Human Pluripotent Stem Cells Research Articles

Enhancing cryopreservation of human induced pluripotent stem cells: Bottom-up versus conventional freezing geometry.

Induced pluripotent stem cells (iPSCs) hold large potential in regenerative medicine due to their pluripotency and unlimited self-renewal capacity without the ethical issues of embryonic stem cells. To provide quality-controlled iPSCs for clinical therapies, it is essential to develop safe cryopreservation protocols for long-term storage, preferably amenable to scale-up and automation. We have compared the impact of two different freezing geometries (bottom-up and conventional radial freezing) on the viability and differentiation potential of human iPSCs. Our results demonstrate that bottom-up freezing under optimized conditions significantly increases iPSC viability, up to 9% for cell membrane integrity and up to 21% for cell metabolic state, compared to conventional freezing. The improvement achieved for bottom-up versus conventional freezing was maintained after scale-up from cryogenic vials to 30 mL bags, highlighting its potential for clinical applications. These findings show that bottom-up freezing can offer a more controlled and scalable cryopreservation strategy for iPSCs, promoting their application in regenerative medicine.

A systematic review and embryological perspective of pluripotent stem cell-derived autonomic postganglionic neuron differentiation for human disease modelling.

Human autonomic neuronal cell models are emerging as tools for modelling diseases such as cardiac arrhythmias. In this systematic review, we compared thirty-three articles applying fourteen different protocols to generate sympathetic neurons and three different procedures to produce parasympathetic neurons. All methods involved the differentiation of human pluripotent stem cells, and none employed permanent or reversible cell immortalization. Almost all protocols were reproduced in multiple pluripotent stem cell lines, and over half show evidence of neural firing capacity. Common limitations in the field are a lack of three-dimensional models and models including multiple cell types. Sympathetic neuron differentiation protocols largely mirrored embryonic development, with the notable absence of migration, axon extension, and target-specificity cues. Parasympathetic neuron differentiation protocols may be improved by including several embryonic cues promoting cell survival, cell maturation, or ion channel expression. Moreover, additional markers to define parasympathetic neurons in vitro may support the validity of these protocols. Nonetheless, four sympathetic neuron differentiation protocols and one parasympathetic neuron differentiation protocol reported more than two thirds of cells expressing autonomic neuron markers. Altogether, these protocols promise to open new research avenues of human autonomic neuron development and disease modelling.

Pluripotent stem cell-derived mesenchymal stem cells for therapeutic applications, developmental study, and cancer research.

Pluripotent stem cell-derived mesenchymal stem cells for therapeutic applications, developmental study, and cancer research.

Cathepsin K inhibition promotes efficient differentiation of human embryonic stem cells to mature cardiomyocytes by mediating glucolipid metabolism and cellular energy homeostasis

Background and aimGeneration of functional cardiomyocytes from human pluripotent stem cells (hPSCs) offers promising applications for cardiac regenerative medicine. Proper control of pluripotency and differentiation is vital for generating high-quality cardiomyocytes and repairing damaged myocardium. Cathepsin K, a lysosomal cysteine protease, is a potential target for cardiovascular disease treatment; however, its role in cardiomyocyte differentiation and regeneration is unclear. This study aims to investigate the effects and mechanisms of cathepsin K inhibition on the differentiation of human embryonic stem cell-induced cardiomyocytes (hESC-CMs) and myocardial generation.MethodsWe cultured H9-hESCs in CDM3 medium to induce myocardial differentiation, adding cathepsin K inhibitor II (1 μM) on days 2, 5 and 8, respectively. Cells were observed and collected 48 h after each treatment. The morphology and contractile clusters of H9-hESCs were tracked with microscopy and video recording. Pluripotency and cardiac markers were assessed at each stage of differentiation. We also examined glucose and lipid metabolism, mitochondrion-related markers, apoptosis and autophagy.ResultsCDM3 medium effectively differentiated high-density H9-hESCs into mature, spontaneously contracting cardiomyocytes. Cathepsin K inhibition accelerates the differentiation of H9-hESCs into cardiac mesoderm and cardiac precursor cells (CPCs) by reducing apoptosis, decreasing glycolysis and fatty acid metabolism at the early and middle stages, and subsequently facilitate the development and differentiation of cardiomyocytes by enhancing glucolipid metabolism and oxidative phosphorylation at the late stage. Meanwhile, cathepsin K inhibition enhanced mitochondrial function and lysosome-related gene transcription during the differentiation process.ConclusionOur study highlights the potential of cathepsin K inhibition for renewable cardiomyocytes and suggests exploring metabolic pathways and signaling to improve cardiac regeneration and organoid development.Graphical abstract

