Induced Pluripotent Stem Cell-Derived Fibroblasts Efficiently Engage Senescence Pathways but Show Increased Sensitivity to Stress Inducers.

The risk of aberrant growth of induced pluripotent stem cell (iPSC)-derived cells in response to DNA damage is a potential concern as the tumor suppressor genes TP53 and CDKN2A are transiently inactivated during reprogramming. Herein, we evaluate the integrity of cellular senescence pathways and DNA double-strand break (DSB) repair in Sendai virus reprogrammed iPSC-derived human fibroblasts (i-HF) compared to their parental skin fibroblasts (HF). Using transcriptomics analysis and a variety of functional assays, we show that the capacity of i-HF to enter senescence and repair DSB is not compromised after damage induced by ionizing radiation (IR) or the overexpression of H-RASV12. Still, i-HF lines are transcriptionally different from their parental lines, showing enhanced metabolic activity and higher expression of p53-related effector genes. As a result, i-HF lines generally exhibit increased sensitivity to various stresses, have an elevated senescence-associated secretory phenotype (SASP), and cannot be immortalized unless p53 expression is knocked down. In conclusion, while our results suggest that i-HF are not at a greater risk of transformation, their overall hyperactivation of senescence pathways may impede their function as a cell therapy product.

Clearance of defective muscle stem cells by senolytics restores myogenesis in myotonic dystrophy type 1.

Muscle stem cells, the engine of muscle repair, are affected in myotonic dystrophy type 1 (DM1); however, the underlying molecular mechanism and the impact on the disease severity are still elusive. Here, we show using patients' samples that muscle stem cells/myoblasts exhibit signs of cellular senescence in vitro and in situ. Single cell RNAseq uncovers a subset of senescent myoblasts expressing high levels of genes related to the senescence-associated secretory phenotype (SASP). We show that the levels of interleukin-6, a prominent SASP cytokine, in the serum of DM1 patients correlate with muscle weakness and functional capacity limitations. Drug screening revealed that the senolytic BCL-XL inhibitor (A1155463) can specifically remove senescent DM1 myoblasts by inducing their apoptosis. Clearance of senescent cells reduced the expression of SASP, which rescued the proliferation and differentiation capacity of DM1 myoblasts in vitro and enhanced their engraftment following transplantation in vivo. Altogether, this study identifies the pathogenic mechanism associated with muscle stem cell defects in DM1 and opens a therapeutic avenue that targets these defective cells to restore myogenesis.

Induced pluripotent stem cells display a distinct set of MHC I-associated peptides shared by human cancers.

Previous reports showed that mouse vaccination with pluripotent stem cells (PSCs) induces durable anti-tumor immune responses via T cell recognition of some elusive oncofetal epitopes. We characterize the MHC I-associated peptide (MAP) repertoire of human induced PSCs (iPSCs) using proteogenomics. Our analyses reveal a set of 46 pluripotency-associated MAPs (paMAPs) absent from the transcriptome of normal tissues and adult stem cells but expressed in PSCs and multiple adult cancers. These paMAPs derive from coding and allegedly non-coding (48%) transcripts involved in pluripotency maintenance, and their expression in The Cancer Genome Atlas samples correlates with source gene hypomethylation and genomic aberrations common across cancer types. We find that several of these paMAPs were immunogenic. However, paMAP expression in tumors coincides with activation of pathways instrumental in immune evasion (WNT, TGF-ß, and CDK4/6). We propose that currently available inhibitors of these pathways could synergize with immune targeting of paMAPs for the treatment of poorly differentiated cancers.

Autologous humanized mouse models of iPSC-derived tumors enable characterization and modulation of cancer-immune cell interactions.

Modeling the tumor-immune cell interactions in humanized mice is complex and limits drug development. Here, we generated easily accessible tumor models by transforming either primary skin fibroblasts or induced pluripotent stem cell-derived cell lines injected in immune-deficient mice reconstituted with human autologous immune cells. Our results showed that fibroblastic, hepatic, or neural tumors were all efficiently infiltrated and partially or totally rejected by autologous immune cells in humanized mice. Characterization of tumor-immune infiltrates revealed high expression levels of the dysfunction markers Tim3 and PD-1 in T cells and an enrichment in regulatory T cells, suggesting rapid establishment of immunomodulatory phenotypes. Inhibition of PD-1 by Nivolumab in humanized mice resulted in increased immune cell infiltration and a slight decrease in tumor growth. We expect that these versatile and accessible cancer models will facilitate preclinical studies and the evaluation of autologous cancer immunotherapies across a range of different tumor cell types.

