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. 2024 Apr 23;15(1):114.
doi: 10.1186/s13287-024-03714-3.

Stepwise combined cell transplantation using mesenchymal stem cells and induced pluripotent stem cell-derived motor neuron progenitor cells in spinal cord injury

Jang-Woon Kim  1   2 , Juryun Kim  3 , Hyunkyung Mo  1   2 , Heeju Han  1   2 , Yeri Alice Rim  4   5 , Ji Hyeon Ju  6   7   8   9
Affiliations

Stepwise combined cell transplantation using mesenchymal stem cells and induced pluripotent stem cell-derived motor neuron progenitor cells in spinal cord injury

Jang-Woon Kim et al. Stem Cell Res Ther. .

Abstract

Background: Spinal cord injury (SCI) is an intractable neurological disease in which functions cannot be permanently restored due to nerve damage. Stem cell therapy is a promising strategy for neuroregeneration after SCI. However, experimental evidence of its therapeutic effect in SCI is lacking. This study aimed to investigate the efficacy of transplanted cells using stepwise combined cell therapy with human mesenchymal stem cells (hMSC) and induced pluripotent stem cell (iPSC)-derived motor neuron progenitor cells (iMNP) in a rat model of SCI.

Methods: A contusive SCI model was developed in Sprague-Dawley rats using multicenter animal spinal cord injury study (MASCIS) impactor. Three protocols were designed and conducted as follows: (Subtopic 1) chronic SCI + iMNP, (Subtopic 2) acute SCI + multiple hMSC injections, and (Main topic) chronic SCI + stepwise combined cell therapy using multiple preemptive hMSC and iMNP. Neurite outgrowth was induced by coculturing hMSC and iPSC-derived motor neuron (iMN) on both two-dimensional (2D) and three-dimensional (3D) spheroid platforms during mature iMN differentiation in vitro.

Results: Stepwise combined cell therapy promoted mature motor neuron differentiation and axonal regeneration at the lesional site. In addition, stepwise combined cell therapy improved behavioral recovery and was more effective than single cell therapy alone. In vitro results showed that hMSC and iMN act synergistically and play a critical role in the induction of neurite outgrowth during iMN differentiation and maturation.

Conclusions: Our findings show that stepwise combined cell therapy can induce alterations in the microenvironment for effective cell therapy in SCI. The in vitro results suggest that co-culturing hMSC and iMN can synergistically promote induction of MN neurite outgrowth.

Keywords: Cell transplantation; Induced pluripotent stem cells; Mesenchymal stem cells; Motor neuron progenitor cells; Spinal cord injury.

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Conflict of interest statement

The authors declare that there is no competing interest. Affiliation 3 is declare that there is no competing interest. J.K. is employee at YiPSCELL, Inc., and J.H.J. is the employer. J.H.J. is the founder of YiPSCELL. Inc. and also works at the Seoul St. Mary’s hospital, Catholic University of Korea. The two groups do not have competing interests.

