Mesodermal Derivatives of Pluripotent Stem Cells Route to Scarless Healing.

Scar formation during normal tissue regeneration in adults may result in noticeable cosmetic and functional defects and have a significant impact on the quality of life. In contrast, fetal tissues in the mid-gestation period are known to be capable of complete regeneration with the restitution of the initial architecture, organization, and functional activity. Successful treatments that are targeted to minimize scarring can be realized by understanding the cellular and molecular mechanisms of fetal wound regeneration. However, such experiments are limited by the inaccessibility of fetal material for comparable studies. For this reason, the molecular mechanisms of fetal regeneration remain unknown. Mesenchymal stromal cells (MSCs) are central to tissue repair because the molecules they secrete are involved in the regulation of inflammation, angiogenesis, and remodeling of the extracellular matrix. The mesodermal differentiation of human pluripotent stem cells (hPSCs) recapitulates the sequential steps of embryogenesis in vitro and provides the opportunity to generate the isogenic cell models of MSCs corresponding to different stages of human development. Further investigation of the functional activity of cells from stromal differon in a pro-inflammatory microenvironment will procure the molecular tools to better understand the fundamental mechanisms of fetal tissue regeneration. Herein, we review recent advances in the generation of clonal precursors of primitive mesoderm cells and MSCs from hPSCs and discuss critical factors that determine the functional activity of MSCs-like cells in a pro-inflammatory microenvironment in order to identify therapeutic targets for minimizing scarring.

Prognostic Analysis of Human Pluripotent Stem Cells Based on Their Morphological Portrait and Expression of Pluripotent Markers.

The ability of human pluripotent stem cells for unlimited proliferation and self-renewal promotes their application in the fields of regenerative medicine. The morphological assessment of growing colonies and cells, as a non-invasive method, allows the best clones for further clinical applications to be safely selected. For this purpose, we analyzed seven morphological parameters of both colonies and cells extracted from the phase-contrast images of human embryonic stem cell line H9, control human induced pluripotent stem cell (hiPSC) line AD3, and hiPSC line HPCASRi002-A (CaSR) in various passages during their growth for 120 h. The morphological phenotype of each colony was classified using a visual analysis and associated with its potential for pluripotency and clonality maintenance, thus defining the colony phenotype as the control parameter. Using the analysis of variance for the morphological parameters of each line, we showed that selected parameters carried information about different cell lines and different phenotypes within each line. We demonstrated that a model of classification of colonies and cells by phenotype, built on the selected parameters as predictors, recognized the phenotype with an accuracy of 70-75%. In addition, we performed a qRT-PCR analysis of eleven pluripotency markers genes. By analyzing the variance of their expression in samples from different lines and with different phenotypes, we identified group-specific sets of genes that could be used as the most informative ones for the separation of the best clones. Our results indicated the fundamental possibility of constructing a morphological portrait of a colony informative for the automatic identification of the phenotype and for linking this portrait to the expression of pluripotency markers.

Aberrant Splicing of INS Impairs Beta-Cell Differentiation and Proliferation by ER Stress in the Isogenic iPSC Model of Neonatal Diabetes.

One of the causes of diabetes in infants is the defect of the insulin gene (INS). Gene mutations can lead to proinsulin misfolding, an increased endoplasmic reticulum (ER) stress and possible beta-cell apoptosis. In humans, the mechanisms underlying beta-cell failure remain unclear. We generated induced pluripotent stem cells (iPSCs) from a patient diagnosed with neonatal diabetes mellitus carrying the INS mutation in the 2nd intron (c.188-31G>A) and engineered isogenic CRISPR/Cas9 mutation-corrected cell lines. Differentiation into beta-like cells demonstrated that mutation led to the emergence of an ectopic splice site within the INS and appearance of the abnormal RNA transcript. Isogenic iPSC lines differentiated into beta-like cells showed a clear difference in formation of organoids at pancreatic progenitor stage of differentiation. Moreover, MIN6 insulinoma cell line expressing mutated cDNA demonstrated significant decrease in proliferation capacity and activation of ER stress and unfolded protein response (UPR)-associated genes. These findings shed light on the mechanism underlying the pathogenesis of monogenic diabetes.

Equilibrium among Inflammatory Factors Determines Human MSC-Mediated Immunosuppressive Effect.

