Application of mesenchymal stem cells derived from human pluripotent stem cells in regenerative medicine

Mesenchymal stem cells(MSCs)represent the most clinically used stem cells in regenerative medicine.However,due to the disadvantages with primary MSCs,such as limited cell proliferative capacity and rarity in the tissues leading to limited MSCs,gradual loss of differentiation during in vitro expansion reducing the efficacy of MSC application,and variation among donors increasing the uncertainty of MSC efficacy,the clinical application of MSCs has been greatly hampered.MSCs derived from human pluripotent stem cells(hPSC-MSCs)can circumvent these problems associated with primary MSCs.Due to the infinite selfrenewal of hPSCs and their differentiation potential towards MSCs,hPSC-MSCs are emerging as an attractive alternative for regenerative medicine.This review summarizes the progress on derivation of MSCs from human pluripotent stem cells,disease modelling and drug screening using hPSC-MSCs,and various applications of hPSC-MSCs in regenerative medicine.In the end,the challenges and concerns with hPSC-MSC applications are also discussed.

Deriving striatal projection neurons from human pluripotent stem cells with Activin A

The striatum is the main input structure of the basal ganglia and is involved in voluntary motor control,habit learning and reward processing.Medium spiny neurons（MSNs）comprise80%and 95%of striatal neurons in primates and rodents,respectively.

Practical choice for robust and efficient differentiation of human pluripotent stem cells

Human pluripotent stem cells(hPSCs)have the distinct advantage of being able to differentiate into cells of all three germ layers.Target cells or tissues derived from hPSCs have many uses such as drug screening,disease modeling,and transplantation therapy.There are currently a wide variety of differentiation methods available.However,most of the existing differentiation methods are unreliable,with uneven differentiation efficiency and poor reproducibility.At the same time,it is difficult to choose the optimal method when faced with so many differentiation schemes,and it is time-consuming and costly to explore a new differentiation approach.Thus,it is critical to design a robust and efficient method of differentiation.In this review article,we summarize a comprehensive approach in which hPSCs are differentiated into target cells or organoids including brain,liver,blood,melanocytes,and mesenchymal cells.This was accomplished by employing an embryoid body-based three-dimensional(3D)suspension culture system with multiple cells co-cultured.The method has high stable differentiation efficiency compared to the conventional 2D culture and can meet the requirements of clinical application.Additionally,ex vivo co-culture models might be able to constitute organoids that are highly similar or mimic human organs for potential organ transplantation in the future.

Human pluripotent stem cells:Towards therapeutic development for the treatment of lifestyle diseases

There are two types of human pluripotent stem cells: Embryonic stem cells(ESCs) and induced pluripotent stem cells(iPSCs),both of which launched themselves on clinical trials after having taken measures to overcome problems: Blocking rejections by immunosuppressants regarding ESCs and minimizing the risk of tumorigenicity by depleting exogenous gene components regarding iP SCs.It is generally assumed that clinical applications of human pluripotent stem cells should be limited to those cases where there are no alternative measures for treatments because of the risk in transplanting those cells to living bodies.Regarding lifestyle diseases,we have already several therapeutic options,and thus,development of human pluripotent stem cell-based therapeutics tends to be avoided.Nevertheless,human pluripotent stem cells can contribute to the development of new therapeutics in this field.As we will show,there is a case where only a short-term presence of human pluripotent stem-derived cells can exert long-term therapeutic effects even after they are rejected.In those cases,immunologically rejections of ESC-or allogenic iP SC-derived cells may produce beneficial outcomes by nullifying the risk of tumorigenesis without deterioration of therapeutic effects.Another utility of human pluripotent stem cells is the provision of an innovative tool for drug discovery that are otherwise unavailable.For example,clinical specimens of human classical brown adipocytes(BAs),which has been attracting a great deal of attention as a new target of drug discovery for the treatment of metabolic disorders,are unobtainable from living individuals due to scarcity,fragility and ethical problems.However,BA can easily be produced from human pluripotent stem cells.In this review,we will contemplate potential contribution of human pluripotent stem cells to therapeutic development for lifestyle diseases.

