Chemical conversion of human fibroblasts into functional Schwann cells

Abstract

Direct transdifferentiation of somatic cells is a promising approach to obtain patient-specific cells for numerous applications. However, conversion across germ-layer borders often requires ectopic gene expression with unpredictable side effects. Here, we present a gene-free approach that allows efficient conversion of human fibroblasts via a transient progenitor stage into Schwann cells, the major glial cell type of peripheral nerves. Using a multikinase inhibitor, we transdifferentiated fibroblasts into transient neural precursors that were subsequently further differentiated into Schwann cells. The resulting induced Schwann cells (iSCs) expressed numerous Schwann cell-specific proteins and displayed neurosupportive and myelination capacity in vitro. Thus, we established a strategy to obtain mature Schwann cells from human postnatal fibroblasts under chemically defined conditions without the introduction of ectopic genes.

Graphical abstract

Figure 1

Identification of a Small Molecule Enhancing Neural Stem Cell Proliferation and Enabling Conversion of Fibroblasts into Neurosphere-like Structures (A) Structure of compound B (CB). (B) CB selectively promotes proliferation of ESC-NSCs. Proliferation was analyzed by ATP assay. Fold increase to control-treated cells is shown. Left: dose-response assay with ESC-NSCs (n = 4). Right: effect of CB (0.5 μM) on proliferation of ESC-NSCs (n = 4) and ESC-MSCs (n = 2). (C) CB inhibits differentiation of ESC-NSCs in a dose-dependent manner. Left: immunostaining of TUJ1 of NSCs treated for 7 days with DMSO (control) or CB at various concentrations. Scale bar, 100 μm. Right: quantification of total length of neurite network upon CB treatment for 7 days. Values are normalized to control-treated cells (n = 3). (D) CB (2 μM)-treated fibroblasts form sphere-like structures in suspension. Scale bars, 200 μm. (E) Kinase selectivity profiling of CB. Orange bars represent kinases inhibited more than 80% at 1 μM. (F) Single or combined inhibition of CB target kinases by other compounds (targets in parentheses) has no or smaller effect on sphere formation. Graphs show proliferation rate as fold change of initial cell number (left) and mean sphere diameter (right) at day 3 of suspension culture (n = 3). ^∗p < 0.05 compared to DMSO control. +, p < 0.05 for CB compared to single inhibitors.

Figure 2

Conversion of Human Fibroblasts into a Transient Neural Precursor Stage (A) Scheme of experimental setup for conversion into induced Schwann cells. (B) Pretreatment with valproic acid (VPA) results in increased sphere size. Columns show mean sphere diameter ± SD at day 3 of suspension culture (n = 4). (C) Secondary spheres at day 11 with bipolar cells growing out of spheres. Cells express neural plate markers SOX1 and NESTIN (top left). No expression of NSC marker SOX2 and of neural crest markers SNAI1, SOX10, FOXD3, and PAX3 was detected. Scale bars, 50 μm. (D) At day 18, cells express neural crest markers SNAI1, SOX10, FOXD3, and PAX3. Scale bars, 20 μm. (E) Flow cytometry of precursors (d18) and fibroblasts (d0) revealed downregulation of fibroblast marker CD29 and upregulation of neural crest marker CD271. Panels show quantification of mean fluorescence intensity (MFI) (n = 3). (F–H) Nonneural differentiation of transient precursors. (F) Adipocyte formation analyzed by oil red O staining. (G) Formation of SMA⁺ smooth muscle cells. (H) Formation of chondrocytes analyzed by Alcian blue staining. Inset shows closeup of chondrogenic pellet. Scale bars, 50 (F), 100 (G), and 20 μm (H, inset). See also Figure S1 and S2.

Figure 3

Differentiation of Transient Precursors into Induced Schwann Cells (A–D) iSCs express Schwann cell markers. Scale bars, 50 μm. (E) Quantification of PLP-positive cells at d31. Few PLP-positive fibroblasts are due to background signal (n = 3). (F) Principal component analysis of whole-transcriptome expression profiles from fibroblasts (d0), transdifferentiated cells at d7 (early tP), d11 (early tP), d18 (late tP), d39 (iSCs), and primary Schwann cells (pSCs). Principal component 1 (x axis) accounts for 27.4% and principal component 2 (y axis) accounts for 16.5% of the variation of the data set. Each stage is represented by at least two data points derived from independent experiments. The clustered transcriptomic profiles at day 39 suggest the robustness of the protocol. (G) Enrichment map of gene sets for cellular signaling pathways (Reactome/NCI Nature PID) derived from GSEA comparing iSCs (d39) with fibroblasts (d0). Red nodes represent gene sets enriched in iSCs, whereas blue nodes represent gene sets enriched in fibroblasts. Nodes are grouped and annotated by their similarity according to related gene sets. Cluster of functionally related nodes were summarized and annotated using WordCloud (n = 3). (H) Whole patch-clamp analysis of iSCs. Voltage-dependent current obtained from a –70 to +40 mV in 10 mV increasing steps protocol from a holding potential Vh = −80 mV. Absence of early inward current confirms the deficiency of voltage-dependent Na⁺ channels, whereas the outward component is consistent with the presence of voltage-dependent K⁺ channels. (I) Maximal voltage-dependent K⁺ currents are significantly higher in iSCs than in fibroblasts. Columns show means ± SD of different cells (FBs: n = 12; iSCs: n = 7) measured in two independent experiments. See also Figure S3 and Table S2.

Figure 4

Functionality of iSCs in Coculture with Neuronal Cells (A) Culture of NSC-derived neurons alone, on cell tracker-labeled fibroblasts and iSCs, respectively. Scale bars, 100 μm. (B) NSC-neurons cultured with iSCs form a more dense and branched network analyzed by MAP2-stained area, neurite number, and total neurite length (n = 3). ^∗p < 0.05. (C) Coculture of iSCs with primary rat DRG neurons. Some iSCs form single myelinated fragments (arrowhead) detected by colocalization of MBP staining (yellow) and neurofilament (NF) staining (magenta). Scale bars, 20 μm. See also Figure S4.