scholarly journals Directed Differentiation of Human Pluripotent Stem Cells towards Corneal Endothelial-Like Cells under Defined Conditions

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 331
Author(s):  
Pyry Grönroos ◽  
Tanja Ilmarinen ◽  
Heli Skottman
Keyword(s):  
Stem Cells ◽  
Retinoic Acid ◽  
Pluripotent Stem Cells ◽  
Fluid Transport ◽  
Transforming Growth Factor ◽  
Specific Inhibitor ◽  
Human Pluripotent Stem Cells ◽  
Differentiated Cells ◽  
Corneal Transplants

The most crucial function of corneal endothelial cells (CEnCs) is to maintain optical transparency by transporting excess fluid out of stroma. Unfortunately, CEnCs are not able to proliferate in vivo in the case of trauma or dystrophy. Visually impaired patients with corneal endothelial deficiencies that are waiting for transplantation due to massive global shortage of cadaveric corneal transplants are in a great need of help. In this study, our goal was to develop a defined, clinically applicable protocol for direct differentiation of CEnCs from human pluripotent stem cells (hPSCs). To produce feeder-free hPSC-CEnCs, we used small molecule induction with transforming growth factor (TGF) beta receptor inhibitor SB431542, GSK-3-specific inhibitor CHIR99021 and retinoic acid to guide differentiation through the neural crest and periocular mesenchyme (POM). Cells were characterized by the morphology and expression of human (h)CEnC markers with immunocytochemistry and RT-qPCR. After one week of induction, we observed the upregulation of POM markers paired-like homeodomain transcription factor 2 (PITX2) and Forkhead box C1 (FOXC1) and polygonal-shaped cells expressing CEnC-associated markers Zona Occludens-1 (ZO-1), sodium-potassium (Na+/K+)-ATPase, CD166, sodium bicarbonate cotransporter 1 (SLC4A4), aquaporin 1 (AQP1) and N-cadherin (NCAD). Furthermore, we showed that retinoic acid induced a dome formation in the cell culture, with a possible indication of fluid transport by the differentiated cells. Thus, we successfully generated CEnC-like cells from hPSCs with a defined, simple and fast differentiation method.

scholarly journals Generation of Retinoic Acid-Dependent Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 35-35
Author(s):  
Stephanie A Luff ◽  
J Philip Creamer ◽  
Carissa Dege ◽  
Rebecca Scarfò ◽  
Samantha Morris ◽  
...  
Keyword(s):  
Stem Cells ◽  
Retinoic Acid ◽  
Pluripotent Stem Cells ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Human Pluripotent Stem Cells ◽  
Human Embryos ◽  
Dependent Manner ◽  
Hematopoietic Stem

The generation of the hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) is a major goal for regenerative medicine. In the embryo, HSCs derive from a HOXA+ population known as hemogenic endothelium (HE) in a retinoic acid (RA)-dependent manner. Using hPSCs, we have previously identified a KDR+CD235a− mesodermal population that gives rise to a clonally multipotent HOXA+ definitive HE. However, this HE lacks HSC-like capacity in the absence of exogenous transgenes and is functionally unresponsive to RA treatment. Thus, the specification of an RA-dependent hematopoietic program from hPSCs has remained elusive. Through single cell RNA-seq (scRNA-seq) analyses, we identified that 2 distinct KDR+CD235a− populations exist prior to HE specification, distinguishable by CXCR4 expression. Interestingly, KDR+CD235a−CXCR4− mesoderm expressed CYP26A1, an RA degrading enzyme, and harbored definitive hematopoietic potential within hPSC differentiation cultures in the absence of RA signaling, indicating the HE specified from CXCR4− mesoderm as RA-independent (RAi). In sharp contrast, KDR+CD235a−CXCR4+ mesoderm exclusively expressed ALDH1A2, the key enzyme in the synthesis of RA, but lacked hematopoietic potential under the same culture conditions. However, the stage-specific application of RA signaling to CXCR4+ mesoderm resulted in the robust specification of CD34+HOXA+ HE with definitive erythroid, myeloid, and lymphoid hematopoietic potential, establishing this HE as RA-dependent (RAd). Furthermore, while RAi HE entirely failed to persist following murine hematopoietic xenografts, RAd HE transiently persisted within the peripheral blood and bone marrow of murine hosts. To assess whether these functionally distinct hPSC mesodermal progenitors are physiologically relevant to human embryonic development, we integrated scRNA-seq datasets from the hPSC mesodermal cultures and a gastrulating human embryo. These analyses revealed that in vivo, distinct KDR+CXCR4−CYP26A1+ and KDR+CXCR4+ALDH1A2+ populations can be found at the stage of emergent mesoderm, following patterning of nascent mesoderm. Additional comparison to later stage human embryos demonstrated that RAd HE has a more fetal-like HOXA expression pattern than RAi HE. Scoring of single fetal HE cells against hPSC-derived HE revealed that while some early fetal HE cells were similar to RAi HE, the late fetal HE cells, which are hypothesized to give rise to HSCs, were more similar to RAd HE. Lastly, as HSC-competent HE is expected to express arterial genes, we found a subset of late fetal HE with this phenotype that were exclusively similar to RAd HE. Collectively, these data represent the first ever characterization of RA-dependent hPSC-derived definitive hematopoiesis and its mesodermal progenitor. Additionally, we provide evidence for in vivo mesodermal and HE correlates for both RAi and RAd hematopoietic programs within human embryos. This novel insight into human hematopoietic development will serve as an important tool for modeling development and ultimately provide the basis for de novo specification of HSCs. Disclosures No relevant conflicts of interest to declare.

