Spherical culture could promote the plasticity and stemness of human corneal stromal cells (hCSCs). Here, we introduce a novel three-dimensional (3D) cell culture system based on a polydimethylsiloxane (PDMS) microwell platform composed of many V-bottom microcavities to generate human corneal stromal cell spheroids and promote cell stemness. We isolated hCSCs from SMILE-derived lenticules and maintained their physiological phenotype by culturing them in a medium supplemented with human corneal stromal extract (hCSE). Utilizing a PDMS microwell platform fabricated through 3D printing technology, we successfully generated 3D corneal stromal cell spheroids (3D-CSC) with uniform size and stable structure, exhibiting increased expression of pluripotency factors, including OCT4, NANOG, SOX2, KLF4, and PAX6. Furthermore, the iPS supernatant of E8-conditioned medium (E8-CM) significantly enhanced the stemness properties of these cells. RNA sequencing and proteomics analyses revealed that 3D-CSCs exhibited superior proliferation, differentiation, cell adhesion, migration, and neurogenesis compared to traditional monolayer cultures, underscoring the role of biophysical cues in promoting hCSCs stemness. In summary, this study presents an effective 3D cell culture platform that mimics the in vivo microenvironment, facilitating the enhancement of stemness properties and providing valuable insights into corneal tissue engineering and regenerative medicine, particularly for treating corneal opacities and diseases.

Stem cells are unique, undifferentiated cells that have the ability to both replicate themselves and develop into specialized cell types. This dual capability makes them valuable in the development of regenerative medicine. Current development in stem cell research has widened their application in cell therapy, drug discovery, reproductive cloning in animals, and cell models for various diseases. Although there are substantial studies revealing the treatment of human degenerative diseases using stem cells, this is yet to be explored in livestock animals. Many diseases in livestock species such as mastitis, laminitis, neuromuscular disorders, autoimmune diseases, and some debilitating diseases are not covered completely by the existing drugs and treatment can be improved by using different types of stem cells like embryonic stem cells, adult stem cells, and induced pluripotent stem cells. This review mainly focuses on the use of stem cells for disease treatment in livestock animals. In addition to the diseases mentioned, the potential of stem cells can be helpful in wound healing, skin disease therapy, and treatment of some genetic disorders. This article explores the potential of stem cells from various sources in the therapy of livestock diseases and also their role in the conservation of endangered species as well as disease model preparation. Moreover, the future perspectives and challenges associated with the application of stem cells in livestock are discussed. Overall, the transformative impact of stem cell research on the livestock sector is comprehensively studied which will help researchers to design future research work on stem cells related to livestock diseases.

In the last few decades, the rates of infertility among women have been on the rise, usually due to complications with the uterus and related tissue. A wide variety of reasons can cause uterine factor infertility and can be congenital or a result of disease. Uterine transplantation is currently used as a means to enable women with fertility issues to have a natural birth. However, multiple risk factors are involved in uterine transplantation that threaten the lives of the growing fetus and the mother, as a result of which the procedure is not prominently practiced. Uterine tissue engineering provides a potential solution to infertility through the regeneration of replacement of damaged tissue, thus allowing healing and restoration of reproductive capacity. It involves the use of stem cells from the patient incorporated within biocompatible scaffolds to regenerate the entire tissue. This manuscript discusses the need for uterine tissue engineering, giving an overview of the biological and organic material involved in the process. There are numerous existing animal models in which this procedure has been actualized, and the observations from them have been compiled here. These models are used to develop a further understanding of the integration of engineered tissues and the scope of tissue engineering as a treatment for uterine disorders. Additionally, this paper examines the scope and limitations of the procedure.

This comprehensive overview of the historical milestones in cell culture underscores key breakthroughs that have shaped the field over time. It begins with Wilhelm Roux's seminal experiments in the 1880s, followed by the pioneering efforts of Ross Granville Harrison, who initiated groundbreaking experiments that fundamentally shaped the landscape of cell culture in the early 20th century. Carrel's influential contributions, notably the immortalization of chicken heart cells, have marked a significant advancement in cell culture techniques. Subsequently, Johannes Holtfreter, Aron Moscona, and Joseph Leighton introduced methodological innovations in three-dimensional (3D) cell culture, initiated by Alexis Carrel, laying the groundwork for future consolidation and expansion of the use of 3D cell culture in different areas of biomedical sciences. The advent of induced pluripotent stem cells by Takahashi and Yamanaka in 2006 was revolutionary, enabling the reprogramming of differentiated cells into a pluripotent state. Since then, recent innovations have included spheroids, organoids, and organ-on-a-chip technologies, aiming to mimic the structure and function of tissues and organs in vitro, pushing the boundaries of biological modeling and disease understanding. In this review, we overview the history of cell culture shedding light on the main discoveries, pitfalls and hurdles that were overcome during the transition from 2D to 3D cell culture techniques. Finally, we discussed the future directions for cell culture research that may accelerate the development of more effective and personalized treatments.

The central nervous system (CNS) plays a role in regulating heart rate and myocardial contractility through sympathetic and parasympathetic nerves, and the heart can impact the functional equilibrium of the CNS through feedback signals. Although heart and brain diseases often coexist and mutually influence each other, the potential links between heart and brain diseases remain unclear due to a lack of reliable models of these relationships. Induced pluripotent stem cells (iPSCs), which can differentiate into multiple functional cell types, stem cell biology and regenerative medicine may offer tools to clarify the mechanisms of these relationships and facilitate screening of effective therapeutic agents. Because calcium ions play essential roles in regulating both the cardiovascular and nervous systems, this review addresses how recent iPSC disease models reveal how dysregulation of intracellular calcium might be a common pathological factor underlying the relationships between heart and brain diseases.

