Enrichment of retinal ganglion and Müller glia progenitors from retinal organoids derived from human induced pluripotent stem cells-possibilities and current limitations

BACKGROUND Retinal organoids serve as excellent human-specific disease models for conditions affecting otherwise inaccessible retinal tissue from patients.They permit the isolation of key cell types affected in various eye diseases including retinal ganglion cells(RGCs)and Müller glia.AIM To refine human-induced pluripotent stem cells(hiPSCs)differentiated into threedimensional(3D)retinal organoids to generate sufficient numbers of RGCs and Müller glia progenitors for downstream analyses.METHODS In this study we described,evaluated,and refined methods with which to generate Müller glia and RGC progenitors,isolated them via magnetic-activated cell sorting,and assessed their lineage stability after prolonged 2D culture.Putative progenitor populations were characterized via quantitative PCR and immunocytochemistry,and the ultrastructural composition of retinal organoid cells was investigated.RESULTS Our study confirms the feasibility of generating marker-characterized Müller glia and RGC progenitors within retinal organoids.Such retinal organoids can be dissociated and the Müller glia and RGC progenitor-like cells isolated via magnetic-activated cell sorting and propagated as monolayers.CONCLUSION Enrichment of Müller glia and RGC progenitors from retinal organoids is a feasible method with which to study cell type-specific disease phenotypes and to potentially generate specific retinal populations for cell replacement therapies.

Semi-3D cultures using Laminin 221 as a coating material for human induced pluripotent stem cells

It was previously believed that human induced pluripotent stem cells(hiPSCs)did not show adhesion to the coating material Laminin 221,which is known to have specific affinity for cardiomyocytes.In this study,we report that human mononuclear cell-derived hiPSCs,established with Sendai virus vector,form peninsular-like colonies rather than embryonic stem cell-like colonies;these peninsular-like colonies can be passaged more than 10 times after establishment.Additionally,initializationdeficient cells with residual Sendai virus vector adhered to the coating material Laminin 511 but not to Laminin 221.Therefore,the expression of undifferentiated markers tended to be higher in hiPSCs established on Laminin 221 than on Laminin 511.On Laminin 221,hiPSCs15M66 showed a semi-floating colony morphology.The expression of various markers of cell polarity was significantly lower in hiPSCs cultured on Laminin 221 than in hiPSCs cultured on Laminin 511.Furthermore,201B7 and 15M66 hiPSCs showed 3D cardiomyocyte differentiation on Laminin 221.Thus,the coating material Laminin 221 provides semi-floating culture conditions for the establishment,culture and induced differentiation of hiPSCs.

Role of induced pluripotent stem cells in diagnostic cardiology

Ethical concerns about stem cell-based research have delayed important advances in many areas of medicine,including cardiology.The introduction of induced pluripotent stem cells(iPSCs)has supplanted the need to use human stem cells for most purposes,thus eliminating all ethical controversies.Since then,many new avenues have been opened in cardiology research,not only in approaches to tissue replacement but also in the design and testing of antiarrhythmic drugs.This methodology has advanced to the point where induced human cardiomyocyte cell lines can now also be obtained from commercial sources or tissue banks.Initial studies with readily available iPSCs have generally confirmed that their behavioral characteristics accurately predict the behavior of beating cardiomyocytes in vivo.As a result,iPSCs can provide new ways to study arrhythmias and heart disease in general,accelerating the development of new,more effective antiarrhythmic drugs,clinical diagnoses,and personalized medical care.The focus on producing cardiomyocytes that can be used to replace damaged heart tissue has somewhat diverted interest in a host of other applications.This manuscript is intended to provide non-specialists with a brief introduction and overview of the research carried out in the field of heart rhythm disorders.

Patient-derived induced pluripotent stem cells with a MERTK mutation exhibit cell junction abnormalities and aberrant cellular differentiation potential

BACKGROUND Human induced pluripotent stem cell(hiPSC)technology is a valuable tool for generating patient-specific stem cells,facilitating disease modeling,and invest-igating disease mechanisms.However,iPSCs carrying specific mutations may limit their clinical applications due to certain inherent characteristics.AIM To investigate the impact of MERTK mutations on hiPSCs and determine whether hiPSC-derived extracellular vesicles(EVs)influence anomalous cell junction and differentiation potential.METHODS We employed a non-integrating reprogramming technique to generate peripheral blood-derived hiPSCs with and hiPSCs without a MERTK mutation.Chromo-somal karyotype analysis,flow cytometry,and immunofluorescent staining were utilized for hiPSC identification.Transcriptomics and proteomics were employed to elucidate the expression patterns associated with cell junction abnormalities and cellular differentiation potential.Additionally,EVs were isolated from the supernatant,and their RNA and protein cargos were examined to investigate the involvement of hiPSC-derived EVs in stem cell junction and differentiation.RESULTS The generated hiPSCs,both with and without a MERTK mutation,exhibited normal karyotype and expressed pluripotency markers;however,hiPSCs with a MERTK mutation demonstrated anomalous adhesion capability and differentiation potential,as confirmed by transcriptomic and proteomic profiling.Furthermore,hiPSC-derived EVs were involved in various biological processes,including cell junction and differentiation.CONCLUSION HiPSCs with a MERTK mutation displayed altered junction characteristics and aberrant differentiation potential.Furthermore,hiPSC-derived EVs played a regulatory role in various biological processes,including cell junction and differentiation.

