Human neural progenitors derived from integration-free iPSCs for SCI therapy

Abstract

As a potentially unlimited autologous cell source, patient induced pluripotent stem cells (iPSCs) provide great capability for tissue regeneration, particularly in spinal cord injury (SCI). However, despite significant progress made in translation of iPSC-derived neural progenitor cells (NPCs) to clinical settings, a few hurdles remain. Among them, non-invasive approach to obtain source cells in a timely manner, safer integration-free delivery of reprogramming factors, and purification of NPCs before transplantation are top priorities to overcome. In this study, we developed a safe and cost-effective pipeline to generate clinically relevant NPCs. We first isolated cells from patients' urine and reprogrammed them into iPSCs by non-integrating Sendai viral vectors, and carried out experiments on neural differentiation. NPCs were purified by A2B5, an antibody specifically recognizing a glycoganglioside on the cell surface of neural lineage cells, via fluorescence activated cell sorting. Upon further in vitro induction, NPCs were able to give rise to neurons, oligodendrocytes and astrocytes. To test the functionality of the A2B5+ NPCs, we grafted them into the contused mouse thoracic spinal cord. Eight weeks after transplantation, the grafted cells survived, integrated into the injured spinal cord, and differentiated into neurons and glia. Our specific focus on cell source, reprogramming, differentiation and purification method purposely addresses timing and safety issues of transplantation to SCI models. It is our belief that this work takes one step closer on using human iPSC derivatives to SCI clinical settings.

Keywords: Neural repair; Neuroprotection; Spinal cord injury; iPSC.

Fig. 1

Successful generation of iPSCs using cells derived from patients’ urine. (A) Cells derived from patients’ urine resemble the morphology of epithelial cells. (B–E) Phase images of human iPSCs (U-iPSCs) reprogrammed from urine cells from different patients by Sendai viruses. Typical morphology of human iPSC colonies is observed under phase contrast. The flat cells in panel E are MEF feeders. (F–M) iPSCs expresses pluripotent markers OCT4, SSEA4 and TRA1-81. (N) The human iPSCs also maintain a normal karyotype. (O) RT-PCR results of human iPSCs from two representative clones USC-K5 and K7 show that viral components are depleted after passage 10. Bar, 50 μm.

Fig. 2

Differentiation and FACS purification of A2B5+ cells from U-iPSCs. (A) Scheme of our differentiation protocol. (B–C) Before sorting, about 80% of differentiated cells have A2B5 immunoreactivity; after A2B5+ sorting, 95–99% of cells are A2B5+. (D–E) On the contrary, the percentage of TRA1-81+ iPSCs is decreased to 0. (F–H) Before sorting, human iPSCs differentiate along neural lineage express A2B5, and NSC markers SOX1 and PAX6. However, a small percentage of cells express pluripotent marker TRA1-81 (I), indicating contamination of undifferentiated cells. (J–L) After A2B5+ sorting, cells maintain A2B5, SOX1 and NESTIN expression. (M) Most importantly, the undifferentiated TRA1-81+ cells have been depleted. Bar, F, I, J–M, 50 μm; G, H, 20 μm.

Fig. 3

A2B5+ cells derived from patient U-iPSCs give rise to neurons and astrocytes. (A–C) A2B5+ sorted cells continue to proliferate and give rise to a variety of neuronal subtypes including HB9 (MNX1)+ motor neurons and GABA+ neurons, all of which co-express the pan-neuronal marker β3 tubulin. (D–E) A2B5+ sorted cells give rise to NG2+ and PDGFRα+ oligodendrocyte progenitors. (F–H) CD44+ astrocyte progenitors, GFAP+/S100B+ astrocytes are obtained from A2B5+ cells. Bar, A–D, 20 μm; E–H, 50 μm.

Fig. 4

Genome-wide gene expression profiling of U-iPSC derived A2B5 cells and comparison of A2B5 cells to neural progenitor cells. (A) Dendrogram. (B) Heatmap. Global gene expression is compared for human fibroblasts (hFB1, hFB2), patient derived urine cells (USCs, hB35, hK3), iPSCs derived from patient fibroblasts (NR1251 iPS, UTY1 iPS), iPSCs derived from patient urine cells (U-iPSCs, K7 iPS), human embryonic stem cells (WA01 replicates 1, 2, and 3), NPCs derived from hESCs (hN2 NPC), and A2B5+ cells derived from U-iPSCs. Urine cell-derived, Sendai virus reprogrammed U-iPSC K7iPS clusters together with fibroblast-derived, Sendai virus-reprogrammed human iPSC line (NR1251iPS), and fibroblast-derived, retrovirus-reprogrammed human iPSC (UTY1 iPS), as well as previously characterized hESC line WA01 (replicates 1, 2, and 3). iPSC-derived A2B5+ NPCs, patient derived urine cells hB35 and hK3 cluster much closer to the human fibroblasts (hFB1, and hFB2) than to iPSCs or hESCs. Most importantly, A2B5+ NPCs cluster together with previously characterized hESC derived NPCs (hN2 NPC).

Fig. 5

A2B5+ sorted cells derived from patient U-iPSCs give rise to neurons and astrocytes after being grafted to SCI mouse model. FACS purified A2B5+ cells are grafted into the contused thoracic spinal cord of adult mice. Eight weeks after transplantation, grafted human cells survive and integrate into the injured spinal cord as shown by the human nuclei staining (HuNA) (A, E, I). Some have matured to neurons and start to express neuronal marker NFM and MAP2 (C, H, K) some differentiate into GFAP+ astrocytes (B, F, J). Bar, A–D, 500 μm; E–H and I–L, 20 μm. E–H represent higher magnification images of the boxed areas in A–D.