Cardiovascular disease comprises a group of disorders affecting or originating within tissues and organs of the cardiovascular system; most, if not all, will eventually result in cardiomyocyte dysfunction or death, negatively impacting cardiac function. Effective models of cardiac disease are thus important for understanding crucial aspects of disease progression, while recent advancements in stem cell biology have allowed for the use of stem cell populations to derive such models. These include three-dimensional (3D) models such as stem cell-based models of embryos (SCME) as well as organoids, many of which are frequently derived from embryoid bodies (EB). Not only can they recapitulate 3D form and function, but the developmental programs governing the self-organization of cell populations into more complex tissues as well. Many different organoids and SCME constructs have been generated in recent years to recreate cardiac tissue and the complex developmental programs that give rise to its cellular composition and unique tissue morphology. It is thus the purpose of this narrative literature review to describe and summarize many of the recently derived cardiac organoid models as well as their use for the recapitulation of genetic and acquired disease. Owing to the cellular composition of the models examined, this review will focus on disease and tissue injury associated with embryonic/fetal tissues.

Previous studies have implicated epithelial-mesenchymal transition (EMT) in salamander limb regeneration. In this review, we describe putative roles for EMT during each stage of limb regeneration in axolotls and other salamanders. We hypothesize that EMT and EMT-like gene expression programs may regulate three main cellular processes during limb regeneration: (1) keratinocyte migration during wound closure; (2) transient invasion of the stump by epithelial cells undergoing EMT; and (3) use of EMT-like programs by non-epithelial blastemal progenitor cells to escape the confines of their niches. Finally, we propose nontraditional roles for EMT during limb regeneration that warrant further investigation, including alternative EMT regulators, stem cell activation, and fibrosis induced by aberrant EMT.

Human epithelium-derived stem cells and induced pluripotent stem cells (hiPSCs) possess the capability to support tooth formation and differentiate into functional enamel-secreting ameloblasts, making them promising epithelial-component substitutes for future human tooth regeneration. However, current tissue recombination approaches are not only technically challenging, requiring precise induction procedures and sophisticated microsurgery, but also exhibit low success rates in achieving tooth formation and ameloblastic differentiation.

Suspended human keratinocyte stem cells (hKSCs) or cells from three hiPSC lines were directly mixed with dissociated embryonic mouse dental mesenchymal cells (mDMCs) that possess odontogenic potential in different proportions and reaggregated them to construct bioengineered tooth germs. The success rates of tooth formation and ameloblastic differentiation were confirmed after subrenal culture. The sorting capability, sequential development, and ameloblastic differentiation of stem cells were examined via GFP tracing, RT-PCR, and histological analysis, respectively.

Our reaggregation approach achieved an impressive success rate of more than 90% in tooth formation and 100% in ameloblastic differentiation when the chimeric tooth germs contained 1%~10% hKSCs or 5% hiPSCs. In addition, we observed that hiPSCs, upon exposure to mDMCs, initially transformed into epidermal cells, as indicated by KRT14 and CD29 expression, before progressing into dental epithelial cells, as indicated by SP6 and SHH expression. We also found that epithelial-derived hiPSCs, when reaggregated with mDMCs, were more favorable for tooth formation than their mesenchymal-derived counterparts.

This study establishes a simplified yet highly effective cell-cell reaggregation strategy for inducing stem cells to support tooth formation and differentiate into functional ameloblasts, paving the way for novel approaches for the development of stem cell-based tooth organoids and bioengineered tooth germs in vitro.

Diseases of the female reproductive system, especially malignant tumors, pose a serious threat to women's health worldwide. One of the key factors limiting research progress in this area is the lack of representative models. Organoid technology, especially tumor organoids, has been increasingly applied in the study of female reproductive system tumors due to their high heterogeneity, close resemblance to the physiological state, easy acquisition and cultivation advantages. They play a significant role in understanding the origin and causes of tumors, drug screening, and personalized treatment and more. This article reviews the organoid models for the female reproductive system, focusing on the cancer research advancements. It discusses the methods for constructing tumor organoids of the female reproductive tract and summarizes the limitations of current research. The aim is to offer a reference for future development and application of these organoid models, contributing to the advancement of anti-tumor drugs and treatment strategies for female reproductive tract cancer patients.

Animals and plants have developed resilience mechanisms to effectively endure and overcome physical damage and environmental challenges throughout their life span. To sustain their vitality, both animals and plants employ mechanisms to replenish damaged cells, either directly, involving the activity of adult stem cells, or indirectly, via dedifferentiation of somatic cells that are induced to revert to a stem cell state and subsequently redifferentiate. Stem cell research has been a rapidly advancing field in animal studies for many years, driven by its promising potential in human therapeutics, including tissue regeneration and drug development. A major breakthrough was the discovery of induced pluripotent stem cells (iPSCs), which are reprogrammed from somatic cells by expressing a limited set of transcription factors. This discovery enabled the generation of an unlimited supply of cells that can be differentiated into specific cell types and tissues. Equally, a keen interest in the connection between plant stem cells and regeneration has been developed in the last decade, driven by the demand to enhance plant traits such as yield, resistance to pathogens, and the opportunities provided by CRISPR/Cas-mediated gene editing. Here we discuss how knowledge of stem cell biology benefits regeneration technology, and we speculate on the creation of a universal genotype-independent iPSC system for plants to overcome regenerative recalcitrance.

