Research ArticleSpinal Cord Injury

Survival of syngeneic and allogeneic iPSC–derived neural precursors after spinal grafting in minipigs

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Science Translational Medicine  09 May 2018:
Vol. 10, Issue 440, eaam6651
DOI: 10.1126/scitranslmed.aam6651
  • Fig. 1 In vitro–differentiated porcine iPSC-derived neural precursors give rise to functional neurons.

    (A) Schematic diagram of the experimental timeline and design in vitro and in vivo. EGFP, enhanced green fluorescent protein. (B to H) Cultured fibroblasts (B) formed colonies of pluripotent cells (Pluri. colonies) at 10 days after infection (C and D) and expressed pluripotent markers including KLF4 (E), SOX2 (F), OCT4 (G), and Nanog (H). (I) Established iPSC-derived EBs cultured in nonattachment culture flasks. (J) H&E staining of mouse testes at 6 weeks after injection of iPSCs suspended in Matrigel. Development of mature teratoma with identifiable all three germ layer derivatives including neural tube rosettes (1. ectoderm, red arrows; upper inset), smooth muscle (2. mesoderm, red arrows; middle inset), and gut cuboidal epithelium with goblet cells (3. endoderm, red arrows; lower inset) can be seen. (K and L) NPC colonies were manually harvested from the periphery of attached and induced EBs/neural rosettes (K) and further expanded (L). (M to P) Long-term expanded NPCs show expression of markers typical of neural precursors including SOX2 (M), PAX6 (N and P), SOX1 (O), and Nestin (N and O). DAPI, 4′,6-diamidino-2-phenylindole. (Q to T) Staining of induced SYN-EGFP-NPCs (R) with neuronal markers DCX (S and T), NeuN (S), and Tuj1 (U) was seen. A subpopulation of DCX+ neurons was GABA-immunoreactive (T). (U) Fluo-4 AM–loaded, 2 month–induced NPCs showing spontaneous intracellular calcium oscillation in axons (1, 1′) and neuronal soma (2, 2′ and 3, 3′). Scale bars, 500 μm (J), 10 μm (M), 15 μm (N), 10 μm (O), and 10 μm (S to U). a.u. arbitrary units.

  • Fig. 2 Intrastriatal grafting of iPSC-NPCs in immunodeficient rats is associated with robust neuronal differentiation and appearance of mature and functional grafted neurons at 7 to 10 months after grafting.

    (A and B) Schematic representation of the experimental timeline and design. (C and D) Action potential firing (C) (evoked by a depolarizing step of current of 75 pA for 500 ms from resting potential in current clamp at 0 pA) and voltage-gated sodium and potassium currents (D) (evoked by a series of depolarizing steps of 5 mV in voltage clamp at −70 mV) in grafted SYN-EGFP neurons. (E and F) Staining of paraformaldehyde-fixed SYN-EGFP+ grafted striatal section with NeuN (E) and NSE (F) antibodies. (G to I) Immunofluorescence staining with SYN, NeuN, VGAT, and gephyrin (Gephr) antibodies (I) in the core of EGFP+ graft (G and I) and in areas distant from the graft core (medial region of NPC-grafted striatum) (H). Scale bars, 50 μm (E), 10 μm (F), 5 μm (G), and 20 μm (H and I).

  • Fig. 3 iPSC-NPCs grafted into striata of immunodeficient rats acquire genetic signature of mature porcine CNS at 10 months after grafting.

    (A to D) Schematic representation of the experimental timeline and design. (E) Analysis and preprocessing of mixed-species RNA-sequencing (RNA-seq) reads. (F) t-SNE plot showing the degree of correlation in gene expression profiles between the indicated samples.

  • Fig. 4 Spinally grafted iPSC-NPCs show long-term survival and neuronal and glial differentiation in syngeneic recipient in the absence of immunosuppression.

    (A) Immunofluorescence staining showing EGFP fluorescence in the core of the graft. (B and C) Immunofluorescence staining showing NF- and GFAP-stained processes in the same areas as in (A). (D to F) Staining with DCX (D), NeuN (E), and NSE (F) in EGFP+ grafts. (G to I) Immunofluorescence staining showing EGFP/NF+ neurites, in areas cranial and caudal to the borders of the graft. (J and K) Costaining of SYN-EGFP+ grafts with VGAT and gephyrin antibodies in the core of the graft. Scale bars, 30 μm (A), 125 μm (B), 10 μm (C), 40 μm (D), 35 μm (E and F), 50 μm (G to I), and 20 μm (J and K).

  • Fig. 5 Syngeneic iPSC-NPC recipients show no humoral immunity against grafted cells at 3 months after cell transplantation in the absence of immunosuppression.

