy has also been applied for preclinically targeting of Parkinson&#8217;s Disease as NPCs could be successfully employed in the respective animal models. For this, progenitor cells were either derived from fetal brain tissue37 differentiated from pluripotent stem cells38-40 or mesenchymal stem cells41 were used. In 2012 we showed that mouse iNPCs are able to produce proteolipid protein, the major myelin protein component, after transplantation into the brains of myelin-deficient (md) rats22. By that proof-of-principle experiment the therapeutic applicability of iNPCs was firstly proven and soon confirmed by another study32. However, the full potential of therapeutic use of human iNPCs remains to be explored.
Here we show an robust and integrated process of (i) derivation of human primary cells from adult patients via skin biopsy, (ii) direct conversion of human fibroblasts into a neural progenitor state and (iii) the ability of iNPCs to be differentiated into neuronal and glial lineages. Usage of this protocol will help to speed up generation of autologous human cells for therapeutic applications.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Astrocyte media</td>
			<td>ScienCell</td>
			<td align="right">1801</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>LifeTechnologies</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>Peprotech</td>
			<td>450-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Punch</td>
			<td>pfm medical</td>
			<td align="right">48301</td>
			<td></td>
		</tr>
		<tr>
			<td>cAMP</td>
			<td>Sigma</td>
			<td>A6885</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Axon medchem</td>
			<td align="right">1386</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type2</td>
			<td>Worthington Biochemical</td>
			<td>LS004177</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>PAN&#160;</td>
			<td>P10-032100</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>LifeTechnologies</td>
			<td>41966-029</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM F12</td>
			<td>LifeTechnologies</td>
			<td>11320-033</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Roth</td>
			<td align="right">4720</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>LifeTechnologies</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Life Technologies</td>
			<td>PHG0313</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Biochrom AG</td>
			<td align="right">50115</td>
			<td></td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>Peprotech</td>
			<td>450-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Sigma</td>
			<td>G1397</td>
			<td></td>
		</tr>
		<tr>
			<td>GFAP-antibody</td>
			<td>Dako</td>
			<td>Z0334</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>LifeTechnologies</td>
			<td>35050-038</td>
			<td></td>
		</tr>
		<tr>
			<td>hLIF</td>
			<td>Peprotech</td>
			<td>300-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Seralab</td>
			<td>GEM-700-112-P</td>
			<td></td>
		</tr>
		<tr>
			<td>Ki67-antibody</td>
			<td>NeoMarkers</td>
			<td>RM-9106-S</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>LifeTechnologies</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr>
			<td>N2</td>
			<td>LifeTechnologies</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Nestin-antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB1259</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>LifeTechnologies</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acids</td>
			<td>LifeTechnologies</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>NSC freezing media</td>
			<td>Sigma</td>
			<td>C6295</td>
			<td></td>
		</tr>
		<tr>
			<td>Oct4-antibody</td>
			<td>Santa Cruz</td>
			<td>sc9081</td>
			<td></td>
		</tr>
		<tr>
			<td>Pax6-antibody</td>
			<td>Covance</td>
			<td>PRB-278P</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Invivogen</td>
			<td>inh-sb43</td>
			<td></td>
		</tr>
		<tr>
			<td>Sendai virus: CytoTune Sendai Reprogramming Kit</td>
			<td>&#160;LifeTechnologies</td>
			<td>A1378001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodiumpyruvate</td>
			<td>Life Technologies</td>
			<td>11360-039</td>
			<td></td>
		</tr>
		<tr>
			<td>Sox1-antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF3369</td>
			<td></td>
		</tr>
		<tr>
			<td>Sox2-antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB2018</td>
			<td></td>
		</tr>
		<tr>
			<td>T3</td>
			<td>Santa Cruz</td>
			<td>sc-205725</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Life Technologies</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr>
			<td>TUJ1-antibody</td>
			<td>Covance</td>
			<td>MMS-435P-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitamin C</td>
			<td>Sigma</td>
			<td>A4544</td>
			<td></td>
		</tr>
		<tr>
			<td>Y27632</td>
			<td>Calbiochem</td>
			<td>CAS 146986-50-7</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>LifeTechnologies</td>
			<td align="right">21985023</td>
			<td></td>
		</tr>
	</tbody>
</table>

derivation of adult human fibroblasts and their direct conversion into expandable neural progenitor cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Here we present description of an integrated process that allows generation of induced neural progenitor cells (iNPCs) from human fibroblasts that have been obtained by a punch biopsy from skin within less than 8&#160;weeks (Figure 1). Patient-specifc iNPCs can be further differentiated into neuronal and glial lineages and harbor huge potential for cell replacement therapy and disease modeling.
Infection of the fibroblasts with Oct4-, Klf4-, Sox2- and c-myc- Sendai viruses<sup...

Watch this Scientific Journal Video about Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells at JoVE.com

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

derivation-adult-human-fibroblasts-their-direct-conversion-into

Research

JoVE Journal

Neuroscience

15.3K visualizações.  University of Würzburg.  Geração de células estaminais pluripotentes induzidas proporciona perspectivas fascinantes para a derivação de transplantes autólogos. No entanto, a progressão através de um estado pluripotente e trabalhoso re-diferenciação ainda dificulta a tradução clínica. Aqui nós descrevemos a derivação de fibroblastos humanos adultos ea sua conversão direta em células progenitoras neurais induzidos ea subsequente diferenciação em linhagens neuronais. 