Myogenic progenitor cells derived from human induced pluripotent stem cell are immune-tolerated in humanized mice.

It is still unclear if immune responses will compromise the large-scale utilization of human induced pluripotent stem cells (hiPSCs)-derived cell therapies. To answer this question, we used humanized mouse models generated by the adoptive transfer of peripheral blood mononuclear cells or the cotransplantation of hematopoietic stem cells and human thymic tissue. Using these mice, we evaluated the engraftment in skeletal muscle of myoblasts derived either directly from a muscle biopsy or differentiated from hiPSCs or fibroblasts. Our results showed that while allogeneic grafts were mostly rejected and highly infiltrated with human T cells, engraftment of autologous cells was tolerated. We also observed that hiPSC-derived myogenic progenitor cells (MPCs) are not targeted by autologous T cells and natural killer cells in vitro. These findings suggest that the reprogramming and differentiation procedures we used are not immunogenic and that hiPSC-derived MPCs will be tolerated in the presence of a competent human immune system.

Natural Killer Cells Prevent the Formation of Teratomas Derived From Human Induced Pluripotent Stem Cells.

The safe utilization of induced pluripotent stem cell (iPSC) derivatives in clinical use is attributed to the complete elimination of the risk of forming teratomas after transplantation. The extent by which such a risk exists in immune-competent hosts is mostly unknown. Here, using humanized mice reconstituted with fetal hematopoietic stem cells and autologous thymus tissue (bone-liver-thymus humanized mice [Hu-BLT]) or following the adoptive transfer of peripheral blood mononuclear cells(PBMCs) (Hu-AT), we evaluated the capacity of immune cells to prevent or eliminate teratomas derived from human iPSCs (hiPSCs). Our results showed that the injection of hiPSCs failed to form teratomas in Hu-AT mice reconstituted with allogeneic or autologous PBMCs or purified natural killer (NK) cells alone. However, teratomas were observed in Hu-AT mice reconstituted with autologous PBMCs depleted from NK cells. In line with these results, Hu-BLT, which do not have functional NK cells, could not prevent the growth of teratomas. Finally, we found that established teratomas were not targeted by NK cells and instead were efficiently rejected by allogeneic but not autologous T cells in Hu-AT mice. Overall, our findings suggest that autologous hiPSC-derived therapies are unlikely to form teratomas in the presence of NK cells.

Heterotopic bone formation derived from multipotent stromal cells is not inhibited in aged mice.

BACKGROUND AIMS: Decreased bone formation with age is believed to arise, at least in part, because of the influence of the senescent microenvironment. In this context, it is unclear whether multipotent stromal cell (MSC)-based therapies would be effective for the treatment of bone diseases. METHODS: With the use of a heterotopic bone formation model, we investigated whether MSC-derived osteogenesis is impaired in aged mice compared with young mice. RESULTS: We found that bone formation derived from MSCs is not reduced in aged mice. These results are supported by the unexpected finding that conditioned media collected from ionizing radiation-induced senescent MSCs can stimulate mineralization and delay osteoclastogenesis in vitro. CONCLUSIONS: Overall, our results suggest that impaired bone formation with age is mainly cell-autonomous and provide a rationale for the use of MSC-based therapies for the treatment of bone diseases in the elderly.

Targeted Gene Addition of Microdystrophin in Mice Skeletal Muscle via Human Myoblast Transplantation.

Zinc finger nucleases (ZFN) can facilitate targeted gene addition to the genome while minimizing the risks of insertional mutagenesis. Here, we used a previously characterized ZFN pair targeting the chemokine (C-C motif) receptor 5 (CCR5) locus to introduce, as a proof of concept, the enhanced green fluorescent protein (eGFP) or the microdystrophin genes into human myoblasts. Using integrase-defective lentiviral vectors (IDLVs) and chimeric adenoviral vectors to transiently deliver template DNA and ZFN respectively, we achieved up to 40% targeted gene addition in human myoblasts. When the O(6)-methylguanine-DNA methyltransferase(P140K) gene was co-introduced with eGFP, the frequency of cells with targeted integration could be increased to over 90% after drug selection. Importantly, gene-targeted myoblasts retained their mitogenic activity and potential to form myotubes both in vitro and in vivo when injected into the tibialis anterior of immune-deficient mice. Altogether, our results could lead to the development of improved cell therapy transplantation protocols for muscular diseases.Molecular Therapy - Nucleic Acids (2013) 2, e68; doi:10.1038/mtna.2012.55; published online 29 January 2013.