Generating hematopoietic cells from human pluripotent stem cells:approaches,progress and challenges

Human pluripotent stem cells(hPSCs)have been suggested as a potential source for the production of blood cells for clinical application.In two decades,almost all types of blood cells can be successfully generated from hPSCs through various differentiated strategies.Meanwhile,with a deeper understanding of hematopoiesis,higher efficiency of generating progenitors and precursors of blood cells from hPSCs is achieved.However,how to generate large-scale mature functional cells from hPSCs for clinical use is still difficult.In this review,we summarized recent approaches that generated both hematopoietic stem cells and mature lineage cells from hPSCs,and remarked their efficiency and mechanisms in producing mature functional cells.We also discussed the major challenges in hPSC-derived products of blood cells and provided some potential solutions.Our review summarized efficient,simple,and defined methodologies for developing good manufacturing practice standards for hPSC-derived blood cells,which will facilitate the translation of these products into the clinic.

Human pluripotent stem cell-derived kidney organoids:Current progress and challenges

Human pluripotent stem cell(hPSC)-derived kidney organoids share similarities with the fetal kidney.However,the current hPSC-derived kidney organoids have some limitations,including the inability to perform nephrogenesis and lack of a corticomedullary definition,uniform vascular system,and coordinated exit path-way for urinary filtrate.Therefore,further studies are required to produce hPSC-derived kidney organoids that accurately mimic human kidneys to facilitate research on kidney development,regeneration,disease modeling,and drug screening.In this review,we discussed recent advances in the generation of hPSC-derived kidney organoids,how these organoids contribute to the understanding of human kidney development and research in disease modeling.Additionally,the limitations,future research focus,and applications of hPSC-derived kidney organoids were highlighted.

Human pluripotent stem cell-derivedβcells:Truly immature isletβcells for type 1 diabetes therapy?

A century has passed since the Nobel Prize winning discovery of insulin,which still remains the mainstay treatment for type 1 diabetes mellitus(T1DM)to this day.True to the words of its discoverer Sir Frederick Banting,“insulin is not a cure for diabetes,it is a treatment”,millions of people with T1DM are dependent on daily insulin medications for life.Clinical donor islet transplantation has proven that T1DM is curable,however due to profound shortages of donor islets,it is not a mainstream treatment option for T1DM.Human pluripotent stem cell derived insulin-secreting cells,pervasively known as stem cell-derivedβcells(SC-βcells),are a promising alternative source and have the potential to become a T1DM treatment through cell replacement therapy.Here we briefly review how isletβcells develop and mature in vivo and several types of reported SC-βcells produced using different ex vivo protocols in the last decade.Although some markers of maturation were expressed and glucose stimulated insulin secretion was shown,the SC-βcells have not been directly compared to their in vivo counterparts,generally have limited glucose response,and are not yet fully matured.Due to the presence of extra-pancreatic insulin-expressing cells,and ethical and technological issues,further clarification of the true nature of these SC-βcells is required.

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.

One-step cell biomanufacturing platform:porous gelatin microcarrier beads promote human embryonic stem cell-derived midbrain dopaminergic progenitor cell differentiation in vitro and survival after transplantation in vivo

Numerous studies have shown that cell replacement therapy can replenish lost cells and rebuild neural circuitry in animal models of Parkinson’s disease.Transplantation of midbrain dopaminergic progenitor cells is a promising treatment for Parkinson’s disease.However,transplanted cells can be injured by mechanical damage during handling and by changes in the transplantation niche.Here,we developed a one-step biomanufacturing platform that uses small-aperture gelatin microcarriers to produce beads carrying midbrain dopaminergic progenitor cells.These beads allow midbrain dopaminergic progenitor cell differentiation and cryopreservation without digestion,effectively maintaining axonal integrity in vitro.Importantly,midbrain dopaminergic progenitor cell bead grafts showed increased survival and only mild immunoreactivity in vivo compared with suspended midbrain dopaminergic progenitor cell grafts.Overall,our findings show that these midbrain dopaminergic progenitor cell beads enhance the effectiveness of neuronal cell transplantation.

Heart regeneration with human pluripotent stem cells: Prospects and challenges

Cardiovascular disease,ranging from congenital heart disease to adult myocardial infarction,is the leading cause of death worldwide.In pursuit of reliable cardiovascular regenerative medicine,human pluripotent stem cells(hPSCs),including human embryonic stem cells(hESCs)and human induced pluripotent stem cells(hiPSCs),offer plenty of potential cell-based applications.HPSCs are capable of proliferating indefinitely in an undifferentiated state,and are also pluripotent,being able to differentiate into virtually any somatic cell types given specific stepwise cues,thus representing an unlimited source to generate functional cardiovascular cells for heart regeneration.Here we recapitulated current advances in developing efficient protocols to generate hPSCderived cardiovascular cell lineages,including cardiomyocytes,endothelial cells,and epicardial cells.We also discussed applications of hPSC-derived cells in combination with compatible bioactive materials,promising trials of cell transplantation in animal models of myocardial infarction,and potential hurdles to bring us closer to the ultimate goal of cell-based heart repair.