Mesenchymal stem cells (MSCs) are thought to be a promising therapeutic agent due to their multiple paracrine and immunomodulatory properties, providing protection from chronic inflammation and promoting tissue repair. MSCs can regulate the balance of pro-inflammatory and anti-inflammatory factors in inflamed tissues, creating a microenvironment necessary for successful healing; however, their interactions with immune cells are still poorly studied. We examined the temporal and spatial changes in gene regulation and the paracrine milieu accompanying the MSC-mediated immunosuppression effect in mixed cultures with activated peripheral blood mononuclear cells (PBMCs). Our data reveal that the peak of suppression of PBMC proliferation was achieved within 48 h following co-culture with MSCs and subsequently did not undergo a significant change. This effect was accompanied by an increase in COX-2 expression and an induction of IDO synthesis in MSCs. At this point, the expression of IL-1, IL-6, IL-8, IFN-γ, MCP-1, and G-CSF was upregulated in co-cultured cells. On the contrary, we observed a decrease in the concentrations of IL-10, IL-13, IL-5, and MIP-1b in co-culture supernatants compared to intact cultures of activated PBMCs. The regulation of IDO, IL-1, IL-6, and G-CSF production was accomplished with the involvement of direct cell-cell contact between MSCs and PBMCs. These findings provide new insights into the use of potential precondition inducers or their combinations to obtain functionally qualified MSCs for more effective treatment of inflammatory diseases.

A Novel Isogenic Human Cell-Based System for MEN1 Syndrome Generated by CRISPR/Cas9 Genome Editing.

Multiple endocrine neoplasia type 1 (MEN1) is a rare tumor syndrome that manifests differently among various patients. Despite the mutations in the MEN1 gene that commonly predispose tumor development, there are no obvious phenotype-genotype correlations. The existing animal and in vitro models do not allow for studies of the molecular genetics of the disease in a human-specific context. We aimed to create a new human cell-based model, which would consider the variability in genetic or environmental factors that cause the complexity of MEN1 syndrome. Here, we generated patient-specific induced pluripotent stem cell lines carrying the mutation c.1252G>T, D418Y in the MEN1 gene. To reduce the genetically determined variability of the existing cellular models, we created an isogenic cell system by modifying the target allele through CRISPR/Cas9 editing with great specificity and efficiency. The high potential of these cell lines to differentiate into the endodermal lineage in defined conditions ensures the next steps in the development of more specialized cells that are commonly affected in MEN1 patients, such as parathyroid or pancreatic islet cells. We anticipate that this isogenic system will be broadly useful to comprehensively study MEN1 gene function across different contexts, including in vitro modeling of MEN1 syndrome.

Generation of an induced pluripotent stem cell line HPCASRi002-A from a patient with neonatal severe primary hyperparathyroidism caused by a compound heterozygous mutation in the CASR gene.

Neonatal severe primary hyperparathyroidism (NSHPT) is a rare autosomal recessive disorder of calcium homeostasis that manifests shortly after birth with hypercalcemia and bone disease. NSHPT, in most cases, is attributed to mutations in the calcium-sensing receptor (CASR) gene. We reprogrammed dermal fibroblasts derived from a patient with NSHPT carrying a compound heterozygous mutation in the CASR gene into induced pluripotent stem cells (iPSCs). The established iPSCs expressed pluripotency markers, maintained normal karyotype and differentiated into all three germ layers. This line is a valuable resource for modeling of hyperparathyroidism related to CASR mutations.

Generation of an induced pluripotent stem cell line MNDINSi001-A from a patient with neonatal diabetes caused by a heterozygous INS mutation.

Insulin gene (INS) mutations prove to be the second most common cause of permanent neonatal diabetes. Here, we report the generation of iPSC line from a patient, heterozygous for the intronic INS mutation that presumably leads to aberrant splicing. Dermal fibroblasts were reprogrammed using non-integrating RNA-based vector. Derivation and expansion of iPSCs were performed under feeder-free culture conditions. The iPSC line expressed pluripotency markers, had normal karyotype, could differentiate into three germ layers in vitro and retained the disease mutation. This line can be a powerful tool for modeling of diabetes and cell replacement therapy as well.

Locally Delivered Umbilical Cord Mesenchymal Stromal Cells Reduce Chronic Inflammation in Long-Term Nonhealing Wounds: A Randomized Study.