scholarly journals Characterization of Endoplasmic Reticulum (ER) in Human Pluripotent Stem Cells Revealed Increased Susceptibility to Cell Death upon ER Stress

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1078
Author(s):  
Tae Won Ha ◽  
Ji Hun Jeong ◽  
HyeonSeok Shin ◽  
Hyun Kyu Kim ◽  
Jeong Suk Im ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Pluripotent Stem Cells ◽  
Meta Analysis ◽  
Embryonic Stem ◽  
Structural Features ◽  
Human Pluripotent Stem Cells ◽  
Differentiated Cells ◽  
Induced Apoptosis

Human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have a well-orchestrated program for differentiation and self-renewal. However, the structural features of unique proteostatic-maintaining mechanisms in hPSCs and their features, distinct from those of differentiated cells, in response to cellular stress remain unclear. We evaluated and compared the morphological features and stress response of hPSCs and fibroblasts. Compared to fibroblasts, electron microscopy showed simpler/fewer structures with fewer networks in the endoplasmic reticulum (ER) of hPSCs, as well as lower expression of ER-related genes according to meta-analysis. As hPSCs contain low levels of binding immunoglobulin protein (BiP), an ER chaperone, thapsigargin treatment sharply increased the gene expression of the unfolded protein response. Thus, hPSCs with decreased chaperone function reacted sensitively to ER stress and entered apoptosis faster than fibroblasts. Such ER stress-induced apoptotic processes were abolished by tauroursodeoxycholic acid, an ER-stress reliever. Hence, our results revealed that as PSCs have an underdeveloped structure and express fewer BiP chaperone proteins than somatic cells, they are more susceptible to ER stress-induced apoptosis in response to stress.

2022 - IDENTIFICATION OF A RETINOIC ACID-DEPENDENT DEFINITIVE HEMATOPOIETIC PROGENITOR FROM HUMAN PLURIPOTENT STEM CELLS

2019 ◽  
Vol 76 ◽  
pp. S47
Author(s):  
Christopher Sturgeon ◽  
Stephanie Luff ◽  
Carissa Dege ◽  
Rebecca Scarfo ◽  
Sara Maffioletti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Retinoic Acid ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells ◽  
Hematopoietic Progenitor

Synthetic probes for in vitro purification and in vivo tracking of hepatocytes derived from human pluripotent stem cells

Biomaterials ◽  
2019 ◽  
Vol 222 ◽  
pp. 119431 ◽  
Author(s):  
Ji Young Park ◽  
Jiyou Han ◽  
Hyo Sung Jung ◽  
Gyunggyu Lee ◽  
Hyo Jin Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells

scholarly journals Mammalian genes induce partially reprogrammed pluripotent stem cells in non-mammalian vertebrate and invertebrate species

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Ricardo Antonio Rosselló ◽  
Chun-Chun Chen ◽  
Rui Dai ◽  
Jason T Howard ◽  
Ute Hochgeschwender ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Pluripotent Stem Cells ◽  
Mammalian Species ◽  
Embryonic Stem ◽  
Model Organisms ◽  
Differentiated Cells ◽  
Transcription Factor Genes ◽  
Induced Pluripotent

