Characterization of the Nonendocrine Cell Populations in Human Embryonic Stem Cell–Derived (hESC) Islet-Like Clusters Posttransplantation

Islet-like clusters derived from human embryonic stem cells (hESC) hold the potential to cure type 1 diabetes mellitus. Differentiation protocols of islet-like clusters lead to the generation of minor fractions of nonendocrine cells, which are mainly from endodermal and mesodermal lineages, and the risk of implanting these is unclear. In the present study, the histogenesis and the tumorigenicity of nonendocrine cells were investigated in vivo. Immunodeficient mice were implanted under the kidney capsule with islet-like clusters which were derived from differentiation of cells batches with either an intermediate or poor cell purity and followed for 8 or 26 weeks. Using immunohistochemistry and other techniques, it was found that the intermediate differentiated cell implants had limited numbers of small duct-like cysts and nonpancreatic tissue resembling gastrointestinal and retinal pigmented epithelium. In contrast, highly proliferative cystic teratomas were found at a high incidence at the implant site after 8 weeks, only in the animals implanted with the poorly differentiated cells. These findings indicate that the risk for teratoma formation and the amount of nonpancreatic tissue can be minimized by careful in-process characterization of the cells and thus highlights the importance of high purity at transplantation and a thorough ex-vivo characterization during cell product development.

Modaline sulfate promotes Oct4 expression and maintains self-renewal and pluripotency of stem cells through JAK/STAT3 and Wnt signaling pathways

Background Stem cells have been extensively explored for a variety of regenerative medical applications and they play an important role in clinical treatment of many diseases. However, the limited amount of stem cells and their tendency to undergo spontaneous differentiation upon extended propagation in vitro restrict their practical application. Octamer-binding transcription factor-4 (Oct4), a transcription factor belongs to the POU transcription factor family Class V, is fundamental for maintaining self-renewal ability and pluripotency of stem cells. Methods In the present study, we used the previously constructed luciferase reporters driven by the promoter and 3’-UTR of Oct4 respectively to screen potential activators of Oct4. Colony formation assay, sphere-forming ability assay, alkaline phosphatase (AP) activity assay and teratoma-formation assay were used to assess the role of modaline sulfate (MDLS) in promoting self-renewal and reinforcing pluripotency of P19 cells. Immunofluorescence, RT-PCR, and western blotting were used to measure expression changes of stem-related genes and activation of related signaling pathways. Results We screened 480 commercially available small-molecule compounds and discovered that MDLS greatly promoted the expression of Oct4 at both mRNA and protein levels. Moreover, MDLS significantly promoted the self-renewal capacity of P19 cells. Also, we observed that the expression of pluripotency markers and alkaline phosphatase (AP) increased significantly in MDLS-treated colonies. Furthermore, MDLS could promote teratoma formation and enhanced differentiation potential of P19 cells in vivo. In addition, we found that in the presence of LIF, MDLS could replace feeder cells to maintain the undifferentiated state of OG2-mES cells (Oct4-GFP reporter gene mouse embryonic stem cell line), and the MDLS-expanded OG2-mES cells showed an elevated expression levels of pluripotency markers in vitro. Finally, we found that MDLS promoted Oct4 expression by activating JAK/STAT3 and classic Wnt signaling pathways, and these effects were reversed by treatment with inhibitors of corresponding signaling pathways. Conclusions These findings demonstrated, for the first time, that MDLS could maintain self-renewal and pluripotency of stem cells.

Distinctive Clinical and Pathologic Features of Immature Teratomas Arising from Induced Pluripotent Stem Cell Injection in a Patient with type 2 Diabetes: A Case Report

Background: As a new potential cure for diabetes, induced pluripotent stem cells (iPSCs) are characterized by self-renewal capacity and the ability to differentiate into pancreatic islet β-cells, which will hopefully secrete insulin and rebuild blood glucose balance. The safety and validity of iPSC treatment for diabetes remain controversial. One of the most serious risks is teratoma formation arising from undifferentiated stem cells, but clinical reports are rare.Case presentation: Here, we report a distinctive case of immature teratoma after iPSC treatment for diabetes, which was accidentally treated in our soft tissue sarcoma centre. The patient received islet β-cell injection, in which the cells were differentiated from autologous iPSCs, into the deltoideus muscle. Two months later, a mass located in the injection area formed with enlarged axillary lymph nodes. Here, we present the different clinical, radiological and pathological features of this immature teratoma in detail. Distinctive from typical immature teratomas, the tumour was characterized by rapid growth and local lymph node metastasis. The tumour was not sensitive to typical chemotherapy regimens for immature teratomas. MRI scanning showed heterogeneous enhancement and a rich blood supply to the tumour. The histopathology showed immature endoderm, mesoderm and ectoderm tissues composed of osseous, cartilaginous, vascular and adenoid tissues, which have more cellular atypia than typical teratomas. Staining for both Oct-4 and Sox-2 was positive in the tumour cell nucleus by immunofluorescence assay, but insulin staining was negative. Next-generation sequencing showed many missense mutations, but abnormal gene rearrangement or copy number was not observed.Conclusions: More attention should be given to teratoma formation after iPSC treatment for diabetes, which is more aggressive than typical teratomas. The safety and validity of iPSC treatment for diabetes should be confirmed by more standardized clinical trials.