    (A) Schematic diagram of the development of in vivo/ex vivo staining protocol to detect the presence of circulating antibodies against grafted iPSC-NPCs. (B to D) Staining of mature iPSC-NPC grafts in rat striatum and rat spinal cord with serum from previously anti–iPSC-NPC–immunized pig (B), sera harvested from naïve nonimmunized pig (C), or with sera from syngeneic iPSC-NPC–grafted pigs (n = 3) (D) 3 months after grafting. Scale bars, 40 μm (B to D).

  • Fig. 6 Spinally grafted iPSC-NPCs show long-term survival and neuronal and glial differentiation in allogeneic spinally injured pigs with transient immunosuppression.

    (A) Schematic diagram of experimental design. IF, immunofluorescence. (B to F) Costaining of EGFP+ grafts with NeuN, synaptophysin, and GFAP antibodies. HT, host tissue. (G) Costaining with NF and NeuN antibodies in the core of the graft. (H) Costaining of DCX, EGFP, and NSE in the core of the graft. (I) Confocal microscopy image of SYN and VGAT puncta on EGFP+ grafted neurons. (J) Staining of EGFP+ grafted neuron with VGAT and gephyrin antibodies. (K) Costaining with HOMER and NF antibodies of EGFP+ grafted neurons (white arrows indicate HOMER+ puncta on neuronal soma or axons of EGFP+ neurons). (L to N) Confocal images of double-labeled EGFP+/OLIG2+ oligodendrocytes and EGFP+/GFAP+ astrocytes. (O and P) Staining of mature iPSC-NPC-EGFP+ grafts in rat striata with sera from anti–iPSC-NPC–immunized pigs (O) or with sera from iPSC-NPC–grafted transiently immunosuppressed allogeneic pig (n = 3) (P). Scale bars, 300 μm (B), 500 μm (C), 60 μm (D to F), 250 μm (G), 60 μm (H), 10 μm (I), 5 μm (J), 10 μm (K), 10 μm (L and N), and 40 μm (O and P).

  • Fig. 7 Transient immunosuppression (4 weeks) supports long-term graft survival and is associated with progressive decrease in spinal regional inflammatory response in iPSC-NPC–grafted spinally injured allogeneic pig.

    (A to C) Immunofluorescence staining for MHC-II and EGFP around cell injection needle tract in syngeneic pigs (A; n = 3) and in allogeneic continuously immunosuppressed (B; n = 3) or transiently immunosuppressed (C; n = 3) spinally injured pigs 4 weeks or 3.5 months after cell grafting. (D to F) Immunofluorescence staining for Iba1 and EGFP around cell injection needle tract in syngeneic pigs (D) and in allogeneic continuously immunosuppressed (E) or transiently immunosuppressed (F) spinally injured pigs 4 weeks or 3.5 months after cell grafting. (G to I) Immunofluorescence staining for CD45, CD8, and EGFP around cell injection needle tract in syngeneic pigs (G) and in allogeneic continuously immunosuppressed (H) or transiently immunosuppressed (I) spinally injured pigs 4 weeks or 3.5 months after cell grafting. (J) Colocalization of MHC-II with Iba1 in the core of the graft. (K and L) Quantitative analysis of CD45+ and CD8+ leukocytes in EGFP+ grafts and in surrounding host tissue (Student’s t test; ***P < 0.05). Scale bars, 300 μm (A to C and G to I), 20 μm (D to F), and 10 μm (J).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/440/eaam6651/DC1

    Materials and Methods

    Fig. S1. Porcine fibroblast-derived iPSCs generate ectoderm, mesoderm, and endoderm cell derivatives in vitro and in vivo.

    Fig. S2. Previously frozen, in vitro–expanded porcine iPSC-NPCs show stable karyotype and generate neural derivatives (neurons, astrocytes, and oligodendrocytes) after in vitro induction.

    Fig. S3. Long-term grafted (7 to 10 months) porcine iPSC-NPCs in rat striata show protein- and mRNA-defined signature that is consistent with mature porcine CNS tissue.

    Fig. S4. Porcine iPSC-NPCs grafted into rat striata show no tumor formation and incomplete myelinization at 7 months after grafting.

    Fig. S5. Porcine iPSC-NPC-EGFP+ grafts in rat striata show normal vascularization and no changes in tumor suppressors or proto-oncogenes at 7 to 10 months after grafting.

    Fig. S6. iPSC-NPCs grafted into syngeneic pig spinal cord in the absence of immunosuppression show long-term survival and neuronal and glial differentiation at 3 months after transplantation.

    Fig. S7. Porcine iPSC-NPCs grafted spinally in allogeneic, transiently immunosuppressed (1-month immunosuppression) pigs with previous spinal traumatic injury show neuronal and glial differentiation at 3.5 months after grafting.

    Fig. S8. Spinally grafted iPSC-NPCs in allogeneic, spinally injured pig with transient immunosuppression (1 month) show extensive neuronal (NeuN) differentiation at 3.5 months after grafting.