Stepwise differentiation of functional pancreatic β cells from human pluripotent stem cells

Pancreaticβcells differentiated from stem cells provide promise for cell replacement therapy of diabetes.Human pluripotent stem cells could be differentiated into definitive endoderm,followed by pancreatic progenitors,and then subjected to endocrinal differentiation and maturation in a stepwise fashion.Many achievements have been made in making pancreaticβcells from human pluripotent stem cells in last two decades,and a couple of phase I/II clinical trials have just been initiated.Here,we overview the major progresses in differentiating pancreaticβcells from human pluripotent stem cells with the focus on recent technical advances in each differentiation stage,and briefly discuss the current limitations as well.

Early development and functional properties of tryptase/chymase double-positive mast cells from human pluripotent stem cells

Mast cells (MCs) play a pivotal role in the hypersensitivity reaction by regulating the innate and adaptive immune responses. Humans have two types of MCs. The first type, termed MCTC, is found in the skin and other connective tissues and expresses both tryptase and chymase, while the second, termed MCT, which only expresses tryptase, is found primarily in the mucosa. MCs induced from human adult-type CD34+ cells are reported to be of the MCT type, but the development of MCs during embryonic/fetal stages is largely unknown. Using an efficient coculture system, we identified that a CD34+c-kit+ cell population, which appeared prior to the emergence of CD34+CD45+ hematopoietic stem and progenitor cells (HSPCs), stimulated robust production of pure Tryptase+Chymase+ MCs (MCTCs). Single-cell analysis revealed dual development directions of CD34+c-kit+ progenitors, with one lineage developing into erythro-myeloid progenitors (EMP) and the other lineage developing into HSPC. Interestingly, MCTCs derived from early CD34+c-kit+ cells exhibited strong histamine release and immune response functions. Particularly, robust release of IL-17 suggested that these early developing tissue-type MCTCs could play a central role in tumor immunity. These findings could help elucidate the mechanisms controlling early development of MCTCs and have significant therapeutic implications.

Current approaches for efficient genetic editing in human pluripotent stem cells

Human pluripotent stem cells have been much anticipated as a powerful system to study developmental events, model genetic disorders, and serve as a source of autologous cells for cell therapy in genetic disorders. Precise genetic manipulation is crucial to all these applications, and many recent advances have been made in site specific nuclease systems like zinc finger nucleases, TALENs, and CRISPR/Cas. In this review, we address the importance of site-specific genome modification and how this technology can be applied to manipulate human pluripotent stem cells.

Genetic Correction and Hepatic Differentiation of Hemophilia B-specific Human Induced Pluripotent Stem Cells

Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells （iPSCs） derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by sequencing the encoding area of human coagulation factor IX （F IX） gene. Genomic DNA was extracted from the iPSCs, and the primers were designed to amplify the eight exons of F IX. Next, the point mutation in those iPSCs was genetically corrected using CRISPR/Cas9 technology in the presence of a 129-nucleotide homologous repair template that contained two synonymous mutations. Then, top 8 potential off-target sites were subsequently analyzed using Sanger sequencing. Finally, the corrected clones were differentiated into hepatocyte-like cells, and the secretion of F IX was validated by immunocytochemistry and ELISA assay.Results The cell line bore a missense mutation in the 6th coding exon （c.676 C〉T） of F IX gene. Correction of the point mutation was achieved via CRISPR/Cas9 technology in situ with a high efficacy at about 22% （10/45） and no off-target effects detected in the corrected iPSC clones. F IX secretion, which was further visualized by immunocytochemistry and quantified by ELISA in vitro, reached about 6 ng/ml on day 21 of differentiation procedure. Conclusions Mutations in human disease-specific iPSCs could be precisely corrected by CRISPR/Cas9 technology, and corrected cells still maintained hepatic differentiation capability. Our findings might throw a light on iPSC-based personalized therapies in the clinical application, especially for hemophilia B.

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.

