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.

scholarly journals Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells

2015 ◽  
Vol 370 (1680) ◽  
pp. 20140365 ◽  
Author(s):  
Maria Rostovskaya ◽  
Nicholas Bredenkamp ◽  
Austin Smith
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Human Pluripotent Stem Cells ◽  
Type I ◽  
Pancreatic Progenitors ◽  
Stem Cell Lines

Human pluripotent stem cells can in principle be used as a source of any differentiated cell type for disease modelling, drug screening, toxicology testing or cell replacement therapy. Type I diabetes is considered a major target for stem cell applications due to the shortage of primary human beta cells. Several protocols have been reported for generating pancreatic progenitors by in vitro differentiation of human pluripotent stem cells. Here we first assessed one of these protocols on a panel of pluripotent stem cell lines for capacity to engender glucose sensitive insulin-producing cells after engraftment in immunocompromised mice. We observed variable outcomes with only one cell line showing a low level of glucose response. We, therefore, undertook a systematic comparison of different methods for inducing definitive endoderm and subsequently pancreatic differentiation. Of several protocols tested, we identified a combined approach that robustly generated pancreatic progenitors in vitro from both embryo-derived and induced pluripotent stem cells. These findings suggest that, although there are intrinsic differences in lineage specification propensity between pluripotent stem cell lines, optimal differentiation procedures may consistently direct a substantial fraction of cells into pancreatic specification.

scholarly journals Valproic acid Promotes the in Vitro Differentiation of Human Pluripotent Stem Cells Into Spermatogonial Stem Cell-like Cells

2021 ◽  
Author(s):  
Xiaotong Wang ◽  
Mengyuan Qu ◽  
Zili Li ◽  
Yuting Long ◽  
Kai Hong ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Pluripotent Stem Cells ◽  
Wnt Signaling Pathway ◽  
Human Pluripotent Stem Cells ◽  
Sequencing Analysis ◽  
Upregulated Genes

Abstract Background: Studying human germ cell development and male infertility is heavily relied on mouse models. In vitro differentiation of human pluripotent stem cells into spermatogonial stem cell-like cells (SSCLCs) can be used as a model to study human germ cells and infertility. The current study aimed to develop the SSCLC induction protocol and assess the effects of the developed protocol on the SSCLC induction. Methods: We examined the effects of valproic acid (VPA), vitamin C (VC) and the combination of VPA and VC on the SSCLC induction efficiency and determined the expression of spermatogonial genes of differentiated cells. The percentage of haploid cells and cells expressed meiotic and spermatid genes were also detected. RNA-sequencing analysis was performed to compare the transcriptome between cells at 0 and 12 days of differentiation and differently expressed genes were confirmed by RT-qPCR. We further evaluated the alteration in histone marks (H3K9ac and H3K27me3) at 12 days of differentiation. Moreover, the SSCLC induction efficiency of two hiPSC lines of non-obstructive azoospermia (NOA) patients was assessed using different induction protocols.Results: The combination of low concentrations of VPA and VC in the induction medium was most effective to induce SSCLCs expressing several spermatogonial genes from human pluripotent stem cells at 12 days of differentiation. High concentration of VPA was more effective to induce cells expressing meiotic genes and haploid cells. RNA-sequencing analysis revealed that the induction of SSCLC involved the upregulated genes in Wnt signaling pathway, and cells at 12 days of differentiation showed increased H3K9ac and decreased H3K27me3. Additionally, two hiPSC lines of NOA patients showed low SSCLC induction efficiency and the expression of genes in Wnt signaling pathway. Conclusions: VPA robustly promotes the differentiation of human pluripotent stem cell lines into SSCLCs, which involved the upregulated genes in Wnt signaling pathway and epigenetic changes. hiPSCs from NOA patients showed decreased SSCLC induction efficiency and Wnt signaling pathway gene expression, suggesting that inactivation of Wnt signaling pathway might be a cause of SSC depletion in azoospermia testes. Our developed SSCLC induction protocol provides a reliable tool and model to study human germ cell development and male infertility.