Inflammation is part of a complex biological response to injury that mediates a rapid mobilization of cells and triggers the restoration of tissue homeostasis. The systemic diseases of the connective tissues, repetitive strain injuries, neuropathy, and vascular impairment lead to the development of a chronic inflammatory state. In such cases, a forced intervention is required to trigger tissue regeneration. Mesenchymal stromal cells (MSCs) have been considered a perspective tool for regenerative medicine because of their ability to change the expression and secretory profile under the influence of signals from the microenvironment to perform a regulatory function at the site of tissue damage. In this study, MSCs were isolated from the human umbilical cord (UCMSCs). The ability of UCMSCs to regulate chronic inflammation was investigated in a randomized placebo-controlled pilot study to assess the efficacy and safety of UCMSC therapy in patients with nonhealing wounds. A total of 108 patients with chronic wounds of different etiologies were randomly divided into two groups according to the criteria of inclusion and exclusion. The group (n = 59) that was treated with a single local subcutaneous infusion of UCMSCs around the wound periphery showed a pronounced growth of granulation tissue, improved blood microcirculation, and reduction in wound size compared to the placebo group (n = 49). No prominent adverse events were detected in patients from the UCMSC group during the 1-year follow-up period. This research has demonstrated that locally delivered allogeneic UCMSCs can contribute to chronic wound repair and provide an additional support toward new therapeutic strategies. Registration certificate №FS2006/341 was issued by the Federal Service for Surveillance in Healthcare.

Patient-Specific iPSC-Based Models of Huntington's Disease as a Tool to Study Store-Operated Calcium Entry Drug Targeting.

Neurodegenerative pathologies are among the most serious and socially significant problems of modern medicine, along with cardiovascular and oncological diseases. Several attempts have been made to prevent neuronal death using novel drugs targeted to the cell calcium signaling machinery, but the lack of adequate models for screening markedly impairs the development of relevant drugs. A potential breakthrough in this field is offered by the models of hereditary neurodegenerative pathologies based on endogenous expression of mutant proteins in neurons differentiated from patient-specific induced pluripotent stem cells (iPSCs). Here, we study specific features of store-operated calcium entry (SOCE) using an iPSCs-based model of Huntington's disease (HD) and analyze the pharmacological effects of a specific drug targeted to the calcium channels. We show that SOCE in gamma aminobutyric acid-ergic striatal medium spiny neurons (GABA MSNs) was mediated by currents through at least two different channel groups, ICRAC and ISOC. Both of these groups were upregulated in HD neurons compared with the wild-type neurons. Thapsigargin-induced intracellular calcium store depletion in GABA MSNs resulted in predominant activation of either ICRAC or ISOC. The potential anti-HD drug EVP4593, which was previously shown to have neuroprotective activity in different HD models, affected both ICRAC and ISOC.

Epigenetic reprogramming by naïve conditions establishes an irreversible state of partial X chromosome reactivation in female stem cells.

Female human pluripotent stem cells (PSCs) have variable X-chromosome inactivation (XCI) status. One of the X chromosomes may either be inactive (Xi) or display some active state markers. Long-term cultivation of PSCs may lead to an erosion of XCI and partial X reactivation. Such heterogeneity and instability of XCI status might hamper the application of human female PSCs for therapy or disease modeling. We attempted to address XCI heterogeneity by reprogramming human embryonic stem cells (hESCs) to the naïve state. We propagated five hESC lines under naïve culture conditions. PSCs acquired naïve cells characteristics although these changes were not uniform for all of the hESC lines. Transition to the naïve state was accompanied by a loss of XIST expression, loss of Xi H3K27me3 enrichment and a switch in Xi replication synchronously with active X, except for two regions. This pattern of Xi reactivation was observed in all cells in two hESC lines. However, these cells were unable to undergo classical XCI upon spontaneous differentiation. We conclude that naïve culture conditions do not resolve the variability in XCI status in female human ESC lines and establish an irreversible heterogeneous pattern of partial X reactivation.

Mitochondrial distribution violation and nuclear indentations in neurons differentiated from iPSCs of Huntington's disease patients.