Ocular stem cells:a narrative review of current clinical trials

·Stem cells are undifferentiated cells showcasing a remarkable capacity of self-replenishing and differentiating into mature cells.Their ability to proliferate connotes that a designated stem cell source is capable of generating an unrestricted number of mature cells.The ever-increasing comprehension of position,activity,and function of ocular stem cells has led to rapid progress and incessant improvement of possible procedures and therapies.A narrative review was conducted to summarize the current evidence on clinical trials and respective literature,regarding current evolution in the field of ocular regenerative medicine.We tried to ascertain the safety of experimental and clinical procedures,their effectiveness,and the ethical repercussion of their use.

Extracellular vesicles from iPSC-MSCs alleviate chemotherapy-induced mouse ovarian damage via the ILK-PI3K/AKT pathway

Chemotherapy can significantly reduce follicle counts in ovarian tissues and damage ovarian stroma,causing endocrine disorder,reproductive dysfunction,and primary ovarian insufficiency(POI).Recent studies have suggested that extracellular vesicles(EVs)secreted from mesenchymal stem cells(MSCs)exert therapeutic effects in various degenerative diseases.In this study,transplantation of EVs from human induced pluripotent stem cell-derived MSCs(iPSC-MSC-EVs)resulted in significant restoration of ovarian follicle numbers,improved granulosa cell proliferation,and inhibition of apoptosis in chemotherapy-damaged granulosa cells,cultured ovaries,and in vivo ovaries in mice.Mechanistically,treatment with i PSC-MSC-EVs resulted in up-regulation of the integrinlinked kinase(ILK)-PI3K/AKT pathway,which is suppressed during chemotherapy,most likely through the transfer of regulatory microRNAs(miRNAs)targeting ILK pathway genes.This work provides a framework for the development of advanced therapeutics to ameliorate ovarian damage and POI in female chemotherapy patients.

Rebuilding insight into the pathophysiology of Alzheimer’s disease through new blood-brain barrier models

The blood-brain barrier is a unique function of the microvasculature in the brain parenchyma that maintains homeostasis in the central nervous system.Blood-brain barrier breakdown is a common pathology in various neurological diseases,such as Alzheimer’s disease,stroke,multiple sclerosis,and Parkinson’s disease.Traditionally,it has been considered a consequence of neuroinflammation or neurodegeneration,but recent advanced imaging techniques and detailed studies in animal models show that blood-brain barrier breakdown occurs early in the disease process and may precede neuronal loss.Thus,the blood-brain barrier is attractive as a potential therapeutic target for neurological diseases that lack effective therapeutics.To elucidate the molecular mechanism underlying blood-brain barrier breakdown and translate them into therapeutic strategies for neurological diseases,there is a growing demand for experimental models of human origin that allow for functional assessments.Recently,several human induced pluripotent stem cell-derived blood-brain barrier models have been established and various in vitro blood-brain barrier models using microdevices have been proposed.Especially in the Alzheimer’s disease field,the human evidence for blood-brain barrier dysfunction has been demonstrated and human induced pluripotent stem cell-derived blood-brain barrier models have suggested the putative molecular mechanisms of pathological blood-brain barrier.In this review,we summarize recent evidence of blood-brain barrier dysfunction in Alzheimer’s disease from pathological analyses,imaging studies,animal models,and stem cell sources.Additionally,we discuss the potential future directions for blood-brain barrier research.

A novel xeno-free and feeder-cell-free system for human pluripotent stem cell culture

While human induced pluripotent stem cells(hiPSCs)have promising applications in regenerative medicine,most of the hiPSC lines available today are not suitable for clinical applications due to contamination with nonhuman materials,such as sialic acid,and potential pathogens from animal-product-containing cell culture systems.Although several xeno-free cell culture systems have been established recently,their use of human fibroblasts as feeders reduces the clinical potential of hiPSCs due to batch-to-batch variation in the feeders and time-consuming preparation processes.In this study,we have developed a xeno-free and feeder-cell-free human embryonic stem cell(hESC)/hiPSC culture system using human plasma and human placenta extracts.The system maintains the self-renewing capacity and pluripotency of hESCs for more than 40 passages.Human iPSCs were also derived from human dermal fibroblasts using this culture system by overexpressing three transcription factors—Oct4,Sox2 and Nanog.The culture system developed here is inexpensive and suitable for large scale production.

