On-Chip Neural Induction Boosts Neural Stem Cell Commitment: Toward a Pipeline for iPSC-Based Therapies.

The clinical translation of induced pluripotent stem cells (iPSCs) holds great potential for personalized therapeutics. However, one of the main obstacles is that the current workflow to generate iPSCs is expensive, time-consuming, and requires standardization. A simplified and cost-effective microfluidic approach is presented for reprogramming fibroblasts into iPSCs and their subsequent differentiation into neural stem cells (NSCs). This method exploits microphysiological technology, providing a 100-fold reduction in reagents for reprogramming and a ninefold reduction in number of input cells. The iPSCs generated from microfluidic reprogramming of fibroblasts show upregulation of pluripotency markers and downregulation of fibroblast markers, on par with those reprogrammed in standard well-conditions. The NSCs differentiated in microfluidic chips show upregulation of neuroectodermal markers (ZIC1, PAX6, SOX1), highlighting their propensity for nervous system development. Cells obtained on conventional well plates and microfluidic chips are compared for reprogramming and neural induction by bulk RNA sequencing. Pathway enrichment analysis of NSCs from chip showed neural stem cell development enrichment and boosted commitment to neural stem cell lineage in initial phases of neural induction, attributed to a confined environment in a microfluidic chip. This method provides a cost-effective pipeline to reprogram and differentiate iPSCs for therapeutics compliant with current good manufacturing practices.

Deficiency of the Heterogeneous Nuclear Ribonucleoprotein U locus leads to delayed hindbrain neurogenesis.

Genetic variants affecting Heterogeneous Nuclear Ribonucleoprotein U (HNRNPU) have been identified in several neurodevelopmental disorders (NDDs). HNRNPU is widely expressed in the human brain and shows the highest postnatal expression in the cerebellum. Recent studies have investigated the role of HNRNPU in cerebral cortical development, but the effects of HNRNPU deficiency on cerebellar development remain unknown. Here, we describe the molecular and cellular outcomes of HNRNPU locus deficiency during in vitro neural differentiation of patient-derived and isogenic neuroepithelial stem cells with a hindbrain profile. We demonstrate that HNRNPU deficiency leads to chromatin remodeling of A/B compartments, and transcriptional rewiring, partly by impacting exon inclusion during mRNA processing. Genomic regions affected by the chromatin restructuring and host genes of exon usage differences show a strong enrichment for genes implicated in epilepsies, intellectual disability, and autism. Lastly, we show that at the cellular level HNRNPU downregulation leads to an increased fraction of neural progenitors in the maturing neuronal population. We conclude that the HNRNPU locus is involved in delayed commitment of neural progenitors to differentiate in cell types with hindbrain profile.

Crosstalk between astrocytes and microglia results in increased degradation of α-synuclein and amyloid-ß aggregates.

BACKGROUND: Alzheimer's disease (AD) and Parkinson's disease (PD) are characterized by brain accumulation of aggregated amyloid-beta (Aß) and alpha-synuclein (αSYN), respectively. In order to develop effective therapies, it is crucial to understand how the Aß/αSYN aggregates can be cleared. Compelling data indicate that neuroinflammatory cells, including astrocytes and microglia, play a central role in the pathogenesis of AD and PD. However, how the interplay between the two cell types affects their clearing capacity and consequently the disease progression remains unclear. METHODS: The aim of the present study was to investigate in which way glial crosstalk influences αSYN and Aß pathology, focusing on accumulation and degradation. For this purpose, human-induced pluripotent cell (hiPSC)-derived astrocytes and microglia were exposed to sonicated fibrils of αSYN or Aß and analyzed over time. The capacity of the two cell types to clear extracellular and intracellular protein aggregates when either cultured separately or in co-culture was studied using immunocytochemistry and ELISA. Moreover, the capacity of cells to interact with and process protein aggregates was tracked using time-lapse microscopy and a customized "close-culture" chamber, in which the apical surfaces of astrocyte and microglia monocultures were separated by a <1 mm space. RESULTS: Our data show that intracellular deposits of αSYN and Aß are significantly reduced in co-cultures of astrocytes and microglia, compared to monocultures of either cell type. Analysis of conditioned medium and imaging data from the "close-culture" chamber experiments indicate that astrocytes secrete a high proportion of their internalized protein aggregates, while microglia do not. Moreover, co-cultured astrocytes and microglia are in constant contact with each other via tunneling nanotubes and other membrane structures. Notably, our live cell imaging data demonstrate that microglia, when attached to the cell membrane of an astrocyte, can attract and clear intracellular protein deposits from the astrocyte. CONCLUSIONS: Taken together, our data demonstrate the importance of astrocyte and microglia interactions in Aß/αSYN clearance, highlighting the relevance of glial cellular crosstalk in the progression of AD- and PD-related brain pathology.