Abstract 010: A Separable Upstream Promoter of the Slc8a1 (NCX1) Gene Efficiently Marks Cardiac Cell Populations Derived from Human Pluripotent Stem Cells

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Alejandro Hidalgo-Gonzalez ◽  
Dmitry A Ovchinnikov ◽  
James Hudson ◽  
Justin Cooper-White ◽  
Wolvetang Ernst
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Human Pluripotent Stem Cells ◽  
Cardiac Cells ◽  
Protein Quantification ◽  
Egfp Expression ◽  
Cardiac Cell

The sarcolemmal Na+/Ca2+ exchanger SLC8A1(NCX) regulates intracellular Ca+ in cardiomyocytes from early developmental stages. The upstream-most SLC8A1(NCX1) promoter is well conserved amongst the homoeothermic animals and contains putative binding sites for transcription factors of the NKX, GATA, STAT and CDX families. We hypothesized that functional cardiac cells with mature cardiac structural markers will express the sarcolemmal Na+/Ca2+ calcium antiporting channel, important for proper functional contractivity of in vitro differentiated human pluripotent stem cells. Pseudotyped lentiviral particles delivering NCX1cp-EGFP reporter cassette were used to confirm the efficiency and specificity of the reporter in rodent foetal cardiac cell isolates, and to establish stable human pluripotent stem cell lines. Cells were differentiated using a 2D induction protocol, and gene expression analysis and protein quantification carried at day 16. Initial NCX1cp-EGFP expression was observed from day 10-11 of cardiac differentiation. Beating foci were visualized 1-2 day after initial NCXCP-EGFP expression, reporter expression was confined to the grouped and individual beating cells, and highly correlated with the efficiency of spontaneously contractile cell production. At later stages, NCX1cp-EGFP expression correlated with clusters of formed spontaneously contractile units harbouring essentially all cardiomyocytes present in cultures, as evidenced by colocalization of high levels of cardiac troponin T (cTnT) and α-actinin proteins. The EGFP+ sorted fraction of differentiated cultures was found to be highly enriched in both early (ISL1, TBX5) and late (cTnT, MYH6) cardiomyocyte markers when compared to the EGFP- fraction. We conclude that a ~3 kb genomic fragment of the distal cardiac-specific promoter of the SLC8A1(NCX1) containing the upstream-most exon of the gene is sufficient to drive the expression of a lentiviral reporter in both rodent heart-derived primary and human (embryonic and induced) pluripotent stem cell-derived cardiac cells. Isolation of a homogenous and functional cardiomyogenic population represents one of the key objectives for cardiac tissue engineering, and in particular in vitro drug screening applications.

scholarly journals Human pluripotent stem cell-derived organoids and their potential to reduce the use of animal models in research

2021 ◽  
Vol 4 (s1) ◽  
Author(s):  
Salvatore Simmini ◽  
Allen C. Eaves ◽  
Sharon A. Louis ◽  
Wing Chang
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Animal Models ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Human Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cell ◽  
Tissue Specific ◽  
Organoid Cultures

Efficient and reproducible generation of tissue-specific organoids from Human Pluripotent Stem Cells (hPSCs) represents one of the key tools for reducing the use of animals in research. STEMCELL Technologies is committed to optimizing workflows that efficiently support the generation and maintenance of multiple types of organoid cultures derived from hPSCs.

Evi-1 Regulates Myelopoiesis and Hematopoietic Stem Cell Development in Zebrafish and Human Pluripotent Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1281-1281
Author(s):  
Martina Konantz ◽  
Matthias Grauer ◽  
Sarah Grzywna ◽  
Martijn Brugman ◽  
Lothar Kanz ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Pluripotent Stem Cells ◽  
Zebrafish Embryo ◽  
Human Pluripotent Stem Cells ◽  
Hematopoietic Stem ◽  
Hematopoietic Development