A soluble granulocyte colony stimulating factor decoy receptor as a novel tool to increase hematopoietic cell homing and reconstitution in mice.

The relative ineffectiveness of hematopoietic stem cells in reaching the bone marrow upon transplantation combined with the limited number of these cells available is a major reason for graft failure and delayed hematopoietic recovery. Hence, the development of strategies that could enhance homing is of high interest. Here, we provide evidence that homing is severely impaired postexposure to ionizing radiation (IR) in mice, an effect we found was time dependent and could be partially rescued using mesenchymal stromal cell (MSC) therapy. In an attempt to further increase homing, we took advantage of our observation that the granulocyte colony stimulating factor (G-CSF), a cytokine known to induce cell mobilization, is increased in the marrow of mice shortly after their exposure to IR. As such, we developed a truncated, yet functional, soluble G-CSF receptor (solG-CSFR), which we hypothesized could act as a decoy and foster homing. Using MSCs or conditioned media as delivery vehicles, we show that an engineered solG-CSFR has the potential to increase homing and hematopoietic reconstitution in mice. Altogether, our results provide novel findings at the interplay of IR and stromal cell therapy and present the regulation of endogenous G-CSF as an innovative proof-of-concept strategy to manipulate hematopoietic cell homing.

Targeted gene addition to human mesenchymal stromal cells as a cell-based plasma-soluble protein delivery platform.

BACKGROUND AIMS: Gene-modified mesenchymal stromal cells (MSC) provide a promising tool for cell and gene therapy-based applications by potentially acting as a cellular vehicle for protein-replacement therapy. However, to avoid the risk of insertional mutagenesis, targeted integration of a transgene into a 'safe harbor' locus is of great interest. METHODS: We sought to determine whether zinc finger nuclease (ZFN)-mediated targeted addition of the erythropoietin (Epo) gene into the chemokine [C-C motif] receptor 5 (CCR5) gene locus, a putative safe harbor locus, in MSC would result in stable transgene expression in vivo. RESULTS: Whether derived from bone marrow (BM), umbilical cord blood (UCB) or adipose tissue (AT), 30-40% of human MSC underwent ZFN-driven targeted gene addition, as determined by a combination of fluorescence-activated cell sorting (FACS)- and polymerase chain reaction (PCR)-based analyzes. An enzyme-linked immunosorbent assay (ELISA)-based analysis of gene-targeted MSC expressing Epo from the CCR5 locus showed that these modified MSC were found to secrete a significant level of Epo (c. 2 IU/10(6)cells/24 h). NOD/SCID/gammaC mice injected with ZFN-modified MSC expressing Epo exhibited significantly higher hematocrit and Epo plasma levels for several weeks post-injection, compared with mice receiving control MSC. CONCLUSIONS: These data demonstrate that MSC modified by ZFN-driven targeted gene addition may represent a cellular vehicle for delivery of plasma-soluble therapeutic factors.

A new pro-migratory activity on human myogenic precursor cells for a synthetic peptide within the E domain of the mechano growth factor.

Duchenne muscular dystrophy (DMD) is an inherited disease that leads to progressive muscle wasting. Myogenic precursor cell transplantation is an approach that can introduce the normal dystrophin gene in the muscle fibers of the patients. Unfortunately, these myogenic precursor cells do not migrate well in the muscle and thus many injections have to be done to enable a good graft success. Recent reports have shown that there is extensive splicing of the IGF-1 gene in muscles. The MGF isoform contains a C-terminal 24 amino acids peptide in the E domain (MGF-Ct24E) that has intrinsic properties. It can promote the proliferation while delaying the differentiation of C(2)C(12) cells. Here, we demonstrated that this synthetic peptide is a motogenic factor for human precursor myogenic cells in vitro and in vivo. Indeed, MGF-Ct24E peptide can modulate members of the fibrinolytic and metalloproteinase systems, which are implicated in the migration of myogenic cells. MGF-Ct24E peptide enhances the expression of u-PA, u-PAR and MMP-7 while reducing PAI-1 activity. Moreover, it has no effect on the gelatinases MMP-2 and -9. Those combined effects can favour cell migration. Finally, we present some results suggesting that the MGF-Ct24E peptide induces these cell responses through a mechanism that does not involve the IGF-1 receptor. Thus, this MGF-Ct24E peptide has a new pro-migratory activity on human myogenic precursor cells that may be helpful in the treatment of DMD. Those results reinforce the possibility that the IGF-1Ec isoform may produce an E domain peptide that can act as a cytokine.