Partial Monosomy 21 Mirrors Gene Expression of Trisomy 21 in a Patient-Derived Neuroepithelial Stem Cell Model.

Induced pluripotent stem cells (iPSCs) from patients are an attractive disease model to study tissues with poor accessibility such as the brain. Using this approach, we and others have shown that trisomy 21 results in genome-wide transcriptional dysregulations. The effects of loss of genes on chromosome 21 is much less characterized. Here, we use patient-derived neural cells from an individual with neurodevelopmental delay and a ring chromosome 21 with two deletions spanning 3.8 Mb at the terminal end of 21q22.3, containing 60 protein-coding genes. To investigate the molecular perturbations of the partial monosomy on neural cells, we established patient-derived iPSCs from fibroblasts retaining the ring chromosome 21, and we then induced iPSCs into neuroepithelial stem cells. RNA-Seq analysis of NESCs with the ring chromosome revealed downregulation of 18 genes within the deleted region together with global transcriptomic dysregulations when compared to euploid NESCs. Since the deletions on chromosome 21 represent a genetic "contrary" to trisomy of the corresponding region, we further compared the dysregulated transcriptomic profile in with that of two NESC lines with trisomy 21. The analysis revealed opposed expression changes for 23 genes on chromosome 21 as well as 149 non-chromosome 21 genes. Taken together, our results bring insights into the effects on the global and chromosome 21 specific gene expression from a partial monosomy of chromosome 21qter during early neuronal differentiation.

Modeling SHH-driven medulloblastoma with patient iPS cell-derived neural stem cells.

Medulloblastoma is the most common malignant brain tumor in children. Here we describe a medulloblastoma model using Induced pluripotent stem (iPS) cell-derived human neuroepithelial stem (NES) cells generated from a Gorlin syndrome patient carrying a germline mutation in the sonic hedgehog (SHH) receptor PTCH1. We found that Gorlin NES cells formed tumors in mouse cerebellum mimicking human medulloblastoma. Retransplantation of tumor-isolated NES (tNES) cells resulted in accelerated tumor formation, cells with reduced growth factor dependency, enhanced neurosphere formation in vitro, and increased sensitivity to Vismodegib. Using our model, we identified LGALS1 to be a GLI target gene that is up-regulated in both Gorlin tNES cells and SHH-subgroup of medulloblastoma patients. Taken together, we demonstrate that NES cells derived from Gorlin patients can be used as a resource to model medulloblastoma initiation and progression and to identify putative targets.

Ataxia in Patients With Bi-Allelic NFASC Mutations and Absence of Full-Length NF186.

The etiology of hereditary ataxia syndromes is heterogeneous, and the mechanisms underlying these disorders are often unknown. Here, we utilized exome sequencing in two siblings with progressive ataxia and muscular weakness and identified a novel homozygous splice mutation (c.3020-1G > A) in neurofascin (NFASC). In RNA extracted from fibroblasts, we showed that the mutation resulted in inframe skipping of exon 26, with a deprived expression of the full-length transcript that corresponds to NFASC isoform NF186. To further investigate the disease mechanisms, we reprogrammed fibroblasts from one affected sibling to induced pluripotent stem cells, directed them to neuroepithelial stem cells and finally differentiated to neurons. In early neurogenesis, differentiating cells with selective depletion of the NF186 isoform showed significantly reduced neurite outgrowth as well as fewer emerging neurites. Furthermore, whole-cell patch-clamp recordings of patient-derived neuronal cells revealed a lower threshold for openings, indicating altered Na+ channel kinetics, suggesting a lower threshold for openings as compared to neuronal cells without the NFASC mutation. Taken together, our results suggest that loss of the full-length NFASC isoform NF186 causes perturbed neurogenesis and impaired neuronal biophysical properties resulting in a novel early-onset autosomal recessive ataxia syndrome.

Single cell analysis of autism patient with bi-allelic NRXN1-alpha deletion reveals skewed fate choice in neural progenitors and impaired neuronal functionality.