Human induced pluripotent stem cells for monogenic disease modelling and therapy

Recent and advanced protocols are now available to derive human induced pluripotent stem cells (hiPSCs) from patients affected by genetic diseases. No curative treatments are available for many of these diseases; thus, hiPSCs represent a major impact on patient’ health. hiPSCs represent a valid model for the in vitro study of monogenic diseases, together with a better comprehension of the pathogenic mechanisms of the pathology, for both cell and gene therapy protocol applications. Moreover, these pluripotent cells represent a good opportunity to test innovative pharmacological treatments focused on evaluating the efficacy and toxicity of novel drugs. Today, innovative gene therapy protocols, especially gene editing-based, are being developed, allowing the use of these cells not only as in vitro disease models but also as an unlimited source of cells useful for tissue regeneration and regenerative medicine, eluding ethical and immune rejection problems. In this review, we will provide an up-to-date of modelling monogenic disease by using hiPSCs and the ultimate applications of these in vitro models for cell therapy. We consider and summarize some peculiar aspects such as the type of parental cells used for reprogramming, the methods currently used to induce the transcription of the reprogramming factors, and the type of iPSC-derived differentiated cells, relating them to the genetic basis of diseases and to their inheritance model.

Human induced pluripotent stem cells labeled with fluorescent magnetic nanoparticles for targeted imaging and hyperthermia therapy for gastric cancer

Objective: Human induced pluripotent stem(i PS) cells exhibit great potential for generating functional human cells for medical therapies. In this paper, we report for use of human i PS cells labeled with fluorescent magnetic nanoparticles(FMNPs) for targeted imaging and synergistic therapy of gastric cancer cells in vivo. Methods: Human i PS cells were prepared and cultured for 72 h. The culture medium was collected, and then was coincubated with MGC803 cells. Cell viability was analyzed by the MTT method. FMNP-labeled human i PS cells were prepared and injected into gastric cancer-bearing nude mice. The mouse model was observed using a small-animal imaging system. The nude mice were irradiated under an external alternating magnetic field and evaluated using an infrared thermal mapping instrument. Tumor sizes were measured weekly. Results: iP S cells and the collected culture medium inhibited the growth of MGC803 cells. FMNP-labeled human iP S cells targeted and imaged gastric cancer cells in vivo, as well as inhibited cancer growth in vivo through the external magnetic field. Conclusion: FMNP-labeled human i PS cells exhibit considerable potential in applications such as targeted dual-mode imaging and synergistic therapy for early gastric cancer.

Changes in human pluripotent stem cell gene expression after genotoxic stress exposures

Human pluripotent stem cells(h PSCs) represent heterogeneous populations, including induced pluripotent stem cells(i PSCs), endogenous plastic somatic cells, and embryonic stem cells(ESCs). Human ESCs are derived from the inner cell mass of the blastocyst, and they are characterized by the abilities to self-renew indefinitely, and to give rise to all cell types of embryonic lineage(pluripotency) under the guidance of the appropriate chemical, mechanical and environmental cues. The combination of these critical features is unique to h ESCs, and set them apart from other human cells. The expectations are high to utilize h ESCs for treating injuries and degenerative diseases; for modeling of complex illnesses and development; for screening and testing of pharmacological products; and for examining toxicity, mutagenicity, teratogenicity, and potential carcinogenic effects of a variety of environmental factors, including ionizing radiation(IR). Exposures to genotoxic stresses, such as background IR, are unavoidable; moreover, IR is widely used in diagnostic and therapeutic procedures in medicine on a routine basis. One of the key outcomes of cell exposures to IR is the change in gene expression, which may underlie the ultimate h ESCs fate after such a stress. However, gaps in our knowledge about basic biology of h ESCs impose a serious limitation to fully realize the potential of h ESCs in practice. The purpose of this review is to examine the available evidence of alterations in gene expression in human pluripotent stem cells after genotoxic stress, and to discuss strategies for future research in this important area.

Skeletal muscle generated from induced pluripotent stem cells-induction and application

Human induced pluripotent stem cells(hiPS cells or hiPSCs) can be derived from cells of patients with severe muscle disease. If skeletal muscle induced from patientiPSCs shows disease-specific phenotypes, it can be useful for studying the disease pathogenesis and for drug development. On the other hand, human iPSCs from healthy donors or hereditary muscle diseaseiPSCs whose genomes are edited to express normal protein are expected to be a cell source for cell therapy. Several protocols for the derivation of skeletal muscle from human iPSCs have been reported to allow the development of efficient treatments for devastating muscle diseases. In 2017, the focus of research is shifting to another stage:(1) the establishment of mature myofibers that are suitable for study of the pathogenesis of muscle disease;(2) setting up a highthroughput drug screening system; and(3) the preparation of highly regenerative, non-oncogenic cells in large quantities for cell transplantation, etc.

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.