    Fig. S9. iPSC-NPCs grafted spinally in allogeneic pig with previous spinal injury do not form tumors and show incomplete myelination at 3.5 months after grafting.

    Fig. S10. Reprogramming factors (OCT4 and KLF4) are silenced in mature iPSC-NPC grafts in rat striata or spinal cord in allogeneic pig with previous spinal traumatic injury.

    Fig. S11. Long-term grafted iPSC-NPCs in rat striata or spinal cord of allogeneic pig show only occasional presence of Sendai virus–associated protein in grafted cells and show no change in expression of immunogenic genes.

    Table S1. Quantitative analysis of neuronal and glial differentiation in EGFP grafts.

    Table S2. Electrophysiological properties of three transplanted iPSC neurons into the striatum of a rat at 8 months after grafting.

    Table S3. mRNA-sequencing species sorting quantification.

    Table S4. SLA genotypes of the iPSC-NPC donor and the allogeneic graft recipients.

    Table S5. Antibodies used for flow cytometry and immunofluorescence staining.

    Appendix S1. Porcine gene list.

  • Supplementary Material for:

    Survival of syngeneic and allogeneic iPSC–derived neural precursors after spinal grafting in minipigs

    Jan Strnadel, Cassiano Carromeu, Cedric Bardy, Michael Navarro, Oleksandr Platoshyn, Andreas N. Glud, Silvia Marsala, Jozef Kafka, Atsushi Miyanohara, Tomohisa Kato Jr., Takahiro Tadokoro, Michael P. Hefferan, Kota Kamizato, Tetsuya Yoshizumi, Stefan Juhas, Jana Juhasova, Chak-Sum Ho, Taba Kheradmand, PeiXi Chen, Dasa Bohaciakova, Marian Hruska-Plochan, Andrew J. Todd, Shawn P. Driscoll, Thomas D. Glenn, Samuel L. Pfaff, Jiri Klima, Joseph Ciacci, Eric Curtis, Fred H. Gage, Jack Bui, Kazuhiko Yamada, Alysson R. Muotri, Martin Marsala*

    *Corresponding author. Email: mmarsala{at}ucsd.edu

    Published 9 May 2018, Sci. Transl. Med. 10, eaam6651 (2018)
    DOI: 10.1126/scitranslmed.aam6651

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Porcine fibroblast-derived iPSCs generate ectoderm, mesoderm, and endoderm cell derivatives in vitro and in vivo.
    • Fig. S2. Previously frozen, in vitro–expanded porcine iPSC-NPCs show stable karyotype and generate neural derivatives (neurons, astrocytes, and oligodendrocytes) after in vitro induction.
    • Fig. S3. Long-term grafted (7 to 10 months) porcine iPSC-NPCs in rat striata show protein- and mRNA-defined signature that is consistent with mature porcine CNS tissue.
    • Fig. S4. Porcine iPSC-NPCs grafted into rat striata show no tumor formation and incomplete myelinization at 7 months after grafting.
    • Fig. S5. Porcine iPSC-NPC-EGFP+ grafts in rat striata show normal vascularization and no changes in tumor suppressors or proto-oncogenes at 7 to 10 months after grafting.
    • Fig. S6. iPSC-NPCs grafted into syngeneic pig spinal cord in the absence of immunosuppression show long-term survival and neuronal and glial differentiation at 3 months after transplantation.
    • Fig. S7. Porcine iPSC-NPCs grafted spinally in allogeneic, transiently immunosuppressed (1-month immunosuppression) pigs with previous spinal traumatic injury show neuronal and glial differentiation at 3.5 months after grafting.
    • Fig. S8. Spinally grafted iPSC-NPCs in allogeneic, spinally injured pig with transient immunosuppression (1 month) show extensive neuronal (NeuN) differentiation at 3.5 months after grafting.
    • Fig. S9. iPSC-NPCs grafted spinally in allogeneic pig with previous spinal injury do not form tumors and show incomplete myelination at 3.5 months after grafting.
    • Fig. S10. Reprogramming factors (OCT4 and KLF4) are silenced in mature iPSC-NPC grafts in rat striata or spinal cord in allogeneic pig with previous spinal traumatic injury.
    • Fig. S11. Long-term grafted iPSC-NPCs in rat striata or spinal cord of allogeneic pig show only occasional presence of Sendai virus–associated protein in grafted cells and show no change in expression of immunogenic genes.
    • Table S1. Quantitative analysis of neuronal and glial differentiation in EGFP grafts.
    • Table S2. Electrophysiological properties of three transplanted iPSC neurons into the striatum of a rat at 8 months after grafting.
    • Table S3. mRNA-sequencing species sorting quantification.
    • Table S4. SLA genotypes of the iPSC-NPC donor and the allogeneic graft recipients.
    • Table S5. Antibodies used for flow cytometry and immunofluorescence staining.

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    Other Supplementary Material for this manuscript includes the following:

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