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Blood has an important role in the healthcare system, particularly in blood transfusions and immunotherapy. However, the occurrence of outbreaks of infectious diseases worldwide and seasonal fluctuations, blood shortages are becoming a major challenge. Moreover, the narrow specificity of immune cells hinders the widespread application of immune cell therapy. To address this issue, researchers are actively developing strategies for differentiating induced pluripotent stem cells (iPSCs) into blood cells in vitro . The establishment of iPSCs from terminally differentiated cells such as fibroblasts and blood cells is a straightforward process. However, there is need for further refinement of the protocols for differentiating iPSCs into immune cells and red blood cells to ensure their clinical applicability. This review aims to provide a comprehensive overview of the strategies and challenges facing the generation of iPSC-derived immune cells and red blood cells.

Generation of thick vascularized tissues that fully match the patient still remains an unmet challenge in cardiac tissue engineering. Here, a simple approach to 3D‐print thick, vascularized, and perfusable cardiac patches that completely match the immunological, cellular, biochemical, and anatomical properties of the patient is reported. To this end, a biopsy of an omental tissue is taken from patients. While the cells are reprogrammed to become pluripotent stem cells, and differentiated to cardiomyocytes and endothelial cells, the extracellular matrix is processed into a personalized hydrogel. Following, the two cell types are separately combined with hydrogels to form bioinks for the parenchymal cardiac tissue and blood vessels. The ability to print functional vascularized patches according to the patient's anatomy is demonstrated. Blood vessel architecture is further improved by mathematical modeling of oxygen transfer. The structure and function of the patches are studied in vitro, and cardiac cell morphology is assessed after transplantation, revealing elongated cardiomyocytes with massive actinin striation. Finally, as a proof of concept, cellularized human hearts with a natural architecture are printed. These results demonstrate the potential of the approach for engineering personalized tissues and organs, or for drug screening in an appropriate anatomical structure and patient‐specific biochemical microenvironment. A small biopsy of an omental tissue is taken from patients. While the cells are reprogrammed to induce pluripotent stem cells and differentiate to cardiac and endothelial cells, the extra‐cellular matrix is processed into a thermoresponsive, hydrogel‐based bioink. These components are used to 3D‐print functional vascularized cardiac patches and even small scale, whole cellularized human hearts.

Induced pluripotent stem cell (iPSC) technologies have provided in vitro models of inaccessible human cell types, yielding new insights into disease mechanisms especially for neurological disorders. However, without due consideration, the thousands of new human iPSC lines generated in the past decade will inevitably affect the reproducibility of iPSC-based experiments. Differences between donor individuals, genetic stability and experimental variability contribute to iPSC model variation by impacting differentiation potency, cellular heterogeneity, morphology, and transcript and protein abundance. Such effects will confound reproducible disease modelling in the absence of appropriate strategies. In this Review, we explore the causes and effects of iPSC heterogeneity, and propose approaches to detect and account for experimental variation between studies, or even exploit it for deeper biological insight.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin disorder caused by mutations in the COL7A1 gene encoding type VII collagen (C7). The spectrum of severity depends on the type of mutation in the COL7A1 gene. C7 is the major constituent of anchoring fibrils (AFs) at the basement membrane zone (BMZ). Patients with RDEB lack functional C7 and have severely impaired dermal–epidermal stability, resulting in extensive blistering and open wounds on the skin that greatly affect the patient’s quality of life. There are currently no therapies approved for the treatment of RDEB. Here, we demonstrated the correction of mutations in exon 19 (c.2470insG) and exon 32 (c.3948insT) in the COL7A1 gene through homology-directed repair (HDR).We used the clustered regulatory interspaced short palindromic repeats (CRISPR) Cas9-gRNAs system to modify induced pluripotent stem cells (iPSCs) derived from patients with RDEB in both the heterozygous and homozygous states. Three-dimensional human skin equivalents (HSEs) were generated from gene-corrected iPSCs, differentiated into keratinocytes (KCs) and fibroblasts (FBs), and grafted onto immunodeficient mice, which showed normal expression of C7 at the BMZ as well as restored AFs 2 mo postgrafting. Safety assessment for potential offtarget Cas9 cleavage activity did not reveal any unintended nuclease activity. Our findings represent a crucial advance for clinical applications of innovative autologous stem cell-based therapies for RDEB.

Induced pluripotent stem cell-derived mesenchymal stem cells (iMSC) and primary MSC comparison: to show the advantages and applications of iMSC. Mesenchymal stem cells (MSC) isolated from different tissue sources exhibit multiple biological effects and have shown promising therapeutic effects in a broad range of diseases. In order to fulfill their clinical applications in context of precision medicine, however, more detailed molecular characterization of diverse subgroups and standardized scalable production of certain functional subgroups would be highly desired. Thus far, the generation of induced pluripotent stem cell (iPSC)-derived MSC (iMSC) seems to provide the unique opportunity to solve most obstacles that currently exist to prevent the broad application of MSC as an advanced medicinal product. The features of iMSC include their single cell clone origins, and defined and controllable cultural conditions for their derivation and proliferation. Still, comprehensive research of the molecular and functional heterogeneity of iMSC, just like MSC from any other tissue types, would be required. Furthered on previous efforts on iMSC differentiation and expansion platform and transcriptomic studies, advantages of single cell multi-omics analysis and other up-to-dated technologies would be taken in order to elucidate the molecular origin and regulation of heterogeneity and to obtain iMSC subgroups homogeneous enough for particular clinical conditions. In this perspective, the current obstacles in MSC applications, the advantages of iMSC over MSC and their implications for biological research and clinical applications will be discussed.