The advent of induced pluripotent stem cell (iPSC) technology has brought about transformative advancements in regenerative medicine, offering novel avenues for disease modeling, drug testing, and cell-based therapies. Patient-specific iPSC-based treatments hold the promise of mitigating immune rejection risks. However, the intricacies and costs of producing autologous therapies present commercial challenges. The hair follicle is a multi-germ layered versatile cell source that can be harvested at any age. It is a rich source of keratinocytes, fibroblasts, multipotent stromal cells, and the newly defined Hair Follicle-Associated Pluripotent Stem Cells (HAP). It can also be obtained non-invasively and transported via regular mail channels, making it the ideal starting material for an autologous biobank. In this study, cryopreserved hair follicle-derived iPSC lines (HF-iPS) were established through integration-free vectors, encompassing a diverse cohort. These genetically stable lines exhibited robust expression of pluripotency markers, and showcased tri-lineage differentiation potential. The HF-iPSCs effectively differentiated into double-positive cKIT+/CXCR4+ definitive endoderm cells and NKX6.1+/PDX1+ pancreatic progenitor cells, affirming their pluripotent attributes. We anticipate that the use of plucked hair follicles as an accessible, non-invasive cell source to obtain patient cells, in conjunction with the use of episomal vectors for reprogramming, will improve the future generation of clinically applicable pancreatic progenitor cells for the treatment of Type I Diabetes.

Neuronal injuries, as one of the consequences of sports-related incidents, exert a profound influence on the athletes' future, potentially leading to complete immobility and impeding their athletic pursuits. In cases of severe damage inflicted upon the spinal cord (SC) and peripheral nervous systems (PNS), the regenerative process is notably compromised, rendering it essentially inefficient. Among the pivotal therapeutic approaches for the enhancement and prevention of secondary SC injuries (SCI), stem cell transplantation (SCT) stands out prominently. Stem cells, whether directly involved in replacement and reconstruction or indirectly through modification and secretion of crucial bioenvironmental factors, engage in the intricate process of tissue regeneration. Stem cells, through the secretion of neurotrophic factors (NTFs) (aiming to modulate the immune system), reduction of inflammation, axonal growth stimulation, and myelin formation, endeavor to facilitate the regeneration of damaged SC tissue. The fundamental challenges of this approach encompass the proper selection of suitable stem cell candidates for transplantation and the establishment of an appropriate microenvironment conducive to SC repair. In this article, an attempt has been made to explore sports-related injuries, particularly SCI, to comprehensively review innovative methods for treating SCI, and to address the existing challenges. Additionally, some of the stem cells used in neural injuries and the process of their utilization have been discussed.

When skin is damaged or affected by diseases, it often undergoes irreversible scar formation, leading to aesthetic concerns and psychological distress for patients. In cases of extensive skin defects, the patient's life can be severely compromised. In recent years, 3D printing technology has emerged as a groundbreaking approach to skin tissue engineering, offering promising solutions to various skin-related conditions. 3D bioprinting technology enables the precise fabrication of structures by programming the spatial arrangement of cells within the skin tissue and subsequently printing skin replacements either in a 3D bioprinter or directly at the site of the defect. This study provides a comprehensive overview of various biopolymer-based inks, with a particular emphasis on chitosan (CS), starch, alginate, agarose, cellulose, and fibronectin, all of which are natural polymers belonging to the category of biomacromolecules. Additionally, it summarizes artificially synthesized polymers capable of enhancing the performance of these biomacromolecule-based bioinks, thereby composing hybrid biopolymer inks aimed at better application in skin tissue engineering endeavors. This review paper examines the recent advancements, characteristics, benefits, and limitations of biological 3D bioprinting techniques for skin tissue engineering. By utilizing bioinks containing seed cells, hydrogels with bioactive factors, and biomaterials, complex structures resembling natural skin can be accurately fabricated in a layer-by-layer manner. The importance of biological scaffolds in promoting skin wound healing and the role of 3D bioprinting in skin tissue regeneration processes is discussed. Additionally, this paper addresses the challenges and constraints associated with current 3D bioprinting technologies for skin tissue and presents future perspectives. These include advancements in bioink formulations, full-thickness skin bioprinting, vascularization strategies, and skin appendages bioprinting.

This review delves into the groundbreaking impact of induced pluripotent stem cells (iPSCs) and three-dimensional organoid models in propelling forward neuropathology research. With a focus on neurodegenerative diseases, neuromotor disorders, and related conditions, iPSCs provide a platform for personalized disease modeling, holding significant potential for regenerative therapy and drug discovery. The adaptability of iPSCs, along with associated methodologies, enables the generation of various types of neural cell differentiations and their integration into three-dimensional organoid models, effectively replicating complex tissue structures in vitro. Key advancements in organoid and iPSC generation protocols, alongside the careful selection of donor cell types, are emphasized as critical steps in harnessing these technologies to mitigate tumorigenic risks and other hurdles. Encouragingly, iPSCs show promising outcomes in regenerative therapies, as evidenced by their successful application in animal models.