Dual human iPSC-derived cardiac lineage cell-seeding extracellular matrix patches promote regeneration and long-term repair of infarcted hearts

Human pluripotent stem cell-derived cardiovascular progenitor cells (hCVPCs) and cardiomyocytes (hCMs) possess therapeutic potential for infarcted hearts;however, their efficacy needs to be enhanced. Here we tested the hypotheses that the combination of decellularized porcine small intestinal submucosal extracellular matrix (SIS-ECM) with hCVPCs, hCMs, or dual of them (Mix, 1:1) could provide better therapeutic effects than the SIS alone, and dual hCVPCs with hCMs would exert synergic effects in cardiac repair. The data showed that the SIS patch well supported the growth of hCVPCs and hCMs. Epicardially implanted SIS-hCVPC, SIS-hCM, or SIS-Mix patches at 7-day post-myocardial infarction significantly ameliorated functional worsening, ventricular dilation and scar formation at 28- and 90-day post-implantation in C57/B6 mice, whereas the SIS only mildly improved function at 90-day post-implantation. Moreover, the SIS and SIS-cell patches improved vascularization and suppressed MI-induced cardiomyocyte hypertrophy and expression of Col1 and Col3, but only the SIS-hCM and the SIS-Mix patches increased the ratio of collagen III/I fibers in the infarcted hearts. Further, the SIS-cell patches stimulated cardiomyocyte proliferation via paracrine action. Notably, the SIS-Mix had better improvements in cardiac function and structure, engraftments, and cardiomyocyte proliferation. Proteomic analysis showed distinct biological functions of exclusive proteins secreted from hCVPCs and hCMs, and more exclusive proteins secreted from co-cultivated hCVPCs and hCMs than mono-cells involving in various functional processes essential for infarct repair. These findings are the first to demonstrate the efficacy and mechanisms of mono- and dual-hCVPC- and hCM-seeding SIS-ECM for repair of infarcted hearts based on the side-by-side comparison.

Profiling the responsiveness of focal adhesions of human cardiomyocytes to extracellular dynamic nano-topography

Focal adhesion complexes function as the mediators of cell-extracellular matrix interactions to sense and transmit the extracellular signals.Previous studies have demonstrated that cardiomyocyte focal adhesions can be modulated by surface topographic features.However,the response of focal adhesions to dynamic surface topographic changes remains underexplored.To study this dynamic responsiveness of focal adhesions,we utilized a shape memory polymer-based substrate that can produce a flat-to-wrinkle surface transition triggered by an increase of temperature.Using this dynamic culture system,we analyzed three proteins(paxillin,vinculin and zyxin)from different layers of the focal adhesion complex in response to dynamic extracellular topographic change.Hence,we quantified the dynamic profile of cardiomyocyte focal adhesion in a time-dependent manner,which provides new understanding of dynamic cardiac mechanobiology.

Human embryoid bodies to hepatocyte-like clusters:Preparing for translation

End-stage liver disease and acute liver failure are some of the most common causes of death worldwide,affecting over 40,000 patients in the United States,for most of whom liver transplantation is the only treatment.Transplantable livers are obtained primarily from deceased donors in the west and living donors in the east,with demand outstripping supply,leading to thousands of deaths each year for those on the transplant waiting lists.As a bridge to liver transplantation,human primary hepatocytes have been transplanted with low success,owing to their inability to grow and expand in vitro,their high sensitivity to cold storage-induced damage,and their dedifferentiation following two-dimensional culture.In the past decade,human induced pluripotent stem cells(hiPSCs)have been studied as a potential alternative to liver and primary hepatocyte transplantation through their differentiation into hepatocyte-like cells(HLCs).Differentiation of hiPSCs into HLCs is limited by the low percentage of differentiated cells that reach a mature hepatic phenotype,poor reproducibility of existing differentiation protocols,and inadequate long-term viability and function in vitro and in vivo.In this review,we will discuss the mechanisms of the various techniques that aim to improve the hepatic differentiation of hiPSCs into mature and genotypically stable HLCs for use in drug studies,as a potential bridge for liver transplantation after liver failure or as therapy for liver regeneration and replacement.

Brain organoid-on-chip system to study the effects of breast cancer derived exosomes on the neurodevelopment of brain

Early human brain development can be affected by multiple prenatal factors that involve chemical exposures in utero,maternal health characteristics such as psychiatric disorders,and cancer.Breast cancer is one of the most common cancers worldwide arising pregnancy.However,it is not clear whether the breast cancer might influence the brain development of fetus.Exosomes secreted by breast cancer cells play a critical role in mediating intercellular communication and interplay between different organs.In this work,we engineered human induced pluripotent stem cells(hiPSCs)-derived brain organoids in an array of micropillar chip and probed the influences of breast cancer cell(MCF-7)derived-exosomes on the early neurodevelopment of brain.The formed brain organoids can recapitulate essential features of embryonic human brain at early stages,in terms of neurogenesis,forebrain regionalization,and cortical organization.Treatment with breast cancer cell derived-exosomes,brain organoids exhibited enhanced expression of stemness-related marker OCT4 and forebrain marker PAX6.RNA-seq analysis reflected several activated signaling pathways associated with breast cancer,medulloblastoma and neurogenesis in brain organoids induced by tumor-derived exosomes.These results suggested that breast cancer cell-derived exosomes might lead to the impaired neurodevelopment in the brain organoids and the carcinogenesis of brain organoids.It potentially implies the fetus of pregnant women with breast cancer has the risk of impaired neurodevelopmental disorder after birth.