T cells derived from gene-edited induced pluripotent stem cells (iPSCs) are a promising alternative cell source for universal T-cell immunotherapy. However, current preclinical evaluations of iPSC-derived T cells (iPSC-T cells) rely on immunodeficient mouse models, which limit the assessment of immune-related adverse events and in vivo immune cell interactions. To overcome these limitations, we developed a preclinical nonhuman primate model to evaluate the safety and cellular kinetics of iPSC-T cells. iPSCs were generated from peripheral blood T cells obtained from two rhesus macaques and redifferentiated in vitro into CD8αβ CD3 innate-like T cells. Phenotypic and functional characterization was performed using flow cytometric analyses. The proliferative capacity of iPSC-T cells was assessed by repeated stimulation with phytohemagglutinin (PHA), and cytotoxic function was evaluated through co-culture assays with target cells. The cells were further transduced with GFP using retroviral vectors during expansion. Autologous GFP⁺ iPSC-T cells were administered to the donor macaques in three separate infusions to assess in vivo safety and cellular kinetics. The iPSC-T cells exhibited both antigen-dependent and antigen-independent cytotoxicity and demonstrated robust proliferative capacity upon repeated stimulation. Stable GFP expression was maintained during cell expansion. Following autologous infusion, no safety concerns were observed for up to one year after the first administration. Cellular kinetic analyses revealed that the infused iPSC-T cells trafficked to the alveolar space and were no longer detectable in peripheral circulation by seven days post-infusion. These findings establish a unique immunocompetent primate model for assessing the safety and behavior of iPSC-T cells. This platform enables more physiologically relevant preclinical evaluation and supports the development of iPSC-derived T-cell immunotherapies for cancer and autoimmune diseases.

With the world’s population ageing, the incidence of Parkinson’s disease (PD) is on the rise. In recent years, inflammatory processes have emerged as prominent contributors to the pathology of PD. There is great evidence that microglia have a significant neuroprotective role, and that impaired and over activated microglial phenotypes are present in brains of PD patients. Thereby, PD progression is potentially driven by a vicious cycle between dying neurons and microglia through the instigation of oxidative stress, mitophagy and autophagy dysfunctions, a-synuclein accumulation, and pro-inflammatory cytokine release. Hence, investigating the involvement of microglia is of great importance for future research and treatment of PD. The purpose of this review is to highlight recent findings concerning the microglia-neuronal interplay in PD with a focus on human postmortem immunohistochemistry and single-cell studies, their relation to animal and iPSC-derived models, newly emerging technologies, and the resulting potential of new anti-inflammatory therapies for PD.

Induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) serve as a unique source for cell therapy. We investigated whether exosomes from iMSCs promote the proliferation of human keratinocytes (HaCaT) and human dermal fibroblasts (HDFs). iPSCs were established from human Wharton’s jelly MSCs and were allowed to differentiate into iMSCs. Exosomes were collected from the culture supernatant of MSCs (MSC-exo) and iMSCs (iMSC-exo), and their characteristics were investigated. Both exosome types possessed basic characteristics of exosomes and were taken up by skin cells in vitro and in vivo. A significant increase in HaCaT proliferation was observed with iMSC-exo, although both exosomes increased the viability and cell cycle progression in HaCaT and HDFs. No significant difference was observed in the closure of wound scratch and the expression of reparative genes between cells treated with the two exosome types. Both exosomes enhanced the secretion of collagen in HaCaT and HDFs; however, an increase in fibronectin level was observed only in HaCaT, and this effect was better with iMSC-exo treatment. Only iMSC-exo increased the phosphorylation of extracellular signal-regulated kinase (ERK)-1/2. Our results indicate that iMSC-exo promote the proliferation of skin cells by stimulating ERK1/2 and highlight the application of iMSCs for producing exosomes.

Background Microglia, the principle immune cells of the brain, play important roles in neuronal development, homeostatic function and neurodegenerative disease. Recent genetic studies have further highlighted the importance of microglia in neurodegeneration with the identification of disease risk polymorphisms in many microglial genes. To better understand the role of these genes in microglial biology and disease, we, and others, have developed methods to differentiate microglia from human induced pluripotent stem cells (iPSCs). While the development of these methods has begun to enable important new studies of microglial biology, labs with little prior stem cell experience have sometimes found it challenging to adopt these complex protocols. Therefore, we have now developed a greatly simplified approach to generate large numbers of highly pure human microglia. Results iPSCs are first differentiated toward a mesodermal, hematopoietic lineage using commercially available media. Highly pure populations of non-adherent CD43 + hematopoietic progenitors are then simply transferred to media that includes three key cytokines (M-CSF, IL-34, and TGFβ-1) that promote differentiation of homeostatic microglia. This updated approach avoids the prior requirement for hypoxic incubation, complex media formulation, FACS sorting, or co-culture, thereby significantly simplifying human microglial generation. To confirm that the resulting cells are equivalent to previously developed iPSC-microglia, we performed RNA-sequencing, functional testing, and transplantation studies. Our findings reveal that microglia generated via this simplified method are virtually identical to iPS-microglia produced via our previously published approach. To also determine whether a small molecule activator of TGFβ signaling (IDE1) can be used to replace recombinant TGFβ1, further reducing costs, we examined growth kinetics and the transcriptome of cells differentiated with IDE1. These data demonstrate that a microglial cell can indeed be produced using this alternative approach, although transcriptional differences do occur that should be considered. Conclusion We anticipate that this new and greatly simplified protocol will enable many interested labs, including those with little prior stem cell or flow cytometry experience, to generate and study human iPS-microglia. By combining this method with other advances such as CRISPR-gene editing and xenotransplantation, the field will continue to improve our understanding of microglial biology and their important roles in human development, homeostasis, and disease.