Atrial fibrillation (AF), the most common arrhythmia, has been associated with different electrophysiological, molecular, and structural alterations in atrial cardiomyocytes. Therefore, more studies are required to elucidate the genetic and molecular basis of AF. Various genome-wide association studies (GWAS) have strongly associated different single nucleotide polymorphisms (SNPs) with AF. One of these GWAS identified the rs13376333 risk SNP as the most significant one from the 1q21 chromosomal region. The rs13376333 risk SNP is intronic to the KCNN3 gene that encodes for small conductance calcium-activated potassium channels type 3 (SK3). However, the functional electrophysiological effects of this variant are not known. SK channels represent a unique family of K+ channels, primarily regulated by cytosolic Ca2+ concentration, and different studies support their critical role in the regulation of atrial excitability and consequently in the development of arrhythmias like AF. Since different studies have shown that both upregulation and downregulation of SK3 channels can lead to arrhythmias by different mechanisms, an important goal is to elucidate whether the rs13376333 risk SNP is a gain-of-function (GoF) or a loss-of-function (LoF) variant. A better understanding of the functional consequences associated with these SNPs could influence clinical practice guidelines by improving genotype-based risk stratification and personalized treatment. Although research using native human atrial cardiomyocytes and animal models has provided useful insights, each model has its limitations. Therefore, there is a critical need to develop a human-derived model that represents human physiology more accurately than existing animal models. In this context, research with human induced pluripotent stem cells (hiPSC) and subsequent generation of cardiomyocytes derived from hiPSC (hiPSC-CMs) has revealed the underlying causes of various cardiovascular diseases and identified treatment opportunities that were not possible using in vitro or in vivo studies with animal models. Thus, the ability to generate atrial cardiomyocytes and atrial tissue derived from hiPSCs from human/patients with specific genetic diseases, incorporating novel genetic editing tools to generate isogenic controls and organelle-specific reporters, and 3D bioprinting of atrial tissue could be essential to study AF pathophysiological mechanisms. In this review, we will first give an overview of SK-channel function, its role in atrial fibrillation and outline pathophysiological mechanisms of KCNN3 risk SNPs. We will then highlight the advantages of using the hiPSC-CM model to investigate SNPs associated with AF, while addressing limitations and best practices for rigorous hiPSC studies.

Proteins targeted by the ubiquitin proteasome system (UPS) are identified for degradation by the proteasome, which has been implicated in the development of neurodegenerative diseases. Major histocompatibility complex (MHC) molecules present peptides broken down by the proteasome and are involved in neuronal plasticity, regulating the synapse number and axon regeneration in the central or peripheral nervous system during development and in brain diseases. The mechanisms governing these effects are mostly unknown, but evidence from different compartments of the cerebral cortex indicates the presence of immune-like MHC receptors in the central nervous system.

We used human induced pluripotent stem cells (iPSCs) differentiated into neural stem cells and then into motor neurons as a developmental model to better understand the structure of the proteasome in developing motor neurons. We performed a proteomic analysis of starting human skin fibroblasts, their matching iPSCs, differentiated neural stem cells and motor neurons that highlighted significant differences in the constitutive proteasome and immunoproteasome subunits during development toward motor neurons from iPSCs.

The proteomic analysis showed that the catalytic proteasome subunits expressed in fibroblasts differed from those in the neural stem cells and motor neurons. Western blot analysis confirmed the proteomic data, particularly the decreased expression of the β5i (PSMB8) subunit immunoproteasome in MNs compared to HFFs and increased β5 (PSMB5) in MNs compared to HFFs.

The constitutive proteasome subunits are upregulated in iPSCs and NSCs from HFFs. Immunoproteasome subunit β5i expression is higher in MNs than NSCs; however, overall, there is more of a constitutive proteasome structure in MNs when comparing HFFs to MNs. The proteasome composition may have implications for motor neuron development and neurodevelopmental diseases that warrant further investigation.

Various factors are known to contribute to the diversity of human induced pluripotent stem cells (hiPSCs). Among these are the donor's genetic background and family history, the somatic cell source, the iPSC reprogramming method, and the culture system of choice. Moreover, variability is seen even in iPSC clones, generated in a single reprogramming event, where the donor, somatic cell type, and reprogramming platform are the same. The diversity seen in iPSC lines often translates to epigenetic differences, as well as to differences in the expansion rate, iPSC line culture robustness, and their ability to differentiate into specific cell types. As such, the diversity of iPSCs presents a hurdle to standardizing iPSC-based cell therapy manufacturing. In this review, we will expand on the various factors that impact iPSC diversity and the strategies and tools that could be taken by the industry to overcome the differences amongst various iPSC lines, therefore enabling robust and reproducible iPSC-based cell therapy manufacturing processes.

In situ physiological signals of in vitro neural disease models are essential for studying pathogenesis and drug screening. Currently, an increasing number of in vitro neural disease models are established using human-induced pluripotent stem cell (hiPSC) derived neurons (hiPSC-DNs) to overcome interspecific gene expression differences. Microelectrode arrays (MEAs) can be readily interfaced with two-dimensional (2D), and more recently, three-dimensional (3D) neural stem cell-derived in vitro models of the human brain to monitor their physiological activity in real time. Therefore, MEAs are emerging and useful tools to model neurological disorders and disease in vitro using human iPSCs. This is enabling a real-time window into neuronal signaling at the network scale from patient derived. This paper provides a comprehensive review of MEA's role in analyzing neural disease models established by hiPSC-DNs. It covers the significance of MEA fabrication, surface structure and modification schemes for hiPSC-DNs culturing and signal detection. Additionally, this review discusses advances in the development and use of MEA technology to study in vitro neural disease models, including epilepsy, autism spectrum developmental disorder (ASD), and others established using hiPSC-DNs. The paper also highlights the application of MEAs combined with hiPSC-DNs in detecting in vitro neurotoxic substances. Finally, the future development and outlook of multifunctional and integrated devices for in vitro medical diagnostics and treatment are discussed.