AIM: Huntington's disease (HD) is an inherited disease caused by an expansion of cytosine-adenine-guanine (CAG) repeats in the huntingtin gene (HTT) that ultimately leads to neurodegeneration. To study the molecular basis of this disease, induced pluripotent stem cells (iPSCs) generated from patients' fibroblasts were used to investigate axonal mitochondrial trafficking and the nature of nuclear indentations. METHODS: Pathological and control iPSCs generated from patients with a low number of repeats were differentiated in striatal neurons of the brain. Mitochondrial density was measured along the axon using tubulin beta 3 co-staining in pathological and control neurons. To investigate the connection of nuclear roundness with calcium dysregulation, several calcium inhibitors were used. Proteasome system inhibition was applied to mimic premature neuronal ageing. RESULTS: We found that the mitochondrial density was approximately 7.6 ± 0.2 in neurites in control neurons but was only 5.3 ± 0.2 in mutant neurons with 40-44 CAG repeats (p-value <0.005). Neuronal ageing induced by proteasome inhibitor MG132 significantly decreased the mitochondrial density by 15% and 25% in control and mutant neurons to 6.5 ± 0.1 (p-value < 0.005) and 4.0 ± 0.3 (p-value < 0.005), respectively. Thus, for the first time, an impairment of mitochondrial trafficking in pathological neurons with endogenous mutant huntingtin was demonstrated. We found that inhibiting the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), the ryanodine-receptor (RyR) or the inositol 1,4,5-trisphosphate receptor (IP3R) by specific inhibitors did not specifically affect the nuclear roundness or survival of pathological neurons differentiated from patient iPSCs. Therefore, nuclear calcium homeostasis is not directly associated with HD pathology. CONCLUSION: Identifying HD iPSCs and differentiating from them neurons provide a unique system for modelling the disease in vitro. Impairments of mitochondrial trafficking and nuclear roundness manifest long before the disease onset, while premature neuronal ageing enhances differences in mitochondrial distribution.

Manifestation of Huntington's disease pathology in human induced pluripotent stem cell-derived neurons.

BACKGROUND: Huntington's disease (HD) is an incurable hereditary neurodegenerative disorder, which manifests itself as a loss of GABAergic medium spiny (GABA MS) neurons in the striatum and caused by an expansion of the CAG repeat in exon 1 of the huntingtin gene. There is no cure for HD, existing pharmaceutical can only relieve its symptoms. RESULTS: Here, induced pluripotent stem cells were established from patients with low CAG repeat expansion in the huntingtin gene, and were then efficiently differentiated into GABA MS-like neurons (GMSLNs) under defined culture conditions. The generated HD GMSLNs recapitulated disease pathology in vitro, as evidenced by mutant huntingtin protein aggregation, increased number of lysosomes/autophagosomes, nuclear indentations, and enhanced neuronal death during cell aging. Moreover, store-operated channel (SOC) currents were detected in the differentiated neurons, and enhanced calcium entry was reproducibly demonstrated in all HD GMSLNs genotypes. Additionally, the quinazoline derivative, EVP4593, reduced the number of lysosomes/autophagosomes and SOC currents in HD GMSLNs and exerted neuroprotective effects during cell aging. CONCLUSIONS: Our data is the first to demonstrate the direct link of nuclear morphology and SOC calcium deregulation to mutant huntingtin protein expression in iPSCs-derived neurons with disease-mimetic hallmarks, providing a valuable tool for identification of candidate anti-HD drugs. Our experiments demonstrated that EVP4593 may be a promising anti-HD drug.

An integrative analysis of reprogramming in human isogenic system identified a clone selection criterion.

The pluripotency of newly developed human induced pluripotent stem cells (iPSCs) is usually characterized by physiological parameters; i.e., by their ability to maintain the undifferentiated state and to differentiate into derivatives of the 3 germ layers. Nevertheless, a molecular comparison of physiologically normal iPSCs to the "gold standard" of pluripotency, embryonic stem cells (ESCs), often reveals a set of genes with different expression and/or methylation patterns in iPSCs and ESCs. To evaluate the contribution of the reprogramming process, parental cell type, and fortuity in the signature of human iPSCs, we developed a complete isogenic reprogramming system. We performed a genome-wide comparison of the transcriptome and the methylome of human isogenic ESCs, 3 types of ESC-derived somatic cells (fibroblasts, retinal pigment epithelium and neural cells), and 3 pairs of iPSC lines derived from these somatic cells. Our analysis revealed a high input of stochasticity in the iPSC signature that does not retain specific traces of the parental cell type and reprogramming process. We showed that 5 iPSC clones are sufficient to find with 95% confidence at least one iPSC clone indistinguishable from their hypothetical isogenic ESC line. Additionally, on the basis of a small set of genes that are characteristic of all iPSC lines and isogenic ESCs, we formulated an approach of "the best iPSC line" selection and confirmed it on an independent dataset.

Reactivation of Х chromosome upon reprogramming leads to changes in the replication pattern and 5hmC accumulation.