Distinctive Clinical and Pathologic Features of Immature Teratomas Arising From Induced Pluripotent Stem Cell Injection in a Patient With Type 2 Diabetes

As a new potential cure for diabetes, induced pluripotent stem cells (iPSCs) are characterized by self-renewal capacity and the ability to differentiate into pancreatic islet β-cells, which will hopefully secrete insulin and rebuild blood glucose balance. The safety and validity of iPSC treatment for diabetes remain controversial. One of the most serious risks is teratoma formation arising from undifferentiated stem cells, but clinical reports are rare. Here, we report a distinctive case of immature teratoma after iPSC treatment for diabetes, which was accidentally treated in our soft tissue sarcoma centre. The patient received islet β-cell injection, in which the cells were differentiated from autologous iPSCs, into the deltoideus muscle. Two months later, a mass located in the injection area formed with enlarged axillary lymph nodes. Here, we present the different clinical, radiological and pathological features of this immature teratoma in detail. Distinctive from typical immature teratomas, the tumour was characterized by rapid growth and local lymph node metastasis. The tumour was not sensitive to typical chemotherapy regimens for immature teratomas. MRI scanning showed heterogeneous enhancement and a rich blood supply to the tumour. The histopathology showed immature endoderm, mesoderm and ectoderm tissues composed of osseous, cartilaginous, vascular and adenoid tissues, which have more cellular atypia than typical teratomas. Staining for both Oct-4 and Sox-2 was positive in the tumour cell nucleus by immunofluorescence assay, but insulin staining was negative. Next-generation sequencing showed many missense mutations, but abnormal gene rearrangement or copy number was not observed. In conclusion, more attention should be given to teratoma formation after iPSC treatment for diabetes, which is more aggressive than typical teratomas. The safety and validity of iPSC treatment for diabetes should be confirmed by more standardized clinical trials.

GP2-enriched pancreatic progenitors give rise to functional beta cells in vivo and eliminate the risk of teratoma formation

Human pluripotent stem cell (hPSC)-derived pancreatic progenitors (PPs) can be differentiated into beta-like cells in vitro and in vivo, and therefore have therapeutic potential for type 1 diabetes (T1D) treatment. However, the purity of PPs varies across different hPSC lines, differentiation protocols and laboratories. The uncommitted cells may give rise to non-pancreatic endodermal, mesodermal, or ectodermal derivatives in vivo, hampering the safety of hPSC-derived PPs for clinical applications. Recently, proteomics and transcriptomics analyses identified glycoprotein 2 (GP2) as a PP-specific cell surface marker. The GP2-enriched PPs generate higher percentages of beta-like cells in vitro compared to unsorted and GP2- fractions, but their potential in vivo remains to be elucidated. Here, we demonstrate that the GP2-enriched-PPs give rise to all pancreatic cells in vivo, including functional beta-like cells. Remarkably, GP2 enrichment eliminated the formation of teratoma in vivo. This study establishes that the GP2-enriched PPs represent a safe option for T1D treatment.

Stability of Imprinting and Differentiation Capacity in Naïve Human Cells Induced by Chemical Inhibition of CDK8 and CDK19