The present study investigates the impact of two endocrine disruptors, namely Bisphenols (BPs) and Perfluoroalkyls (PFs), on human stem cells. These chemicals leach from plastic, and when ingested through contaminated food and water, they interfere with endogenous hormone signaling, causing various diseases. While the ability of BPs and PFs to cross the placental barrier and accumulate in fetal serum has been documented, the exact consequences for human development require further elucidation. The present research work explored the effects of combined exposure to BPs (BPA or BPS) and PFs (PFOS and PFOA) on human placenta (fetal membrane mesenchymal stromal cells, hFM-MSCs) and amniotic fluid (hAFSCs)-derived stem cells. The effects of the xenobiotics were assessed by analyzing cell proliferation, mitochondrial functionality, and the expression of genes involved in pluripotency and epigenetic regulation, which are crucial for early human development. Our findings demonstrate that antenatal exposure to BPs and/or PFs may alter the biological characteristics of perinatal stem cells and fetal epigenome, with potential implications for health outcomes at birth and in adulthood. Further research is necessary to comprehend the full extent of these effects and their long-term consequences.

The ELMOD3 gene is implicated in causing autosomal recessive/dominant non-syndromic hearing loss in humans. However, the etiology has yet to be completely elucidated. In this study, we generated a patient-derived iPSC line carrying ELMOD3 c.512A>G mutation. In addition, the patient-derived iPSC line was corrected by CRISPR/Cas9 genome editing system. Then we applied RNA sequencing profiling to compare the patient-derived iPSC line with different controls, respectively (the healthy sibling-derived iPSCs and the CRISPR/Cas9 corrected iPSCs). Functional enrichment and PPI network analysis revealed that differentially expressed genes (DEGs) were enriched in the gene ontology, such as sensory epithelial development, intermediate filament cytoskeleton organization, and the regulation of ion transmembrane transport. Our current work provided a new tool for studying how disruption of ELMOD3 mechanistically drives hearing loss.

Identifying cancer type-specific genes that define cell states is important to develop effective therapies for patients and methods for detection, early diagnosis, and prevention. While molecular mechanisms that drive malignancy have been identified for various cancers, the identification of cell-type defining transcription factors (TFs) that distinguish normal cells from cancer cells has not been fully elucidated. Here, we utilized a network biology framework, which assesses the fidelity of cell fate conversions, to identify cancer type-specific gene regulatory networks (GRN) for 17 types of cancer. Through an integrative analysis of a compendium of expression data, we elucidated core TFs and GRNs for multiple cancer types. Moreover, by comparing normal tissues and cells to cancer type-specific GRNs, we found that the expression of key network-influencing TFs can be utilized as a survival prognostic indicator for a diverse cohort of cancer patients. These findings offer a valuable resource for exploring cancer type-specific networks across a broad range of cancer types.

The emergence and development of induced pluripotent stem cells (iPSCs) provides an approach to understand the regulatory mechanisms of cell pluripotency and demonstrates the great potential of iPSCs in disease modeling. Acute myelitis defines a group of inflammatory diseases that cause acute nerve damage in the spinal cord; however, its pathophysiology remains to be elusive. In this study, we derived skin fibroblasts from a patient with acute myelitis (P-HAF) and then reprogrammed P-HAF cells to iPSCs using eight exogenous factors (namely, OCT4, SOX2, c-MYC, KLF4, NANOG, LIN28, RARG, and LRH1). We performed transcriptomic analysis of the P-HAF and compared the biological characteristics of the iPSCs derived from the patient (P-iPSCs) with those derived from normal individuals in terms of pluripotency, transcriptomic characteristics, and differentiation ability toward the ectoderm. Compared to the control iPSCs, the P-iPSCs displayed similar features of pluripotency and comparable capability of ectoderm differentiation in the specified culture. However, when tested in the common medium, the P-iPSCs showed attenuated potential for ectoderm differentiation. The transcriptomic analysis revealed that pathways enriched in P-iPSCs included those involved in Wnt signaling. To this end, we treated iPSCs and P-iPSCs with the Wnt signaling pathway inhibitor IWR1 during the differentiation process and found that the expression of the ectoderm marker Sox1 was increased significantly in P-iPSCs. This study provides a novel approach to investigating the pathogenesis of acute myelitis.

Pluripotent stem cells offer unique avenues to study human-specific aspects of disease and are a highly versatile tool in cancer research. Oncogenic processes and developmental programs often share overlapping transcriptomic and epigenetic signatures, which can be reactivated in induced pluripotent stem cells. With the emergence of brain organoids, the ability to recapitulate brain development and structure has vastly improved, making in vitro models more realistic and hence more suitable for biomedical modeling. This review highlights recent research and current challenges in human pluripotent stem cell modeling of brain and neural crest neoplasms, and concludes with a call for more rigorous quality control and for the development of models for rare tumor subtypes.

