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Byline: R. Negi, A. Srivastava, A. Srivastava, Abhishek. Pandeya, P. Vatsa, U. Ansari, A. Pant BACKGROUND AND OBJECTIVES: Induced pluripotent stem cells (iPSCs) derived three-dimensional (3D) model for rare neurodegenerative [...]

Limited access to embryos has hampered the study of human embryogenesis and disorders that occur during early pregnancy. Human pluripotent stem cells provide an alternative means to study human development in a dish.sup.1-7. Recent advances in partial embryo models derived from human pluripotent stem cells have enabled human development to be examined at early post-implantation stages.sup.8-14. However, models of the pre-implantation human blastocyst are lacking. Starting from naive human pluripotent stem cells, here we developed an effective three-dimensional culture strategy with successive lineage differentiation and self-organization to generate blastocyst-like structures in vitro. These structures--which we term 'human blastoids'--resemble human blastocysts in terms of their morphology, size, cell number, and composition and allocation of different cell lineages. Single-cell RNA-sequencing analyses also reveal the transcriptomic similarity of blastoids to blastocysts. Human blastoids are amenable to embryonic and extra-embryonic stem cell derivation and can further develop into peri-implantation embryo-like structures in vitro. Using chemical perturbations, we show that specific isozymes of protein kinase C have a critical function in the formation of the blastoid cavity. Human blastoids provide a readily accessible, scalable, versatile and perturbable alternative to blastocysts for studying early human development, understanding early pregnancy loss and gaining insights into early developmental defects. An in vitro culture strategy enables the generation of blastocyst-like structures termed human blastoids from naive human pluripotent stem cells, providing a model for studying human embryogenesis., Author(s): Leqian Yu [sup.1] [sup.2] , Yulei Wei [sup.1] [sup.3] [sup.4] , Jialei Duan [sup.5] , Daniel A. Schmitz [sup.1] [sup.2] , Masahiro Sakurai [sup.1] , Lei Wang [sup.5] , [...]

Islets derived from stem cells hold promise as a therapy for insulin-dependent diabetes, but there remain challenges towards achieving this goal.sup.1-6. Here we generate human islet-like organoids (HILOs) from induced pluripotent stem cells and show that non-canonical WNT4 signalling drives the metabolic maturation necessary for robust ex vivo glucose-stimulated insulin secretion. These functionally mature HILOs contain endocrine-like cell types that, upon transplantation, rapidly re-establish glucose homeostasis in diabetic NOD/SCID mice. Overexpression of the immune checkpoint protein programmed death-ligand 1 (PD-L1) protected HILO xenografts such that they were able to restore glucose homeostasis in immune-competent diabetic mice for 50 days. Furthermore, ex vivo stimulation with interferon-[gamma] induced endogenous PD-L1 expression and restricted T cell activation and graft rejection. The generation of glucose-responsive islet-like organoids that are able to avoid immune detection provides a promising alternative to cadaveric and device-dependent therapies in the treatment of diabetes. Metabolically-mature human islet-like organoids generated from induced pluripotent stem cells are able to recapitulate insulin-responsive pancreatic islet function and avoid immunologic cell death in diabetic mouse transplantation models., Author(s): Eiji Yoshihara [sup.1] [sup.5] [sup.6] , Carolyn O'Connor [sup.2] , Emanuel Gasser [sup.1] , Zong Wei [sup.1] [sup.7] , Tae Gyu Oh [sup.1] , Tiffany W. Tseng [sup.1] , [...]

Clinical trials using adult stem cells to regenerate damaged heart tissue continue to this day.sup.1,2, despite ongoing questions of efficacy and a lack of mechanistic understanding of the underlying biological effect.sup.3. The rationale for these cell therapy trials is derived from animal studies that show a modest but reproducible improvement in cardiac function in models of cardiac ischaemic injury.sup.4,5. Here we examine the mechanistic basis for cell therapy in mice after ischaemia-reperfusion injury, and find that--although heart function is enhanced--it is not associated with the production of new cardiomyocytes. Cell therapy improved heart function through an acute sterile immune response characterized by the temporal and regional induction of CCR2.sup.+ and CX3CR1.sup.+ macrophages. Intracardiac injection of two distinct types of adult stem cells, cells killed by freezing and thawing or a chemical inducer of the innate immune response all induced a similar regional accumulation of CCR2.sup.+ and CX3CR1.sup.+ macrophages, and provided functional rejuvenation to the heart after ischaemia-reperfusion injury. This selective macrophage response altered the activity of cardiac fibroblasts, reduced the extracellular matrix content in the border zone and enhanced the mechanical properties of the injured area. The functional benefit of cardiac cell therapy is thus due to an acute inflammatory-based wound-healing response that rejuvenates the infarcted area of the heart. Cardiac stem cell therapy in mouse models of ischaemia-reperfusion injury demonstrates that improvement in heart function is linked to an immune response characterized by the induction of CCR2.sup.+ and CX3CR1.sup.+ macrophages., Author(s): Ronald J. Vagnozzi [sup.1] , Marjorie Maillet [sup.1] , Michelle A. Sargent [sup.1] , Hadi Khalil [sup.1] , Anne Katrine Z. Johansen [sup.1] , Jennifer A. Schwanekamp [sup.2] , [...]