Once set, the inactive status of the X chromosome in female somatic cells is preserved throughout subsequent cell divisions. The inactive status of the X chromosome is characterized by many features, including late replication. In contrast to induced pluripotent stem cells (iPSCs) in mice, the X chromosome in human female iPSCs usually remains inactive after reprogramming of somatic cells to the pluripotent state, although recent studies point to the possibility of reactivation of the X chromosome. Here, we demonstrated that, during reprogramming, the inactive X chromosome switches from late to synchronous replication, with restoration of the transcription of previously silenced genes. This process is accompanied by accumulation of a new epigenetic mark or intermediate of the DNA demethylation pathway, 5-hydroxymethylcytosine (5hmC), on the activated X chromosome. Our results indicate that the active status of the X chromosome is better confirmed by early replication and the reappearance of 5hmC, rather than by appearance of histone marks of active chromatin, removal of histone marks of inactive chromatin, or an absence of XIST coating.

Current progress and potential practical application for human pluripotent stem cells.

Pluripotent stem cells are able to give rise to all cell types of the organism. There are two sources for human pluripotent stem cells: embryonic stem cells (ESCs) derived from surplus blastocysts created for in vitro fertilization and induced pluripotent stem cells (iPSCs) generated by reprogramming of somatic cells. ESCs have been an area of intense research during the past decade, and two clinical trials have been recently approved. iPSCs were created only recently, and most of the research has been focused on the iPSC generation protocols and investigation of mechanisms of direct reprogramming. The iPSC technology makes possible to derive pluripotent stem cells from any patient. However, there are a number of hurdles to be overcome before iPSCs will find a niche in practice. In this review, we discuss differences and similarities of the two pluripotent cell types and assess prospects for application of these cells in biomedicine.

Error-prone nonhomologous end joining repair operates in human pluripotent stem cells during late G2.

Genome stability of human embryonic stem cells (hESC) is an important issue because even minor genetic alterations can negatively impact cell functionality and safety. The incorrect repair of DNA double-stranded breaks (DSBs) is the ultimate cause of the formation of chromosomal aberrations. Using G2 radiosensitivity assay, we analyzed chromosomal aberrations in pluripotent stem cells and somatic cells. The chromatid exchange aberration rates in hESCs increased manifold 2 hours after irradiation as compared with their differentiated derivatives, but the frequency of radiation-induced chromatid breaks was similar. The rate of radiation-induced chromatid exchanges in hESCs and differentiated cells exhibited a quadratic dose response, revealing two-hit mechanism of exchange formation suggesting that a non-homologous end joining (NHEJ) repair may contribute to their formation. Inhibition of DNA-PK, a key NHEJ component, by NU7026 resulted in a significant decrease in radiation-induced chromatid exchanges in hESCs but not in somatic cells. In contrast, NU7026 treatment increased the frequency of radiation-induced breaks to a similar extent in pluripotent and somatic cells. Thus, DNA-PK dependent NHEJ efficiently participates in the elimination of radiation-induced chromatid breaks during the late G2 in both cell types and DNA-PK activity leads to a high level of misrejoining specifically in pluripotent cells.

Induction of pluripotency in human endothelial cells resets epigenetic profile on genome scale.

Reprogramming of a limited number of human cell types has been achieved through ectopic expression of four transcription factors to yield induced pluripotent stem (iPS) cells that closely resemble human embryonic stem cells (ESCs). Here, we determined functional and epigenetic properties of iPS cells generated from human umbilical vein endothelial cells (HUVEC) by conventional method of direct reprogramming. Retroviral overexpression of four transcription factors resets HUVEC to the pluripotency. Human endothelial cell-derived iPS (endo-iPS) cells were similar to human ESCs in morphology, gene expression, in vitro and in vivo differentiation capacity. Endo-iPS cells were efficiently differentiated in vitro into endothelial cells. Using genome-wide methylation profiling we show that promoter elements of endothelial specific genes were methylated following reprogramming whereas pluripotency-related gene promoters were hypomethylated similar to levels observed in ESCs. Genome-wide methylation analysis of CpG sites located in the functional regions of over than 14,000 genes indicated that human endo-iPS cells were highly similar to human ES cells, although differences in methylation levels of 46 genes were found. Overall CpG methylation of promoter regions in the pluripotent cells was higher than in somatic. We also show that during reprogramming female human endo-iPS cells exhibited reactivation of the somatically silenced X chromosome. Our findings demonstrate that iPS cells can be generated from human endothelial cells and reprogramming resets epigenetic status of endothelial cells to pluripotency.