Degenerative musculoskeletal diseases (DMDs), including osteoporosis, osteoarthritis, degenerative disc disease, and sarcopenia, present major challenges in the aging population. Patients with DMDs present with pain, functional decline, and reduced exercise tolerance, which result in long-term or permanent deficits in their ability to perform daily activities. Current strategies for dealing with this cluster of diseases focus on relieving pain, but they have a limited capacity to repair function or regenerate tissue. Cell-based therapies have attracted considerable attention in recent years owing to their unique mechanisms of action and remarkable effects on regeneration. In this review, current experimental attempts to use cell-based therapies for DMDs are highlighted, and the modes of action of different cell types and their derivatives, such as exosomes, are generalized. In addition, the latest findings from state-of-the-art clinical trials are reviewed, approaches to improve the efficiency of cell-based therapies are summarized, and unresolved questions and potential future research directions for the translation of cell-based therapies are identified.

Cardiovascular diseases (CVDs) are pointed out by the World Health Organization (WHO) as the leading cause of death, contributing to a significant and growing global health and economic burden. Despite advancements in clinical approaches, there is a critical need for innovative cardiovascular treatments to improve patient outcomes. Therapies based on adult stem cells (ASCs) and embryonic stem cells (ESCs) have emerged as promising strategies to regenerate damaged cardiac tissue and restore cardiac function. Moreover, the generation of human induced pluripotent stem cells (iPSCs) from somatic cells has opened new avenues for disease modeling, drug discovery, and regenerative medicine applications, with fewer ethical concerns than those associated with ESCs. Herein, we provide a state-of-the-art review on the application of human pluripotent stem cells in CVD research and clinics. We describe the types and sources of stem cells that have been tested in preclinical and clinical trials for the treatment of CVDs as well as the applications of pluripotent stem-cell-derived in vitro systems to mimic disease phenotypes. How human stem-cell-based in vitro systems can overcome the limitations of current toxicological studies is also discussed. Finally, the current state of clinical trials involving stem-cell-based approaches to treat CVDs are presented, and the strengths and weaknesses are critically discussed to assess whether researchers and clinicians are getting closer to success.

Induced pluripotent stem cells (iPSCs) have entered an unprecedented state of development since they were first generated. They have played a critical role in disease modeling, drug discovery, and cell replacement therapy, and have contributed to the evolution of disciplines such as cell biology, pathophysiology of diseases, and regenerative medicine. Organoids, the stem cell-derived 3D culture systems that mimic the structure and function of organs in vitro, have been widely used in developmental research, disease modeling, and drug screening. Recent advances in combining iPSCs with 3D organoids are facilitating further applications of iPSCs in disease research. Organoids derived from embryonic stem cells, iPSCs, and multi-tissue stem/progenitor cells can replicate the processes of developmental differentiation, homeostatic self-renewal, and regeneration due to tissue damage, offering the potential to unravel the regulatory mechanisms of development and regeneration, and elucidate the pathophysiological processes involved in disease mechanisms. Herein, we have summarized the latest research on the production scheme of organ-specific iPSC-derived organoids, the contribution of these organoids in the treatment of various organ-related diseases, in particular their contribution to COVID-19 treatment, and have discussed the unresolved challenges and shortcomings of these models.

Over 40,000 patients in the United States are estimated to suffer from end-stage liver disease and acute hepatic failure, for which liver transplantation is the only available therapy. Human primary hepatocytes (HPH) have not been employed as a therapeutic tool due to the difficulty in growing and expanding them in vitro, their sensitivity to cold temperatures, and tendency to dedifferentiate following two-dimensional culture. The differentiation of human-induced pluripotent stem cells (hiPSCs) into liver organoids (LO) has emerged as a potential alternative to orthotropic liver transplantation (OLT). However, several factors limit the efficiency of liver differentiation from hiPSCs, including a low proportion of differentiated cells capable of reaching a mature phenotype, the poor reproducibility of existing differentiation protocols, and insufficient long-term viability in vitro and in vivo. This review will analyze various methodologies being developed to improve hepatic differentiation from hiPSCs into liver organoids, paying particular attention to the use of endothelial cells as supportive cells for their further maturation. Here, we demonstrate why differentiated liver organoids can be used as a research tool for drug testing and disease modeling, or employed as a bridge for liver transplantation following liver failure.

Tissue engineering uses cells and biomaterials to develop bioartificial tissue substitutes for different purposes. For example, although several skin models have been developed for pharmaceutical and cosmetic research and skin wound healing, there are few studies on 3D cultures of keratinocytes in 3D printed scaffolds. So, this work aimed to develop a 3D-printed hydrogel scaffold to promote human keratinocyte growth. Mesh 3D scaffolds were printed using an extrusion-based method with a 20% gelatin/5% alginate hydrogel, where HaCaT cells were cultured for 7 days. Scaffolds kept their structure for over 1 week, and their stiffness only decreased after 7 days, showing good mechanical and structural characteristics and biodegradability (27% weight lost). Viable keratinocytes (MTT assay) are aggregated into spheroids, a 3D model capable of mimicking in vivo cell properties and phenotypes. Spheroids were formed on 47% of scaffolds pores and grew over time, showing promising cell proliferation. F-actin staining showed cells' irregular and interconnected shapes and organization over time. This method offers an easy and inexpensive solution for keratinocyte spheroid formation, which may be helpful in tissue engineering as a cell delivery system, for pharmacological or basic research, or wound healing medical applications.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are cells with promising applications. However, their immaturity has restricted their use in cell therapy, disease modeling, and other studies. Therefore, the current study focused on inducing the maturation of CMs. We supplemented hiPSC-CMs with fatty acids (FAs) to promote their phenotypic maturity. Proteomic sequencing was performed to identify regulators critical for promoting the maturation of hiPSC-CMs. AKAP1 was found to be significantly increased in FA-treated hiPSC-CMs, and the results were verified. Therefore, we inhibited AKAP1 expression in the FA-treated cells and analyzed the outcomes. FA supplementation promoted the morphological and functional maturation of the hiPSC-CMs, which was accompanied by the development of a mitochondrial network. Proteomic analysis results revealed that AKAP1 expression was significantly higher in FA-treated hiPSC-CMs than in control cells. In addition, increased phosphorylation of the mitochondrial dynamin Drp1 and an increased mitochondrial fusion rate were found in FA-treated hiPSC-CMs. After AKAP1 was knocked down, the level of DRP1 phosphorylation in the cell was decreased, and the mitochondrial fusion rate was reduced. FA supplementation effectively promoted the maturation of hiPSC-CMs, and in these cells, AKAP1 regulated mitochondrial dynamics, possibly playing a significant role.