Establishment of vascularized human retinal organoids from induced pluripotent stem cells.

Stem cell-derived retinal organoids (ROs) have been investigated for applications in regenerative medicine, retinal disease models, and compound safety evaluation. Although the development of 3D organoids has provided novel opportunities for innovation, some unresolved limitations continue to exist in organoid research. The passive diffusion of oxygen and nutrients limits the growth and functional gain of organoids. Vascularization may circumvent these problems because it allows oxygen and nutrients to enter the organoid core. In the present study, ROs and vascular organoids (VOs) were generated from healthy human induced pluripotent stem cells. We attempted to create vascular-like structures in ROs by co-culturing them with VO-derived vascular endothelial cells/pericytes. Our vascularized retinal organoids (vROs) contained type IV collagen- and CD31-positive vascular-like structures. The expression of the mature neuronal marker SMI-32 and SNCG was markedly higher in the vROs than in the ROs. When vROs were cultured under conditions that mimicked diabetes, their size and the number of retinal ganglion cells were significantly decreased. In conclusion, the co-culture of ROs with VO-derived cells enabled the production of ROs with vascular-like structures, and the vROs responded to severe diabetic retinopathy conditions. In summary, our findings underscore the potential of vROs as invaluable tools for elucidating disease mechanisms and screening therapeutic interventions for retinal vascular disorders, thereby paving the way for personalized medicine approaches in ophthalmology.

Phenotypic similarity of spheroids of native chondrocytes and spheroids of chondrocytes differentiated from human induced pluripotent stem cells using recombinant factors TGF-β1 and BMP2

BACKGROUND: Chronic joint diseases represent a significant medical and social problem due to their prevalence, frequent disability of patients, and associated economic losses. Possible therapeutic approaches include cell-based technologies using the patient's chondrocytes, which are obtained surgically and cultured in vitro before transplantation. Recently, chondrocytes have increasingly been cultured using 3D cultivation techniques - in the form of spheroids, which allows better preservation of the functional state of chondrocytes and provides conditions for more optimal maturation of cartilage tissue. However, the quantity and quality of the patient's chondrocytes may be insufficient to produce enough cellular material to compensate for extensive articular cartilage defects fully. Chondrocytes obtained by differentiation of induced pluripotent stem cells (iPSCs) may be an alternative source of cells. AIM: This study aimed to compare, at the immunophenotypic level, spheroids of native chondrocytes with spheroids of chondrocytes derived from human iPSCs. MATERIALS AND METHODS: This article presents detailed protocols for obtaining spheroids of native chondrocytes and spheroids of chondrocytes differentiated from iPSCs using mini-bioreactors and recombinant factors TGF-β1 and BMP2. Using immunocytochemical (ICX) staining and quantitative RT-PCR, we compared spheroids of native chondrocytes and spheroids of chondrocytes differentiated from iPSCs by the expression of the primary chondrocyte markers: aggrecan (ACAN), collagens (COL1A2 and COL2A1) and the key transcription factor SOX9. RESULTS: iPSC-derived chondrocyte spheroids showed expression profiles nearly identical to those of native chondrocyte spheroids, with the notable exception of significantly elevated type I collagen (COL1A2) expression. CONCLUSION: These findings suggest that chondrocyte spheroids generated from iPSCs hold promise as a viable therapeutic approach for treating chronic joint diseases.