Exercise improves the success of myoblast transplantation in mdx mice.

Transplantation of normal muscle precursor cells is a potential approach to restore dystrophin expression within dystrophin [deficient] mdx mice, a model of Duchenne Muscular Dystrophy. This study aims to evaluate whether exercise could improve graft success and hybrid fiber distribution within mdx muscle. eGFP(+) Muscle precursor cells were transplanted into tibialis anterior muscles of mdx mice using a single injection trajectory. During the following weeks, muscle fiber breaks were induced by making mdx mice swim. To evaluate fiber damage, Evans blue solution was injected intraperitoneally to mice 16h before their sacrifice. Tibialis anterior muscles were then harvested and eGFP, dystrophin and Evans blue labeling were analyzed by fluorescent microscopy. Twenty minutes of exercise (i.e., swimming) were used to induce damage in about 30% of TA muscle fibers. Graft success, evaluated as the percentage of hybrid fibers which are eGFP(+), was improved by 1.9-fold after swimming 3 times per week during 4 weeks and by 1.8-fold after daily swimming. Hybrid muscle fiber transversal and longitudinal distribution were also increased after repeated physical efforts. Exercise induced fiber breaks, which improved MPC recruitment and fusion and increased long-term graft success and also transverse and longitudinal distribution of hybrid fibers.

Real-time imaging of myoblast transplantation using the human sodium iodide symporter.

The quantification of the graft success is a key element to evaluate the efficiency of cellular therapies for several pathologies such as Duchenne muscular dystrophy. This study describes an approach to evaluate the success of myoblast transplantation (i.e., survival of the transplanted cells and the muscle fibers formed) by real-time imaging. C2C12 myoblasts were first transfected with a plasmid containing the human sodium iodide symporter (hNIS) gene. Specific uptake of the radioactive sodium pertechnetate (Na99mTcO4) by the hNIS-positive myoblasts was demonstrated in vitro, while only background level of Na99mTcO4 was observed within the control cells. The cells were then transplanted into the tibialis anterior (TA) muscle of mdx (X-linked dystrophic) mice. Following intraperitoneal administration of Na99mTcO4, scintigraphies were performed to detect hNIS-dependent Na99mTcO4 uptake within the TA. This approach permitted to evaluate the progression of the transplantation and the graft success without having to biopsy the animals during the follow-up period.

Myoblast survival enhancement and transplantation success improvement by heat-shock treatment in mdx mice.

BACKGROUND: Duchenne muscular dystrophy is a disease caused by the incapacity to synthesize dystrophin, which is implicated in the maintenance of the sarcolemma integrity. Myoblast transplantation is a potential treatment of this disease. However, most of the transplanted cells die very rapidly after their injection. Heat-shock proteins (HSPs) are over-expressed when cells undergo various types of stresses. Our goal was thus to investigate whether the expression of HSPs (HSP70 in particular) could protect myoblasts from death after intramuscular injection. METHODS: HSP70 expression was induced by warming the cells at 42 degrees C for 60 minutes. HSP70 over-expression was quantified by Western blot analysis. The in vitro effect of HSPs on cell survival was evaluated by fluorescence-activated cell sorter analysis using the Hoescht/propidium iodide-labeling technique, and their in vivo effects were investigated by transplanting TnI-LacZ myoblasts labeled with [methyl-14C] thymidine. RESULTS: Western blots indicated a sevenfold over-expression of the HSP70 after the heat-shock treatment. In vitro, the heat-shock treatment protected 18% of the cells from staurosporine- (1 microM) induced apoptosis. HSPs also protected 10% of the cells from death induced by either tumor necrosis factor-alpha (30 ng/mL) or glucose oxydase (0.1 U/mL). In vivo, the treatment improved the cell survival by twofold 5 days after the graft and increased by fourfold the long-term graft success. CONCLUSIONS: The heat-shock treatment is a practical approach for improving the success of myoblast transplantation; in fact, using this kind of treatment, there is no need to genetically modify the cells before their transplantation.