Cells are fundamental units of life, but little is known about evolution of cell states. Induced pluripotent stem cells (iPSCs) are once differentiated cells that have been re-programmed to an embryonic stem cell-like state, providing a powerful platform for biology and medicine. However, they have been limited to a few mammalian species. Here we found that a set of four mammalian transcription factor genes used to generate iPSCs in mouse and humans can induce a partially reprogrammed pluripotent stem cell (PRPSCs) state in vertebrate and invertebrate model organisms, in mammals, birds, fish, and fly, which span 550 million years from a common ancestor. These findings are one of the first to show cross-lineage stem cell-like induction, and to generate pluripotent-like cells for several of these species with in vivo chimeras. We suggest that the stem-cell state may be highly conserved across a wide phylogenetic range.

scholarly journals Volatile compounds emission from teratogenic human pluripotent stem cells observed during their differentiation in vivo

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Rosamaria Capuano ◽  
Paola Spitalieri ◽  
Rosa Valentina Talarico ◽  
Alexandro Catini ◽  
Ana Carolina Domakoski ◽  
...  
Keyword(s):  
Stem Cells ◽  
Volatile Compounds ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells

scholarly journals In Vitro Induction and In Vivo Engraftment of Lung Bud Tip Progenitor Cells Derived from Human Pluripotent Stem Cells

2018 ◽  
Vol 10 (1) ◽  
pp. 101-119 ◽  
Author(s):  
Alyssa J. Miller ◽  
David R. Hill ◽  
Melinda S. Nagy ◽  
Yoshiro Aoki ◽  
Briana R. Dye ◽  
...  
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells ◽  
In Vitro Induction

scholarly journals Retinoic Acid Regulates Hematopoietic Development from Human Pluripotent Stem Cells

2015 ◽  
Vol 4 (2) ◽  
pp. 269-281 ◽  
Author(s):  
Roger E. Rönn ◽  
Carolina Guibentif ◽  
Roksana Moraghebi ◽  
Patricia Chaves ◽  
Shobhit Saxena ◽  
...  
Keyword(s):  
Stem Cells ◽  
Retinoic Acid ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells ◽  
Hematopoietic Development

scholarly journals Transcriptome-based molecular staging of human stem cell-derived retinal organoids uncovers accelerated photoreceptor differentiation by 9-cis retinal

2019 ◽  
Author(s):  
Koray D. Kaya ◽  
Holly Y. Chen ◽  
Matthew J. Brooks ◽  
Ryan A. Kelley ◽  
Hiroko Shimada ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Retinoic Acid ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells ◽  
Mitochondrial Morphology ◽  
Rod Photoreceptor ◽  
Molecular Staging ◽  
Organoid Cultures

ABSTRACTRetinal organoids generated from human pluripotent stem cells exhibit considerable variability in temporal dynamics of differentiation. To assess the maturity of neural retina in vitro, we performed transcriptome analyses of developing organoids from human embryonic and induced pluripotent stem cell lines. We show that the developmental variability in organoids was reflected in gene expression profiles and could be evaluated by molecular staging with the human fetal and adult retinal transcriptome data. We also demonstrated that addition of 9-cis retinal, instead of widely-used all-trans retinoic acid, accelerated rod photoreceptor differentiation in organoid cultures, with higher rhodopsin expression and more mature mitochondrial morphology evident by day 120. Our studies thus provide an objective transcriptome-based modality for determining the differentiation state of retinal organoids, which should facilitate disease modeling and evaluation of therapies in vitro.Summary StatementThree-dimensional organoids derived from human pluripotent stem cells have been extensively applied for investigating organogenesis, modeling diseases and development of therapies. However, substantial variations within organoids pose challenges for comparison among different cultures and studies. We generated transcriptomes of multiple distinct retinal organoids and compared these to human fetal and adult retina gene profiles for molecular staging of differentiation state of the cultures. Our analysis revealed the advantage of using 9-cis retinal, instead of the widely-used all-trans retinoic acid, in facilitating rod photoreceptor differentiation. Thus, a transcriptome-based comparison can provide an objective method to uncover the maturity of organoid cultures across different lines and in various study platforms.

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