Approaches in vivo and in vitro for solving the vascularization issue of brain organoids

Organoids are three-dimensional tissue cultures generated using human pluripotent stem cells. It shows great potential in modeling human disorders, organogenesis, and developmental disorders. However, they have limitations in replicating the complexity of the human brain and lack a complete vasculature to support long-term cultivation and endogenous microenvironment simulation. Researchers are exploring strategies to promote vascularization in brain organoids, including in vitro co-culture methods and xenotransplantation into highly vascularized regions of animal hosts. The presence of a physiologically perfused vasculature in organoid models can prevent tissue necrosis, provide essential nutrients, and enable accurate modeling of interactions with non-neuronal cell types. In vivo, xenotransplantation shows advantages over in vitro approaches, such as active blood flow, and demonstrates potential benefits for aiding recovery from stroke by repairing damaged tissue structures and improving sensory-motor deficits.

Engineering Extracellular Vesicles Secreted by Human Brain Organoids with Different Regional Identity.

Extracellular vesicles (EVs) are membrane-bound nanovesicles that show significance in intercellular communications and high therapeutic potential. In this study, a novel type of EV subpopulation, matrix-bound nanovesicles (MBVs), was identified from a decellularized extracellular matrix of brain organoids that were derived from human pluripotent stem cells to compare with supernatant EVs (SuEVs) isolated from spent media. The organoids generated 10-fold more MBVs than did SuEVs. SuEVs contained more enriched microRNA cargo than MBVs, and the microRNA relative abundance changed during organoid maturation. The forebrain and hindbrain organoid SuEVs had a highly overlapped protein cargo based on proteomics analysis. More membrane proteins, including integrins, were identified in MBVs than SuEVs, which could contribute to MBV retention in matrices. Lipidomics data showed that MBVs were enriched in glycerophospholipids and sphingolipids, which affect the lipid membrane rigidity and recruitment of integral membrane proteins. To mimic ischemic stroke, in vitro oxygen and glucose deprivation model results revealed stronger recovery effects of MBVs than SuEVs at the same dose. The effects were exerted by regulating autophagy, reactive oxygen species scavenging, and anti-inflammatory ability. This study laid the foundation for advancing our knowledge of intercellular communication and for developing cell-free based therapies for treating neurological disorders such as ischemic stroke.

Protocol for differentiating hematopoietic progenitor cells from human pluripotent stem cells in chemically defined monolayer culture.

Protocol for differentiating hematopoietic progenitor cells from human pluripotent stem cells in chemically defined monolayer culture.

Engineering human cerebral organoids to explore mechanisms of arsenic-induced developmental neurotoxicity.

Engineering human cerebral organoids to explore mechanisms of arsenic-induced developmental neurotoxicity.

Human striatal progenitor cells that contain inducible safeguards and overexpress BDNF rescue Huntington's disease phenotypes.

Human striatal progenitor cells that contain inducible safeguards and overexpress BDNF rescue Huntington's disease phenotypes.

TLR7/8 signaling activation enhances the potency of human pluripotent stem cell-derived eosinophils in cancer immunotherapy for solid tumors

BackgroundEfficient tumor T-cell infiltration is crucial for the effectiveness of T-cell-based therapies against solid tumors. Eosinophils play crucial roles in recruiting T cells in solid tumors. Our group has previously generated induced eosinophils (iEOs) from human pluripotent stem cells and exhibited synergistic efficacy with CAR-T cells in solid tumor inhibition. However, administrated eosinophils might influx into inflammatory lungs, posing a potential safety risk. Mitigating the safety concern and enhancing efficacy is a promising development direction for further application of eosinophils.MethodsWe developed a new approach to generate eosinophils with enhanced potency from human chemically reprogrammed induced pluripotent stem cells (hCiPSCs) with the Toll-like receptor (TLR) 7/8 signaling agonist R848.ResultsR848-activated iEOs (R-iEOs) showed significantly decreased influx to the inflamed lungs, indicating a lower risk of causing airway disorders. Furthermore, these R-iEOs had enhanced anti-tumor functions, preferably accumulated at tumor sites, and further increased T-cell infiltration. The combination of R-iEOs and CAR-T cells suppressed tumor growth in mice. Moreover, the chemo-trafficking signaling increased in R-iEOs, which may contribute to the decreased lung influx of R-iEOs and the increased tumor recruitment of T cells.ConclusionOur study provides a novel approach to alleviate the potential safety concerns associated with eosinophils while increasing T-cell infiltration in solid tumors. This finding offers a prospective strategy for incorporating eosinophils to improve CAR-T-cell immunotherapy for solid tumors in the future.Graphical