Vesicles secreted by stem cells might give clinicians a safer and simpler alternative to cell therapy, but researchers are still grappling with how best to prepare and study these tiny particles. Vesicles secreted by stem cells might give clinicians a safer and simpler alternative to cell therapy, but researchers are still grappling with how best to prepare and study these tiny particles., Author(s): Michael Eisenstein Author Affiliations: Inside the stem-cell pharmaceutical factory Researchers at the Paracelsus Medical University in Salzburg, Austria, prepare extracellular vesicles. [see PDF for image] Credit: Courtesy of Mario [...]

Bardet-Biedl syndrome (BBS) is a rare autosomal recessive disorder caused by mutations in genes encoding components of the primary cilium and is characterized by hyperphagic obesity. To investigate the molecular basis of obesity in human BBS, we developed a cellular model of BBS using induced pluripotent stem cell-derived (iPSC-derived) hypothalamic arcuate-like neurons. BBS mutations [BBS1.sup.M390R] and BBS[10.sup.C91fsX95] did not affect neuronal differentiation efficiency but caused morphological defects, including impaired neurite outgrowth and longer primary cilia. Single-cell RNA sequencing of [BBS1.sup.M390R] hypothalamic neurons identified several downregulated pathways, including insulin and cAMP signaling and axon guidance. Additional studies demonstrated that [BBS1.sup.M390R] and BBS[10.sup.C91fsX95] mutations impaired insulin signaling in both human fibroblasts and iPSC-derived neurons. Overexpression of intact BBS10 fully restored insulin signaling by restoring insulin receptor tyrosine phosphorylation in BBS[10.sup.C91fsX95] neurons. Moreover, mutations in BBS1 and BBS10 impaired leptin-mediated p-STAT3 activation in iPSC-derived hypothalamic neurons. Correction of the BBS mutation by CRISPR rescued leptin signaling. POMC expression and neuropeptide production were decreased in [BBS1.sup.M390R] and BBS[10.sup.C91fsX95] iPSC-derived hypothalamic neurons. In the aggregate, these data provide insights into the anatomic and functional mechanisms by which components of the BBSome in CNS primary cilia mediate effects on energy homeostasis., Introduction Bardet-Biedl syndrome (BBS) is an autosomal recessive disorder characterized by severe, early-onset obesity, as well as an array of other diagnostic features typical of ciliopathies including polydactyly, retinal degeneration, [...]

Chronic diseases are the primary cause of mortality worldwide, accounting for 67% of deaths. One of the major challenges in developing new treatments is the lack of understanding of the exact underlying biological and molecular mechanisms. Chronic cardiovascular diseases are the single most common cause of death worldwide, and sudden deaths due to cardiac arrhythmias account for approximately 50% of all such cases. Traditional genetic screening for genes involved in cardiac disorders is labourious and frequently fails to detect the mutation that explains or causes the disorder. However, when mutations are identified, human induced pluripotent stem cells (hiPSCs) derived from affected patients make it possible to address fundamental research questions directly relevant to human health. As such, hiPSC technology has recently been used to model human diseases and patient-specific hiPSC-derived cardiomyocytes (hiPSC- CMs) thus offer a unique opportunity to investigate potential disease-causing genetic variants in their natural environment. The purpose of this review is to present the current state of knowledge regarding hiPSC-CMs, including their potential, limitations, and challenges and to discuss future prospects. Key words: hIPSCs, cardiac tissue, electrophysiology, cardiac arrhythmias, cardiomyocytes, sodium channels, [Na.sub.V]. Les maladies chroniques constituent la principale cause de mortalite a l'echelle mondiale: elles sont responsables de 67 % des deces. L'un des principaux enjeux du developpement de nouveaux traitements correspond au manque de connaissances precises quant aux mecanismes biologiques et moleculaires sous-jacents. Les maladies cardiovasculaires chroniques constituent a elles seules la cause la plus frequente de deces a l'echelle mondiale, et les morts subites causees par les arythmies cardiaques sont responsables d'environ 50 % de l'ensemble de ces cas. Le depistage genetique conventionnel de genes jouant un role dans les maladies cardiaques est laborieux et echoue frequemment dans la detection des mutations qui peuvent expliquer ou causer la maladie. Cependant, quand les mutations sont etablies, les cellules souches pluripotentes induites chez l'humain (hiPSC pour <>) qui sont issues de patients atteints permettent de s'attaquer aux questions a l'origine de la recherche fondamentale en lien direct avec la sante humaine. En tant que telle, la technologie des hiPSC a recemment ete utilisee comme modele de maladies humaines, et les cardiomyocytes derives des hiPSC chez des patients specifiques offrent donc une occasion unique d'etudier des variants genetiques pouvant causer des maladies dans leur environnement naturel. Cette synthese a pour but de presenter l'etat actuel des connaissances quant aux cardiomyocytes derives des hiPSC, y compris leur potentiel, leurs limites et les difficultes afferentes ainsi que de discuter des espoirs qu'ils suscitent. [Traduit par la Redaction] Mots-cles: hIPSCs, tissu cardiaque, electrophysiologie, arythmies cardiaques, cardiomyocytes, canaux sodiques, [Na.sub.V]., Introduction Industrialized societies are increasingly confronted with major challenges posed by chronic health-care problems such as cancer, cardiovascular and neurodegenerative diseases, and metabolic, pain, and developmental disorders. Accounting for 50% [...]