We generated human iPS derived neural stem cells and differentiated cells from healthy control individuals and an individual with autism spectrum disorder carrying bi-allelic NRXN1-alpha deletion. We investigated the expression of NRXN1-alpha during neural induction and neural differentiation and observed a pivotal role for NRXN1-alpha during early neural induction and neuronal differentiation. Single cell RNA-seq pinpointed neural stem cells carrying NRXN1-alpha deletion shifting towards radial glia-like cell identity and revealed higher proportion of differentiated astroglia. Furthermore, neuronal cells carrying NRXN1-alpha deletion were identified as immature by single cell RNA-seq analysis, displayed significant depression in calcium signaling activity and presented impaired maturation action potential profile in neurons investigated with electrophysiology. Our observations propose NRXN1-alpha plays an important role for the efficient establishment of neural stem cells, in neuronal differentiation and in maturation of functional excitatory neuronal cells.

Stem cell models of schizophrenia, what have we learned and what is the potential?

Schizophrenia is a complex disorder with clinical manifestations in early adulthood. However, it may start with disruption of brain development caused by genetic or environmental factors, or both. Early deteriorating effects of genetic/environmental factors on neural development might be key to described disease causing mechanisms. Establishing cellular models with cells from affected individual using the induced pluripotent stem cells (iPSC) technology could be used to mimic early neurodevelopment alterations caused by risk genes or environmental stressors. Indeed, cellular models have allowed identification and further study of risk factors and the biological pathways in which they are involved. New advancements in differentiation methods such as defined and robust monolayer protocols and cerebral 3D organoids have made it possible to faithfully mimic neural development and neuronal functionality while CRISPR-editing tools assist to engineer isogenic cell lines to precisely explore genetic variation in polygenic diseases such as schizophrenia. Here we review the current field of iPSC models of schizophrenia and how risk factors can be modelled as well as discussing the common biological pathways involved.

Kindlin-2 Modulates the Survival, Differentiation, and Migration of Induced Pluripotent Cell-Derived Mesenchymal Stromal Cells.

Kindlin-2 is a multidomain intracellular protein that can be recruited to ß-integrin domains to activate signaling, initiate transcriptional programs, and bind to E-cadherin. To explore its involvement in cell fate decisions in mesenchymal cells, we studied the effects of Kindlin-2 modification (overexpression/knockdown) in induced pluripotent cell-derived mesenchymal stromal cells (iPSC-MSCs). Kindlin-2 overexpression resulted in increased proliferation and reduced apoptosis of iPSC-MSCs, as well as inhibition of their differentiation towards osteocytes, adipocytes, and chondrocytes. In contrast, siRNA-mediated Kindlin-2 knockdown induced increased apoptosis and increased differentiation response in iPSC-MSCs. The ability of iPSC-MSCs to adhere to VCAM-1/SDF-1α under shear stress and to migrate in a wound scratch assay was significantly increased after Kindlin-2 overexpression. In contrast, inhibition of mixed lymphocyte reaction (MLR) was generally independent of Kindlin-2 modulation in iPSC-MSCs, except for decreased production of interleukin-2 (IL-2) after Kindlin-2 overexpression in iPS-MSCs. Thus, Kindlin-2 upregulates survival, proliferation, stemness, and migration potential in iPSC-MSCs and may therefore be beneficial in optimizing performance of iPSC-MSC in therapies.

Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase.

Homology directed repair (HDR)-based genome editing via selectable long flanking arm donors can be hampered by local transgene silencing at transcriptionally silent loci. Here, we report efficient bi-allelic modification of a silent locus in patient-derived hiPSC by using Cas9 nickase and a silencing-resistant donor construct that contains an excisable selection/counter-selection cassette. To identify the most active single guide RNA (sgRNA)/nickase combinations, we employed a lentiviral vector-based reporter assay to determine the HDR efficiencies in cella. Next, we used the most efficient pair of sgRNAs for targeted integration of an improved, silencing-resistant plasmid donor harboring a piggyBac-flanked puroΔtk cassette. Moreover, we took advantage of a dual-fluorescence selection strategy for bi-allelic targeting and achieved 100% counter-selection efficiency after bi-allelic excision of the selection/counter-selection cassette. Together, we present an improved system for efficient bi-allelic modification of transcriptionally silent loci in human pluripotent stem cells.

Does metformin improve in vitro maturation and ultrastructure of oocytes retrieved from estradiol valerate polycystic ovary syndrome-induced rats.