HPSCs-derived brain organoids for disease modeling, toxicity testing and drug evaluation.

hPSCs-derived brain organoids for disease modeling, toxicity testing and drug evaluation.

Addressing myelination disorders: Novel strategies using human 3D peripheral nerve model.

Peripheral myelination disorders encompass a variety of disorders that affect myelin sheaths in the peripheral nervous system. The Charcot-Marie-Tooth disease (CMT), the most common inherited peripheral neuropathy, is one of the most prevalent among them. CMT stems from a wide range of genetic causes that disrupt the nerve conduction, leading to progressive muscle weakness and atrophy, sensory loss, and motor function impairment. Historically, the study of these disorders has relied heavily on animal studies, owing to the challenges in accessing human cells. However, the advent of human induced pluripotent stem cell (hiPSC)-derived neuronal cells has addressed these limitations in the realm of peripheral myelination disorders. Despite this, obtaining myelin in these models remains an expensive, time-consuming, and material-intensive process. This study presents a novel, cost-effective method utilizing hiPSC-derived Schwann cells and motor neurons in a three-dimensional culture system. Our method successfully enabled the acquisition of myelin in a control clone within just four weeks, as confirmed by electron microscopy. Furthermore, the utility of these approaches was validated by studying CMT4C, also named AR-CMTde-SH3TC2, the most common recessive demyelinating form of CMT. This revealed defects in Schwann cell support to motor neuron neurite outgrowth and impaired myelination in disease-specific hiPSC-derived lines. This approach offers valuable insights into the pathogenesis of peripheral myelination disorders and provides a platform for testing potential therapeutic strategies.

Establishing of human induced pluripotent stem cell line DMSCi002-A from the hematopoietic stem cells of a healthy male donor.

Establishing of human induced pluripotent stem cell line DMSCi002-A from the hematopoietic stem cells of a healthy male donor.

Focused ultrasound and microbubble-mediated delivery of CRISPR-Cas9 ribonucleoprotein to human induced pluripotent stem cells.

Focused ultrasound and microbubble-mediated delivery of CRISPR-Cas9 ribonucleoprotein to human induced pluripotent stem cells.

Both carvedilol and cimetidine alleviate cisplatin-induced nephrotoxicity via downregulating OCT2.

Both carvedilol and cimetidine alleviate cisplatin-induced nephrotoxicity via downregulating OCT2.

Generation of an induced pluripotent stem cell line from a Kennedy Disease patient with AR mutation.

Generation of an induced pluripotent stem cell line from a Kennedy Disease patient with AR mutation.

Generation of human induced pluripotent stem cell lines from three different male XLRS patients carrying RS1 gene mutation.

Generation of human induced pluripotent stem cell lines from three different male XLRS patients carrying RS1 gene mutation.

Generation, characterization, and validation of two human induced pluripotent stem cell lines from the peripheral blood of young and older adults.

Aging is a leading risk factor for the development of age-related diseases. However, how aging impacts human induced pluripotent stem cell (hiPSC) reprogramming, age-related epigenetic memory post-reprogramming, differentiation, and its potential applicability to cardiovascular regenerative medicine remains underexplored. We generated, characterized, and validated two hiPSC lines from human peripheral blood mononuclear cells (PBMCs) obtained from whole blood of young and older human donors. The two hiPSC lines expressed four pluripotency markers, have normal karyotypes and trilineage differentiation potential, and genetically match parental PBMCs. These lines are invaluable for regenerative medicine and exploring epigenetic-related molecular mechanisms that underlie aging and aging-related diseases.