As the global number of chronic wound patients rises, the financial burden and social pressure on patients increase daily. Stem cells have emerged as promising tissue engineering seed cells due to their enriched sources, multidirectional differentiation ability, and high proliferation rate. However, delivering them in vitro for the treatment of skin injury is still challenging. In addition, bacteria from the wound site and the environment can significantly impact wound healing. In the last decade, 3D bioprinting has dramatically enriched cell delivery systems. The produced scaffolds by this technique can be precisely localized within cells and perform antibacterial actions. In this review, we summarized the 3D bioprinting-based external delivery of stem cells and their antibiosis to improve wound healing.

Initiating from multipotent progenitors, the lineages extrapolated from hematopoietic stem cells are determined by transcription factors specific to each of them. The commitment factors assist in the differentiation of progenitor cells into terminally differentiated cells. B lymphocytes constitute a population of cells that expresses clonally diverse cell surface immunoglobulin (Ig) receptors specific to antigenic epitopes. B cells are a significant facet of the adaptive immune system. The secreted antibodies corresponding to the B cell recognize the antigens via the B cell receptor (BCR). Following antigen recognition, the B cell is activated and thereafter undergoes clonal expansion and proliferation to become memory B cells. The essence of 'cellular reprogramming' has aided in reliably altering the cells to desired tissue type. The potential of reprogramming has been harnessed to decipher and find solutions for various genetically inherited diseases and degenerative disorders. B lymphocytes can be reprogrammed to their initial naive state from where they get differentiated into any lineage or cell type similar to a pluripotent stem cell which can be accomplished by the deletion of master regulators of the B cell lineage. B cells can be reprogrammed into pluripotent stem cells and also can undergo transdifferentiation at the midway of cell differentiation to other cell types. Mandated expression of C/EBP in specialized B cells corresponds to their fast and effective reprogramming into macrophages, reversing the cell fate of these lymphocytes and allowing them to differentiate freshly into other types of cells. The co-expression of C/EBPα and OKSM (Oct4, Sox2, Klf4, c-Myc) amplified the reprogramming efficiency of B lymphocytes. Various human somatic cells including the immune cells are compliant to reprogramming which paves a path for opportunities like autologous tissue grafts, blood transfusion, and cancer immunotherapy. The ability to reprogram B cells offers an unprecedented opportunity for developing a therapeutic approach for several human diseases. Here, we will focus on all the proteins and transcription factors responsible for the developmental commitment of B lymphocytes and how it is harnessed in various applications.

Background: We had shown that cardiomyocytes (CMs) were more efficiently differentiated from human induced pluripotent stem cells (hiPSCs) when the hiPSCs were reprogrammed from cardiac fibroblasts rather than dermal fibroblasts or blood mononuclear cells. Here, we continued to investigate the relationship between somatic-cell lineage and hiPSC-CM production by comparing the yield and functional properties of CMs differentiated from iPSCs reprogrammed from human atrial or ventricular cardiac fibroblasts (AiPSC or ViPSC, respectively). Methods: Atrial and ventricular heart tissues were obtained from the same patient, reprogrammed into AiPSCs or ViPSCs, and then differentiated into CMs (AiPSC-CMs or ViPSC-CMs, respectively) via established protocols. Results: The time-course of expression for pluripotency genes (OCT4, NANOG, and SOX2), the early mesodermal marker Brachyury, the cardiac mesodermal markers MESP1 and Gata4, and the cardiovascular progenitor-cell transcription factor NKX2.5 were broadly similar in AiPSC-CMs and ViPSC-CMs during the differentiation protocol. Flow-cytometry analyses of cardiac troponin T expression also indicated that purity of the two differentiated hiPSC-CM populations (AiPSC-CMs: 88.23% ± 4.69%, ViPSC-CMs: 90.25% ± 4.99%) was equivalent. While the field-potential durations were significantly longer in ViPSC-CMs than in AiPSC-CMs, measurements of action potential duration, beat period, spike amplitude, conduction velocity, and peak calcium-transient amplitude did not differ significantly between the two hiPSC-CM populations. Yet, our cardiac-origin iPSC-CM showed higher ADP and conduction velocity than previously reported iPSC-CM derived from non-cardiac tissues. Transcriptomic data comparing iPSC and iPSC-CMs showed similar gene expression profiles between AiPSC-CMs and ViPSC-CMs with significant differences when compared to iPSC-CM derived from other tissues. This analysis also pointed to several genes involved in electrophysiology processes responsible for the physiological differences observed between cardiac and non-cardiac-derived cardiomyocytes. Conclusion: AiPSC and ViPSC were differentiated into CMs with equal efficiency. Detected differences in electrophysiological properties, calcium handling activity, and transcription profiles between cardiac and non-cardiac derived cardiomyocytes demonstrated that 1) tissue of origin matters to generate a better-featured iPSC-CMs, 2) the sublocation within the cardiac tissue has marginal effects on the differentiation process.