Figures

Fig. 1
Fig. 1
Generation of iMNP and iMN in vitro. a Scheme of MNP and MN differentiation from human iPSCs using a small molecule cocktail. b Representative time-course images of iNEPs after six days of culture media conditions, iMNPs on day 12 under different conditions with RA and Pur, and a representative image of iMNs on day 18. A light microscopy image showing the iMature MNs on day 28 of differentiation. c Fluorescence image showing the expression of SOX1 in iNEP, OLIG2 in iMNP, co-localization of ChAT and HB9 in iMN, and SMI-32 in mature iMN. d Representative images of heatmap activity for plate-wide visualization of spike or beat rates and amplitudes on multi-electrode arrays (iPSCs, n = 3; iNEP, n = 3; iMN, n = 4; iMature MN, n = 4). e Measure of average spike numbers of active electrodes per well. f Measurement of the mean firing rate of active electrodes per well over 28 days of in vitro differentiation. Data are presented as mean ± SEM. Statistical significance was estimated using Kruskal–Wallis test with post hoc analysis and Mann–Whitney (†) test with least significant difference post hoc analysis (*); *, † P < 0.05, Scale bars = 50 μm. iNEP, iPSC-derived neuroepithelial progenitor cells; iMNP, iPSC-derived motor neuron progenitor cells; iMN, iPSC-derived motor neuron cells; iMature MNs, iPSC-derived mature motor neuron cells
Fig. 2
Fig. 2
Transplantation of iMNP in contusive chronic SCI model. a Representative images of Ki-67 positive proliferating cells in each milestone cell type and proliferation were analyzed using the Cell Counting Kit-8 assay. b Experimental scheme of generation of contusive chronic SCI rat model. c The transplanted iMNPs show white and gray matter at the lesion epicenter. d IF stained image of OLIG2 and iMNP in the dorsal portion of the lesional site. e IF stained image of ChAT and iMNP in the gray matter of the lesional site. f Expression of SMI-32 in the transplanted iMNP cells was mostly noted around the ependymal cell layer and posterior part of the lesion. g BBB scores improved at 12 and 14 weeks after SCI (PBS, n = 4; 12 weeks iMNP, n = 3, and 14 weeks iMNP n = 2). Data are presented as mean ± SEM. Scale bars = 50 and 20 μm. iMNP, induced pluripotent stem cell-derived motor neuron progenitor cells; SCI, spinal cord injury; IF, immunofluorescence; ChAT, choline acetyltransferase; BBB, Basso–Beattie–Bresnahan
Fig. 3
Fig. 3
Induced neuronal differentiation and neuroprotection by multiple injections of hMSC at the lesional site. a Schematic of multiple intravenous injections of hMSC in contusive acute spinal cord injury (SCI). Animals were injected with hMSC (1’ MSC = single injection at 24 h post injury, 2’MSC = Dual injection at 24 h and one week post injury). b IF images show merged 4′,6-diamidino-2-phenylindole and hMSC at the lesional site at six weeks. c Multifluorescent, confocal images showing the expression of NeuN and hMSC in the gray matter. d Multi-fluorescent confocal images showing the expression of NGF and hMSC around the gray matter. e MAP-2 positivity indicates the lesional site and gray matter around and within hMSCs in trauma-induced gliosis at the posterior lesional site at six weeks. f WB images of NeuN, NGF and MAP-2 expression in lesional site segments (formula image 1 cm). g WB results of NeuN expression in the lesional site segments. h WB results of NGF expression in the lesional site segments. i WB results of MAP-2 expression in the lesional site segments. Full-length western blot images are presented in Additional file 7: Fig. S7. j BBB scales in the 1’MSC and 2’MSC groups are significantly higher than that in the PBS group. Data are presented as mean ± SEM. Statistical significance was estimated using Kruskal–Wallis test with post hoc analysis and Mann–Whitney (†) test with least significant difference post hoc analysis (*); *, † P < 0.05. (WB analysis: PBS n = 5, 1’MSC n = 4 and 2’MSC n = 5, BBB locomotor scales: PBS n = 7, 1’MSC n = 5, 2’MSC n = 6) Scale bars = 20 μm. hMSC, human mesenchymal stem cells; IF, immunofluorescence; BBB, Basso–Beattie–Bresnahan; WB, Western blotting
Fig. 4
Fig. 4