Pluripotent stem cells can be stabilized in vitro at different developmental states by the use of specific chemicals and soluble factors. The naïve and primed states are the best characterized pluripotency states. Naïve pluripotent stem cells (PSCs) correspond to the early pre-implantation blastocyst and, in mice, constitute the optimal starting state for subsequent developmental applications. However, the stabilization of human naïve PSCs remains challenging because, after short-term culture, most current methods result in karyotypic abnormalities, aberrant DNA methylation patterns, loss of imprinting and severely compromised developmental potency. We have recently developed a novel method to induce and stabilize naïve human PSCs that consists in the simple addition of a chemical inhibitor for the closely related CDK8 and CDK19 kinases (CDK8/19i). Long-term cultured CDK8/19i-naïve human PSCs preserve their normal karyotype and do not show widespread DNA demethylation. Here, we investigate the long-term stability of allele-specific methylation at imprinted loci and the differentiation potency of CDK8/19i-naïve human PSCs. We report that long-term cultured CDK8/19i-naïve human PSCs retain the imprinting profile of their parental primed cells, and imprints are further retained upon differentiation in the context of teratoma formation. We have also tested the capacity of long-term cultured CDK8/19i-naïve human PSCs to differentiate into primordial germ cell (PGC)-like cells (PGCLCs) and trophoblast stem cells (TSCs), two cell types that are accessible from the naïve state. Interestingly, long-term cultured CDK8/19i-naïve human PSCs differentiated into PGCLCs with a similar efficiency to their primed counterparts. Also, long-term cultured CDK8/19i-naïve human PSCs were able to differentiate into TSCs, a transition that was not possible for primed PSCs. We conclude that inhibition of CDK8/19 stabilizes human PSCs in a functional naïve state that preserves imprinting and potency over long-term culture.

MC4R mutant mice develop ovarian teratomas

Teratomas in mice, composed of different tissue types, are derived from primordial germ cells (PGCs) in the foetal gonads. The strongest candidate gene in the testicular teratoma locus (Ter) responsible for testicular teratoma formation was identified as mutation in Dnd1, Dnd1R178*. However, the phenotype of mice with a mutated Dnd1 gene was germ cell loss. This suggests that other genes are involved in teratoma formation. Testicular teratomas can also be induced experimentally (experimentally testicular teratomas: ETTs) in 129/Sv mice by transplanting E12.5 foetal testes into adult testes. Previously, we mapped the ett1 locus, which is the locus responsible for ETT formation on chromosome 18. By exome sequence analysis of the 129 and LTXBJ (LT) strains, we identified a missense mutation in the melanocortin 4 receptor (MC4R) gene among 8 genes in the ett1 region. The missense mutation causes a substitution of glycine 25 by serine. Thus, this gene is a candidate for ETT formation. We established the LT-ett1 congenic strain, which introduced the locus responsible for ETT formation genetically into the genomes of a testicular teratoma non-susceptible strain. In this study, we crossed LT-ett1 and a previously established LT-Ter strain to establish the double congenic strain LT-Ter-ett1. Also, we established a strain with a point mutation in the MC4R gene of the LT strain by genome editing, LT-MC4RG25S. Furthermore, double genetically modified strain LT-Ter-MC4RG25S was established to address the relation between Ter and MC4R. Surprisingly, highly developed ovarian teratomas (OTs), instead of testicular teratomas, appeared not only in the LT-Ter-MC4RG25S and LT-MC4RG25S strains but also in the LT-ett1 and LT-Ter-ett1 strains. The incidence of OT formation was high in double genetically modified strains. The results demonstrated that MC4R is one of the genes responsible for OT formation. It was suggested that the effect of the missense mutation in MC4R on teratoma formation was promoted by abnormal germ cell formation by the mutation in DND1.

Salicylic diamines selectively eliminate residual undifferentiated cells from pluripotent stem cell-derived cardiomyocyte preparations

Clinical translation of pluripotent stem cell (PSC) derivatives is hindered by the tumorigenic risk from residual undifferentiated cells. Here, we identified salicylic diamines as potent agents exhibiting toxicity to murine and human PSCs but not to cardiomyocytes (CMs) derived from them. Half maximal inhibitory concentrations (IC50) of small molecules SM2 and SM6 were, respectively, 9- and 18-fold higher for human than murine PSCs, while the IC50 of SM8 was comparable for both PSC groups. Treatment of murine embryoid bodies in suspension differentiation cultures with the most effective small molecule SM6 significantly reduced PSC and non-PSC contamination and enriched CM populations that would otherwise be eliminated in genetic selection approaches. All tested salicylic diamines exerted their toxicity by inhibiting the oxygen consumption rate (OCR) in PSCs. No or only minimal and reversible effects on OCR, sarcomeric integrity, DNA stability, apoptosis rate, ROS levels or beating frequency were observed in PSC-CMs, although effects on human PSC-CMs seemed to be more deleterious at higher SM-concentrations. Teratoma formation from SM6-treated murine PSC-CMs was abolished or delayed compared to untreated cells. We conclude that salicylic diamines represent promising compounds for PSC removal and enrichment of CMs without the need for other selection strategies.