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

Summary

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.

Abstract

Generation of 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.

Introduction

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 rep....

Protocol

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 Würzburg, Germany (ethical report no: 96/11 dated 10.06.2011).

1. Punch Biopsy

1. 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 sterile 3mm biopsy punch, rinse biopsy with DPBS + 1 µl/1ml Gentamicin and remove fat. Rinse biopsy twice with DPBS + Gentamicin, aspirate DPBS complet....

Results

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 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 viruses²³

Discussion

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 experiments^33,34. For biomedi.......

Materials

List of materials used in this article

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