The enteric nervous system (ENS) of the gastrointestinal tract controls many diverse functions, including motility and epithelial permeability. Perturbations in ENS development or function are common, yet there is no human model for studying ENS-intestinal biology and disease. We used a tissue-engineering approach with embryonic and induced pluripotent stem cells (PSCs) to generate human intestinal tissue containing a functional ENS. We recapitulated normal intestinal ENS development by combining human-PSC-derived neural crest cells (NCCs) and developing human intestinal organoids (HIOs). NCCs recombined with HIOs in vitro migrated into the mesenchyme, differentiated into neurons and glial cells and showed neuronal activity, as measured by rhythmic waves of calcium transients. ENS-containing HIOs grown in vivo formed neuroglial structures similar to a myenteric and submucosal plexus, had functional interstitial cells of Cajal and had an electromechanical coupling that regulated waves of propagating contraction. Finally, we used this system to investigate the cellular and molecular basis for Hirschsprung's disease caused by a mutation in the gene PHOX2B. This is, to the best of our knowledge, the first demonstration of human-PSC-derived intestinal tissue with a functional ENS and how this system can be used to study motility disorders of the human gastrointestinal tract., Author(s): Michael J Workman [1]; Maxime M Mahe [2]; Stephen Trisno [1]; Holly M Poling [2]; Carey L Watson [2]; Nambirajan Sundaram [2]; Ching-Fang Chang [1, 3]; Jacqueline Schiesser [1]; [...]

Microglia, the only lifelong resident immune cells of the central nervous system (CNS), are highly specialized macrophages that have been recognized to have a crucial role in neurodegenerative diseases such as Alzheimer's, Parkinson's and adrenoleukodystrophy (ALD). However, in contrast to other cell types of the human CNS, bona fide microglia have not yet been derived from cultured human pluripotent stem cells. Here we establish a robust and efficient protocol for the rapid production of microglia-like cells from human (h) embryonic stem (ES) and induced pluripotent stem (iPS) cells that uses defined serum-free culture conditions. These in vitro pluripotent stem cell-derived microglia-like cells (termed pMGLs) faithfully recapitulate the expected ontogeny and characteristics of their in vivo counterparts, and they resemble primary fetal human and mouse microglia. We generated these cells from multiple disease-specific cell lines and find that pMGLs derived from an hES model of Rett syndrome are smaller than their isogenic controls. We further describe a platform to study the integration and live behavior of pMGLs in organotypic 3D cultures. This modular differentiation system allows for the study of microglia in highly defined conditions as they mature in response to developmentally relevant cues, and it provides a framework in which to study the long-term interactions of microglia residing in a tissue-like environment., Author(s): Julien Muffat [1]; Yun Li [1]; Bingbing Yuan [1]; Maisam Mitalipova [1]; Attya Omer [1, 2]; Sean Corcoran [1, 3]; Grisilda Bakiasi [1, 4]; Li-Huei Tsai [5]; Patrick Aubourg [...]

Smith-Lemli-Opitz syndrome (SLOS) is a malformation disorder caused by mutations in DHCR7, which impair the reduction of 7-dehydrocholesterol (7DHC) to cholesterol. SLOS results in cognitive impairment, behavioral abnormalities and nervous system defects, though neither affected cell types nor impaired signaling pathways are fully understood. Whether 7DHC accumulation or cholesterol loss is primarily responsible for disease pathogenesis is also unclear. Using induced pluripotent stem cells (iPSCs) from subjects with SLOS, we identified cellular defects that lead to precocious neuronal specification within SLOS derived neural progenitors. We also demonstrated that 7DHC accumulation, not cholesterol deficiency, is critical for SLOS-associated defects. We further identified downregulation of Wnt/β-catenin signaling as a key initiator of aberrant SLOS iPSC differentiation through the direct inhibitory effects of 7DHC on the formation of an active Wnt receptor complex. Activation of canonical Wnt signaling prevented the neural phenotypes observed in SLOS iPSCs, suggesting that Wnt signaling may be a promising therapeutic target for SLOS., Impaired cholesterol synthesis underlies a group of human disorders characterized by cognitive impairment, congenital malformations, and distinct behavioral phenotypes, including autism (1). The most common cholesterol synthesis disorder, Smith-Lemli-Opitz syndrome [...]