The Differentiation Stage of Transplanted Stem Cells Modulates Nerve Regeneration

Abstract

In regenerative medicine applications, the differentiation stage of implanted stem cells must be optimized to control cell fate and enhance therapeutic efficacy. We investigated the therapeutic potential of human induced pluripotent stem cell (iPSC)-derived cells at two differentiation stages on peripheral nerve regeneration. Neural crest stem cells (NCSCs) and Schwann cells (NCSC-SCs) derived from iPSCs were used to construct a tissue-engineered nerve conduit that was applied to bridge injured nerves in a rat sciatic nerve transection model. Upon nerve conduit implantation, the NCSC group showed significantly higher electrophysiological recovery at 1 month as well as better gastrocnemius muscle recovery at 5 months than the acellular group, but the NCSC-SC group didn't. Both transplanted NCSCs and NCSC-SCs interacted with newly-growing host axons, while NCSCs showed better survival rate and distribution. The transplanted NCSCs mainly differentiated into Schwann cells with no teratoma formation, and they secreted higher concentrations of brain-derived neurotrophic factor and nerve growth factor than NCSC-SCs. In conclusion, transplantation of iPSC-NCSCs accelerated functional nerve recovery with the involvement of stem cell differentiation and paracrine signaling. This study unravels the in vivo performance of stem cells during tissue regeneration, and provides a rationale of using appropriate stem cells for regenerative medicine.

Figure 1

Establishment and characterization of human integration-free iPS cell lines and iPSC-derived NCSCs and Schwann cells. (A) Human dermal fibroblasts were reprogrammed with episomal vectors containing Oct4, Sox2, Klf4, and c-Myc genes by using electroporation method. Pluripotent stem cell-like colonies were picked up and expanded to obtain stable iPSC lines. (B) To establish NCSC lines, iPSCs were detached and formed embryoid bodies (EBs) in suspension cultures. EBs were then plated on Matrigel-coating culture plates for up to 2 weeks. Subsequently, cells were dissociated into single cells and cultured as monolayer. To obtain homogeneous NCSC populations, magnetic-activated cell sorting (MACS) were used to select p75 positive cells. Expended p75+ NCSCs were further purified by fluorescence-activated cell sorting (FACS) for HNK-1 positive and SSEA4 negative cells to obtain more homogeneous and stable NCSC line. (C) Established iPSC lines showed typical pluripotent stem cell morphology, positive AP staining, and iPSC markers OCT-4, SSEA4, and TRA-1-60. (D) The iPSC-derived NCSC lines showed positive NCSC markers SOX10, HNK-1, and AP2 and negative iPSC marker SSEA4. (E) In vitro differentiation of iPSC-derived NCSCs into peripheral neural lineages (peripheral neurons, TUJ1; Schwann cells, S100β) and mesenchymal lineages (osteoblasts, Alizarin red; adipocytes, Oil red). Nuclei were stained by Hoechst 33342. Scale bar: 50 μm.

Figure 2

Marker expression of 10-day and 21-day differentiated iPSC-NCSCs in Schwann cell differentiation medium.

Figure 3

Transplantation of NCSCs and NCSC-SCs for peripheral nerve regeneration and in vivo evaluation of functional recovery. (A) Schematic outline of tissue engineering approach by combining NCSCs/NCSC-SCs, collagen/HA hydrogel, and a PLCL nerve conduit. The NCSCs/NCSC-SCs were mixed with collagen-hyaluronic acid (Col-HA) hydrogel, injected in the nerve conduits, and incubate overnight in vitro. The nerve conduits were then used to connect the cut sciatic nerves in nude rats. (B) Compound muscle action potential (CMAP) was measured in vivo at 1-month after surgery. Representative CMAP curves of the acellular, NCSC, and NCSC-SC groups are shown. Recovery rate is the ratio of injured hindlimb’s CMAP to contralateral normal hindlimb’s CMAP of a rat. Bars represent mean ± standard error of mean. ** indicates significant difference (p < 0.01; n = 6) (C) Five months after surgery, gastrocnemius muscle wet weight was measured and compared between injured hindlimb and contralateral normal hindlimb of a rat. Representative images of gastrocnemius muscle are shown for the acellular group, the NCSC group, and the NCSC-SC group. Bars represent mean ± standard error of mean. * indicates significant difference (p < 0.05; n = 5 for acellular group and NCSC group, and n = 6 for NCSC-SC group).

Figure 4

Distribution and behavior of transplanted NCSCs/NCSC-SCs in nerve conduit and NCSC differentiation in vivo. (A) Immunofluorescence staining of axon marker NF-M and human nuclei antigen NuMA was performed in longitudinally cryosectioned nerve conduits of the NCSC group and the NCSC-SC group at 2-week after surgery. Images were taken at 2–3 mm (left images), 5–6 mm (middle images), and 8–9 mm (right images) of conduit from the proximal end, respectiely. (B) Immunofluorescence staining of Schwann cell marker S100β (Green), fibroblast marker FSP1 (Green), NCSC marker HNK-1 (Green), and endothelial marker CD31 (Red) was performed in a longitudinal section of the NCSC group with double staining of human nuclei antigen (NuMA or Lamin A/C). The images in upper row showed marker expression of transplanted NCSC in middle conduit (4–7 mm from proximal end). The bar chart indicated the ratio of S100β/FSP1/HNK-1-positive human cells to total human cells in the examined areas in proximal, middle, and distal conduit. The lower-right image was taken near proximal stump of sciatic nerve in nerve conduit. White arrows pointed the transplanted NCSCs that accompanied the newly growing blood vessels (CD31+). Scale bars are 50μm for all images.

Figure 5

Neurotrophic factors (NFs) Secreted by Transplanted NCSCs/NCSC-SCs. The tissues inside nerve conduits at 2-week after surgery were collected and tested with the expressions of human NGF, BDNF, and CNTF by using sandwich ELISA. Bars represent mean ± standard error of mean. * indicates significant difference from one-way ANOVA (*: p < 0.05; **: P < 0.01; n = 3 for each group).