BACKGROUND: Metformin decreases polycystic ovary syndrome (PCOS) symptoms, induces ovulation, and may improve developmental competence of in vitro oocyte maturation. This study was designed to define the effects of metformin on the characteristics of in vitro oocyte maturation in estradiol valerate (EV) PCOS-induced rats. METHODS: Forty-five adult female Sprague-Dawley rats were randomly divided into control; sham and PCOS-induced (treated by a single dose of estradiol valerate, 4 mg/rat, IM) groups. The body weight was measured weekly for 12 weeks. At the end of week 12, the serum levels of testosterone, estrogen, progesterone, LH, and FSH and blood glucose of all the rats were measured. About 380 cumulus oocyte complexes (control, 125; sham, 122; PCOS-induced rats, 133) were incubated in Ham's F10 in the absence and/or presence of metformin (M 5(-10)) for 12, 24, 36, and 48 h. The cumulus cells expansion and nuclear and cytoplasmic maturation of the oocytes was evaluated using 1 % aceto-orcein staining, and transmission electron microscopy (TEM). RESULTS: No significant differences were observed in the body weight of the rats. The serum level of testosterone was reduced, and progesterone and LH were significantly increased in the PCOS-induced rats (p < 0.05). However, no significant differences were observed in the serum levels of estrogen and FSH among the groups. Blood glucose level was higher in the PCOS-induced rats than control, (p < 0.01). The expansion of cumulus cells was observed in the metformin-treated oocytes. The oocytes retrieved from PCOS-induced rats show a stage of meiotic division (GVBD, MI, A-T, and MII) in 57.12 % of metformin-untreated and fairly significantly increased to 64.28 % in metformin-treated oocytes, (p < 0.05), but no differences were observed in the MII stage within groups. The redistribution of some cytoplasmic organelles throughout the ooplasm, particularly the peripheral cortical granules, was defined in the metformin-treated oocytes. CONCLUSIONS: Single dose of EV can creates a reversible PCO adult rat model. Metformin enhances the COCs to initiate meiotic resumption at the first 6 h of IVM. In our study the metformin inability to show all aspects of in vitro oocyte maturation and may be resulted from deficiency of EV to induce PCOS.

Mesenchymal Stem/Stromal Cells Derived from Induced Pluripotent Stem Cells Support CD34(pos) Hematopoietic Stem Cell Propagation and Suppress Inflammatory Reaction.

Mesenchymal stem/stromal cells (MSCs) represent a promising cell source for research and therapeutic applications, but their restricted ex vivo propagation capabilities limit putative applications. Substantial self-renewing of stem cells can be achieved by reprogramming cells into induced pluripotent stem cells (iPSCs) that can be easily expanded as undifferentiated cells even in mass culture. Here, we investigated a differentiation protocol enabling the generation and selection of human iPSC-derived MSCs exhibiting relevant surface marker expression profiles (CD105 and CD73) and functional characteristics. We generated such iPSC-MSCs from fibroblasts and bone marrow MSCs utilizing two different reprogramming constructs. All such iPSC-MSCs exhibited the characteristics of normal bone marrow-derived (BM) MSCs. In direct comparison to BM-MSCs our iPSC-MSCs exhibited a similar surface marker expression profile but shorter doubling times without reaching senescence within 20 passages. Considering functional capabilities, iPSC-MSCs provided supportive feeder layer for CD34(+) hematopoietic stem cells' self-renewal and colony forming capacities. Furthermore, iPSC-MSCs gained immunomodulatory function to suppress CD4(+) cell proliferation, reduce proinflammatory cytokines in mixed lymphocyte reaction, and increase regulatory CD4(+)/CD69(+)/CD25(+) T-lymphocyte population. In conclusion, we generated fully functional MSCs from various iPSC lines irrespective of their starting cell source or reprogramming factor composition and we suggest that such iPSC-MSCs allow repetitive cell applications for advanced therapeutic approaches.

Therapeutic potential of human induced pluripotent stem cell-derived mesenchymal stem cells in mice with lethal fulminant hepatic failure.

Large-scale production and noninvasive methods for harvesting mesenchymal stem cells (MSCs), particularly in elderly individuals, has prompted researchers to find new patient-specific sources for MSCs in regenerative medicine. This study aims to produce MSCs from human induced pluripotent stem cells (hiPSCs) and to evaluate their therapeutic effects in a CCl4-induced mouse model of fulminant hepatic failure (FHF). hiPSC-MSCs have shown MSC morphology, antigen profile and differentiation capabilities, and improved hepatic function in our model. hiPSC-MSC-transplanted animals provide significant benefit in terms of survival, serum LDH, total bilirubin, and lipid peroxidation. hiPSC-MSC therapy resulted in a one-third reduction of histologic activity index and a threefold increase in the number of proliferating hepatocytes. This was accompanied by a significant decrease in the expression levels of collagen type I, Mmp13, Mmp2, and Mmp9 genes and increase in Timp1 and Timp2 genes in transplanted groups. hiPSC-MSCs secreted hepatocyte growth factor (HGF) in vitro and also expressed HGF in evaluated liver sections. Similar results were observed with human bone marrow (hBM)-derived MSCs. In conclusion, our results have demonstrated that hiPSC-MSCs might be valuable appropriate alternatives for hBM-MSCs in FHF liver repair and support liver function by cell therapy with a large-scale production capacity, patient-specific nature, and no invasive MSC harvesting.

