Stem Cells Engineered During Different Stages of Reprogramming Reveal Varying Therapeutic Efficacies

Abstract

Stem cells are emerging as promising treatment strategies for several brain disorders and pathologies. In this study, we explored the potential of creating induced pluripotent stem cell-derived neural stem cells (ipNSC) by using either unmodified or gene-modified somatic cells and tested their fate and therapeutic efficacies in vitro and in vivo. We show that cells engineered in somatic state lose transgene-expression during the neural induction process, which is partially restored by histone deacetylase inhibitor treatment whereas cells engineered at the ipNSC state have sustained expression of transgenes. In vivo, bimodal mouse and human ipNSCs engineered to express tumor specific death-receptor ligand and suicide-inducing therapeutic proteins have profound anti-tumor efficacy when encapsulated in synthetic extracellular matrix and transplanted in mouse models of resected-glioblastoma. This study provides insights into using somatic cells for treating CNS disorders and presents a receptor-targeted cancer therapeutic approach for brain tumors. Stem Cells 2018;36:932-942.

Keywords: Brain tumors; Cellular engineering; Epigenetic modulation; Pro-apoptotic therapy; Reprogramming; Stem cells; Targeted therapy.

Figure 1. Engineered MEFs derived iPSCs have ES cell like properties and are functional

(a) Flow chart of the reprogramming and differentiation processes including the used plasmids of the steps described in this figure. (b) Light and fluorescence images of plain MEFs and MEFs transduced with LV-GF and LV-TR. (c) Plot showing the percentage of tumor cell viability comparing co-cultures with plain MEFs and MEF-S-TR. (inset) MEF-TR, MEF-GF and plain MEFs were co-cultured 1:1 with Gli36-EvIII-FmC. (d) Plot showing the number of AP stained colonies from ipSC generated from unmodified, LV-GF and LV-TR modified MEFs. (e) RT-PCR analysis of ES cell marker genes in iPSC colonies (f) Light and fluorescence images as well as AP staining (suggestive for an ES cell-like state) of iPSC colonies generated from wild-type and engineered MEF (g) Plots and photomicrographs showing GBM cell viability based on the Fluc signal after 2 days of exposure to the conditioned medium from wild-type iPS, iPS-GF and iPS-TR cells, respectively (h) Plot and representative bioluminescence images showing teratoma formation in mice following intramuscular injection of plain iPS-GF into one flank of nude mice and bioluminescence imaging was performed on days 0, 7, 14, 21 and 24 (i) Histopathological analysis of the teratoma tissue by identification of tissue derivatives of all three germ layers, mesoderm, ectoderm and endoderm from iPS-GFP-Fluc into one flank of nude mice mice and sacrificed after 4 weeks. Scale Bars: 100μM (b,i), 200 μM (c,f,g). (GF: GFP-Fluc; TR: TRAIL)

Figure 2. ipNSCs derived from un-modified and engineered MEFs express NSC markers and lose expression of transgenes that can be restored by HDAC inhibition

(a) Light and fluorescence images of embryoid body (EB) formation from non-modified and transduced iPSC colonies showing transgene expression. (b) Light and fluorescence images of un-modified and modified ipSC differentiated into neural rosette structures upon exposure to appropriate culture conditions. (c) RT-PCR analysis of NSC marker genes. (d) Plot showing viability of various engineered ipNSC lines over time (0h, 24h, 48h) following HDAC inhibition (inset) photomicrographs representing restoration of transgene expression as demonstrated by GFP by addition of 2μM VA, an HDAC inhibitor (e-f) RT-PCR analysis of NSC marker genes post-treatment of ipNSC-GF and ipNSC-TR with different concentrations of HDAC inhibitors: Valproic acid (VA) and CN147. (g-j) Fluorescence photomicrographs showing immunostaining with Nestin (g), GFAP (h) and MAP-2 (i) antibodies. Scale Bars: 100μM (g-j), 200 μM (a,b,d). (GF: GFP-Fluc; TR: TRAIL)

Figure 3. HDAC inhibition leads to apoptotic cell death in reprogrammed mouse ipNSC in vitro.

(a) Photomicrographs of immunostaining depicting expression of Nestin and Sox2 markers in mipNSC following CN147 (HDACi) treatment as compared to untreated cells. (b) Plot showing changes in HDAC activity in ipNSC-GF and ipNSC-TR following 24hrs of CN147 treatment (above) Schematic outlining the HDAC inhibition by CN147. (c) Plot showing changes in Caspase 9 activity in ipNSC-GF and ipNSC-TR following 48hrs of CN147 treatment. (d) Plot showing changes in Caspase 9 activity in ipNSC-GF and ipNSC-TR following CN147 treatment pre-treated with Z-VAD-FMK and controls. Scale Bars: 100μM (a). (GF: GFP-Fluc; TR: TRAIL; Cas Inhi: Caspase Inhibitor Z-VAD-FMK). (*P<0.05 as compared to untreated cells)

Figure 4. Engineered mouse ipNSCs derived from un-modified MEFs have profound anti-tumor efficacy in vivo

(a) Flow chart of the reprogramming and differentiation processes including the used plasmids (above). (below) Photomicrographs showing mouse ipNSCs transduced with lentiviral vectors and expressing GFP in neural rosette structures (magnification x4) (b) Plot showing the therapeutic efficacy of ipNSCs engineered to secrete TR and express TK co-cultured with primary GBM8-FmC and GBM18-FmC. (c) Plot showing the changes in the tumor volumes represented by changes in Fluc intensity post treatment of ipNSC-TRTK on primary GBMs. Mice bearing GBM8 (n=30) were intratumorally injected with ipNSC-GFP, ipNSC- TK, ipNSC-TR or ipNSC-TRTK and followed by bioluminescence imaging. Ganciclovir (GCV) was administered to study the effect of HSV-TK activation. Scale Bars: 100μM (a). (TR: TRAIL; TK: HSV-TK; TRTK: TRAIL/HSV-TK)

Figure 5. Human ipNSC engineered to express therapeutic transgenes eliminate GBM in vivo.

(a) Flow chart of the NSC differentiation processes (b) Photomicrographs show successful differentiation of human iPSCs to NSCs expressing Musashi1, SOX1, Nestin and MAP2. (c) Engineered hipNSCs were co-cultured with primary patient derived GBM and photomicrographs of co-culture assay and an illustration of the experimental setup (above). (d) Plot showing changes in GBM18 tumor volumes following tumor resection and treatment with sECM encapsulated ipNSC-TRTK treated with 10mg/kg of Ganciclovir (e) Kaplan Meier survival curves showing survival benefit in mice treatment following tumor resection and treatment with sECM encapsulated ipNSC-TRTK treated with 10mg/kg of Ganciclovir as compared to controls (f) H&E staining of mouse brain sections showing reduction of tumor burden in mice treated with ipNSC-TRTK + GCV. Scale Bars: 100μM (b), 200 μM (c,f). (TR: TRAIL; TK: HSV-TK; TRTK: TRAIL/HSV-TK)