Enhancement of MN differentiation and maturation by preemptive cell transplantation of hMSC and iMNP in contusive SCI model. a Schematic image of preemptive transplantation of hMSC and iMNP in a contusive SCI model. Multiple intravenous injections of hMSC were administered at 24 h and one week post injury. Six weeks post injury, iMNPs were transplanted to the lesional site using intralesional injection. b BBB scale scores in the hMSC + iMNP group was significantly higher than that in the PBS group. c Transplanted hMSC and iMNP show white and gray matter at the epicenter of the lesion. d SMI-32 differentiation of transplanted iMNP cells is predominant. e Orthogonal view of confocal images showing SMI-32 and transplanted cell expression around the lesional site. f WB results of SMI-32 expression in the hMSC + iMNP group were significantly higher than those in the PBS group. Full-length WB images are presented in Additional file 7: Fig. S7. Data are presented as mean ± SEM. Statistical significance was estimated using Kruskal–Wallis test with post hoc analysis and Mann–Whitney (†) test with least significant difference post hoc analysis (*); *, † P < 0.05. (WB analysis: PBS n = 6, hMSC n = 5, iMNP n = 4 and hMSC + iMNP n = 5, BBB locomotor scales: PBS n = 10, hMSC n = 10, iMNP n = 8 and hMSC + iMNP n = 10) Scale bars = 20 and 10 μm. hMSC, human mesenchymal stem cells; iMNP, induced pluripotent stem cell-derived motor neuron progenitor cells; SCI, spinal cord injury; BBB, Basso–Beattie–Bresnahan; WB, Western blotting
Fig. 5
Fig. 5
Increased axonal regeneration by preemptive hMSC and iMNP transplantation in the SCI lesion. a Confocal images showing synapsin-1 and transplanted cell expression around the lesional site. IF staining of synapsin-1 longitudinal sections. b IF staining of synapsin-1 showing axial sections of the lesional site. Synapsin-1 and transplanted iMNP merged around the lesional site. c WB results of synapsin-1 expression in lesional site segments (formula image 1 cm). WB showed significantly increased expression of synapsin-1 in the MSC + MNP group compared to the PBS group. d IF staining images of MAP-2 showing a longitudinal section. e IF staining images of MAP-2 showing an axial section from the lesional site. MAP-2 and transplanted iMNP merged around the SCI lesional site. f WB results of MAP2 expression in the lesional site segments. Full-length WB images are presented in Additional file 7: Fig. S7. g Transmission electron microscopy images proximal to the lesional site. Data are presented as mean ± SEM. Statistical significance was estimated using Kruskal–Wallis test with post hoc analysis and Mann–Whitney (†) test with least significant difference post hoc analysis (*); *, † P < 0.05. (WB analysis: PBS n = 6, hMSC only n = 5, iMNP only n = 4, and hMSC + iMNP n = 5, Scale bars = 20 μm. hMSC, human mesenchymal stem cells; iMNP, induced pluripotent stem cell-derived motor neuron progenitor cells; SCI, spinal cord injury; IF, immunofluorescence; BBB, Basso–Beattie–Bresnahan; WB, Western blotting
Fig. 6
Fig. 6
Neurite outgrowth induction by 2D and 3D co-culture of hMSC and iMN in vitro. a Schematic of hMSC and iMN 2D and 3D co-culture neurite outgrowth assessment in vitro. b Representative light microscopy images of hMSC and iMN 2D co-culture into mature MN differentiation on day 10. c IF staining images of MAP-2 in 2D co-cultured hMSC and iMN on day 10. d Protein expression of MAP-2 in hMSC and iMN 2D co-culture cell lysates on day 10. Full-length WB images are presented in Additional file 7: Fig. S7. e IF staining of MAP-2 in 3D co-cultured spheroids consisting of hMSC and iMN on day 10. f Representative light microscopy images of neurite outgrowths on day 10 in the 3D co-cultured spheroids. g Representative fluorescence microscopy images of neurite outgrowths on day 10 in the 3D co-cultured spheroids. h Quantification of neurite outgrowth on day 10. Data are presented as mean ± SEM. Statistical significance was estimated using Kruskal–Wallis test with post hoc analysis and Mann–Whitney (†) test with least significant difference post hoc analysis (*); *, † P < 0.05. (WB analysis: hMSC n = 3, iMN n = 3, hMSC + iMN n = 3, Neurite outgrowth assay analysis: hMSC n = 6, iMN n = 6, hMSC + iMN n = 6, Scale bars = 20 μm. hMSC, human mesenchymal stem cells; iMN, induced pluripotent stem cell-derived motor neuron cells; IF, immunofluorescence; WB, Western blotting
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