Differentiation and transplantation of human induced pluripotent stem cell-derived hepatocyte-like cells.

The generation of human induced pluripotent stem cells (hiPSCs) with a high differentiation potential provided a new source for hepatocyte generation not only for drug discovery and in vitro disease models, but also for cell replacement therapy. However, the reported hiPSC-derived hepatocyte-like cells (HLCs) were not well characterized and their transplantation, as the most promising clue of cell function was not reported. Here, we performed a growth factor-mediated differentiation of functional HLCs from hiPSCs and evaluated their potential for recovery of a carbon tetrachloride (CCl4)-injured mouse liver following transplantation. The hiPSC-derived hepatic lineage cells expressed hepatocyte-specific markers, showed glycogen and lipid storage activity, secretion of albumin (ALB), alpha-fetoprotein (AFP), urea, and CYP450 metabolic activity in addition to low-density lipoprotein (LDL) and indocyanin green (ICG) uptake. Similar results were observed with human embryonic stem cell (hESC)-derived HLCs. The transplantation of hiPSC-HLCs into a CCl4-injured liver showed incorporation of the hiPSC-HLCs into the mouse liver which resulted in a significant enhancement in total serum ALB after 1 week. A reduction of total serum LDH and bilirubin was seen when compared with the control and sham groups 1 and 5 weeks post-transplantation. Additionally, we detected human serum ALB and ALB-positive transplanted cells in both the host serum and livers, respectively, which showed functional integration of transplanted cells within the mouse livers. Therefore, our results have opened up a proof of concept that functional HLCs can be generated from hiPSCs, thus improving the general condition of a CCl4-injured mouse liver after their transplantation. These results may bring new insights in the clinical applications of hiPSCs once safety issues are overcome.

Engineered MSCs from Patient-Specific iPS Cells.

Mesenchymal stroma/stem cells (MSCs) represent a heterogenic cell population that can be isolated from various tissues of the body or can be generated from pluripotent stem cells by in vitro differentiation. Various promising pre-clinical and clinical studies suggest that MSCs might stimulate endogenous regeneration and/or act as anti-inflammatory agents, which could be of high therapeutic relevance for a number of diseases, including graft-versus-host disease after allogeneic hematopoietic stem cell transplantation, inflammatory bowel diseases, or some forms of liver failure. Notably, conflicting results of various studies illustrated that the source of MSCs, the cultivation condition, and the way of administration have important effects on the desired clinical effect. Some of the involved molecular pathways have recently been elucidated and an artificial modulation of these pathways by engineered MSCs might result in superfunctional MSCs for enhanced endogenous regeneration or anti-inflammatory response. In this review, we summarize important findings of conventional MSCs for applications in gastroenterology and we describe the state-of-the-art for the generation of patient-derived iPS cells that eventually might provide genetically engineered superfunctional iPS cells for advanced cell therapies.

Cell-based therapeutics for liver disorders.

INTRODUCTION: Due to a lack of adequate liver donors and post-surgical complications, researchers propose that cell therapy should be an alternative treatment for patients with end-stage liver diseases. DATA SOURCES: We performed a literature review on cell-based therapy for liver disorders. AREAS OF AGREEMENT: Due to growing numbers of patients on waiting lists for liver transplantation, a substitute treatment strategy is needed for our patients. Cell therapy can save patients who are in life-threatening situations, enabling them to have more time and increase their chances of survival. Pluripotent stem cells can be a good resource for cell-based therapy after the establishment of efficient differentiation protocols in addition to the settlement of ethical and immunological issues. Cell-based therapy will be applicable after the approval of its efficiency via animal model studies. AREAS OF CONTROVERSY: Transplanted cells cannot integrate into the recipient liver and lose their functionality after a limited time. The rate of homing and transdifferentiation of transplanted cells into hepatocytes is scant. GROWING POINTS: Application of autologous bone marrow mononuclear cells (MNCs), hematopoietic and mesenchymal stem cells (HSCs and MSCs) has improved the general conditions of certain patients. Although this improvement is temporary, new studies have focused on increasing their performance. TIMELY AREAS FOR DEVELOPING RESEARCH: The safety, feasibility and efficacy of applying MNCs, HSCs and MSCs in liver disorders have been proven in clinical trials. Patient-specific cell therapy after the production of induced pluripotent stem cells and new discoveries in somatic cell conversion during transdifferentiation are promising insights.