The search for reliable human blood-brain barrier (BBB) models represents a challenge for the development/testing of strategies aiming to enhance brain delivery of drugs. Human-induced pluripotent stem cells (hiPSCs) have raised hopes in the development of predictive BBB models. Differentiating strategies are thus required to generate endothelial cells (ECs), a major component of the BBB. Several hiPSC-based protocols have reported the generation of in vitro models with significant differences in barrier properties. We studied in depth the properties of iPSCs byproducts from two protocols that have been established to yield these in vitro barrier models. Our analysis/study reveals that iPSCs derivatives endowed with EC features yield high permeability models while the cells that exhibit outstanding barrier properties show principally epithelial cell-like (EpC) features. We found that models containing EpC-like cells express tight junction proteins, transporters/efflux pumps and display a high functional tightness with very low permeability, which are features commonly shared between BBB and epithelial barriers. Our study demonstrates that hiPSC-based BBB models need extensive characterization beforehand and that a reliable human BBB model containing EC-like cells and displaying low permeability is still needed.

Engineered tissues and cell therapies based on human induced pluripotent stem cells (iPSCs) represent a promising approach for novel medicines. However, iPSC-derived cells and tissues may contain residual undifferentiated iPSCs that could lead to teratoma formation after implantation into patients. As a consequence, highly sensitive and specific methods for detecting residual undifferentiated iPSCs are indispensable for safety evaluations of iPSC-based therapies. The present study provides an approach for identifying potential marker genes for iPSC impurities in iPSC-derived cells using RNA sequencing data from iPSCs and various differentiated cell types.

Identifying iPSC marker genes for each cell type individually provided a larger and more specific set of potential marker genes than considering all cell types in the analysis. Thus, the authors focused on identifying markers for iPSC impurities in iPSC-derived cardiomyocytes (iCMs) and validated the selected genes by reverse transcription quantitative polymerase chain reaction. The sensitivity of the candidate genes was determined by spiking different amounts of iPSCs into iCMs and their performance was compared with the previously suggested marker lin-28 homolog A (LIN28A).

Embryonic stem cell-related gene (ESRG), long intergenic non-protein coding RNA 678 (LINC00678), CaM kinase-like vesicle-associated (CAMKV), indoleamine 2,3-dioxygenase 1 (IDO1), chondromodulin (CNMD), LINE1-type transposase domain containing 1 (L1DT1), LIN28A, lymphocyte-specific protein tyrosine kinase (LCK), vertebrae development-associated (VRTN) and zinc finger and SCAN domain containing 10 (ZSCAN10) detected contaminant iPSCs among iCMs with a limit of detection that ranged from 0.001% to 0.1% depending on the gene and iCM batch used.

Using the example of iCMs, the authors provide a strategy for identifying a set of highly specific and sensitive markers that can be used for quality assessment of iPSC-derived products.

Lymphoblastoid cell lines (LCLs) deposited from disease-affected individuals could be a valuable donor cell source for generating disease-specific induced pluripotent stem cells (iPSCs). However, generation of iPSCs from the LCLs is still challenging, as yet no effective gene delivery strategy has been developed.

Here, we reveal an effective gene delivery method specifically for LCLs. We found that LCLs appear to be refractory toward retroviral and lentiviral transduction. Consequently, lentiviral and retroviral transduction of OCT4, SOX2, KFL4 and c-MYC into LCLs does not elicit iPSC colony formation. Interestingly, however we found that transfection of oriP/EBNA-1-based episomal vectors by electroporation is an efficient gene delivery system into LCLs, enabling iPSC generation from LCLs. These iPSCs expressed pluripotency makers (OCT4, NANOG, SSEA4, SALL4) and could form embryoid bodies.

Our data show that electroporation is an effective gene delivery method with which LCLs can be efficiently reprogrammed into iPSCs.

Mutations of the postsynaptic scaffold protein Shank2 lead to autism spectrum disorders (ASD). These patients frequently suffer from higher fracture risk. Here, we investigated whether Shank2 directly regulates bone mass. We show that Shank2 is expressed in bone and that Shank2 levels are increased during osteoblastogenesis. Knockdown of Shank2 by siRNA targeting the encoding regions for PDZ and SAM domain inhibits osteoblastogenesis of primary murine calvarial osteoblasts. Shank2 knockout mice (Shank2 ^-/-) have a decreased bone mass due to reduced osteoblastogenesis and bone formation, whereas bone resorption remains unaffected. Induced pluripotent stem cells (iPSCs)-derived osteoblasts from a loss-of-function Shank2 mutation in a patient showed a significantly reduced osteoblast differentiation potential. Moreover, silencing of known Shank2 interacting proteins revealed that a majority of them promote osteoblast differentiation. From this we conclude that Shank2 and interacting proteins known from the central nervous system are decisive regulators in osteoblast differentiation. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.