Abstract Abstract 1281 The Evi-1 locus was originally identified as a common site of retroviral integration in murine myeloid tumors. Over the last years, Evi-1 evolved as one of the most potent oncogenes associated with human and murine myeloid leukemia. More recent studies in knockout mice suggest also involvement of Evi-1 in the regulation of developmental hematopoiesis, but the role of Evi-1 in this context is poorly understood. Here, we analyzed zebrafish embryo and human pluripotent stem cells (PSC) to understand how Evi-1 modulates early hematopoietic development. We examined the hematopoietic development in zebrafish embryo by in situ hybridization (ISH) for hematopoietic markers. The zebrafish homologue evi-1 was shown to be expressed in co-localization with scl in the posterior blood islands, indicating a role during early blood development. We also performed loss-of-function studies were by injecting morpholino oligonucleotides (MO) in zebrafish zygotes to inhibit evi-1 pre-mRNA splicing. Inhibition of evi-1 was confirmed in MO-injected versus control embryos. N=100 zebrafish embryos were analyzed per experiment in each group. To control for off-target effects, two separate MO were designed and injected. MO mediated evi-1 knockdown severely reduced numbers of circulating blood cells and induced hemorrhages. ISH performed in evi-1 morphants versus control fish revealed strongly impaired formation of myeloid embryonic cells (measured by pu.1 expression), while no changes were observed in primitive erythroid progenitor cells (monitored by gata1 expression) or overall in blood and endothelial precursors in the posterior lateral plate mesoderm (as monitored by scl expression). Moreover, analyses at 36 hours and 5 days post fertilization showed strong reduction of runx1+/cmyb+ cells and rag1+ lymphoid cells, indicating a role of evi-1 in developing hematopoietic stem cells (HSC). Previous reports in adult murine hematopoietic cells suggest that Evi-1 affects hematopoietic stem cell proliferation through regulation of Gata2. To test whether Gata2 is a putative downstream regulator of Evi-1 in our system, we performed a rescue experiment and co-injected gata2 mRNA in evi-1 MO treated fish. Indeed, ectopic gata2 rescued the impaired myeloid phenotype, as shown by re-occurrence of mpo, l-plastin as well as pu.1 expressing cells. To assess whether these molecular interactions are conserved during human developmental hematopoiesis, we surveyed in vitro differentiating human pluripotent stem cells (PSC) genetically modified to suppress EVI-1. EVI-1 expression was detected during differentiation of human PSC in embryoid bodies, especially around day 9 when hematopoietic progenitors start to emerge in this system. Treatment with EVI-1 shRNA strongly reduced the generation of myeloid colonies from human PSC in vitro as well as the numbers of emerging CD34+ and CD45+ cells. Molecularly, EVI-1 suppression inhibited the expression of PU.1 and GATA2 during the course of development, while leaving SCL and GATA1 expression unaltered. Taken together, our data suggest that, in both fish and human, Evi-1 regulates embryonic myelopoiesis through interactions with Gata2 and independently of Gata1 and embryonic erythropoiesis. Moreover, Evi-1 appears crucial for HSC development. Currently ongoing experiments in our laboratory focus on the further elucidation of the molecular mechanisms underlying the Evi-1 effects during developmental hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Mini Review; Differentiation of Human Pluripotent Stem Cells into Oocytes

2020 ◽  
Vol 15 (4) ◽  
pp. 301-307 ◽  
Author(s):  
Gaifang Wang ◽  
Maryam Farzaneh
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Human Pluripotent Stem Cells ◽  
Human Oocyte ◽  
Differentiation Potential ◽  
Cell Lineages ◽  
Cell Based Therapy ◽  
Induced Pluripotent

Primary Ovarian Insufficiency (POI) is one of the main diseases causing female infertility that occurs in about 1% of women between 30-40 years of age. There are few effective methods for the treatment of women with POI. In the past few years, stem cell-based therapy as one of the most highly investigated new therapies has emerged as a promising strategy for the treatment of POI. Human pluripotent stem cells (hPSCs) can self-renew indefinitely and differentiate into any type of cell. Human Embryonic Stem Cells (hESCs) as a type of pluripotent stem cells are the most powerful candidate for the treatment of POI. Human-induced Pluripotent Stem Cells (hiPSCs) are derived from adult somatic cells by the treatment with exogenous defined factors to create an embryonic-like pluripotent state. Both hiPSCs and hESCs can proliferate and give rise to ectodermal, mesodermal, endodermal, and germ cell lineages. After ovarian stimulation, the number of available oocytes is limited and the yield of total oocytes with high quality is low. Therefore, a robust and reproducible in-vitro culture system that supports the differentiation of human oocytes from PSCs is necessary. Very few studies have focused on the derivation of oocyte-like cells from hiPSCs and the details of hPSCs differentiation into oocytes have not been fully investigated. Therefore, in this review, we focus on the differentiation potential of hPSCs into human oocyte-like cells.

scholarly journals Establishing a deeper understanding of the osteogenic differentiation of monolayer cultured human pluripotent stem cells using novel and detailed analyses

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ping Zhou ◽  
Jia-Min Shi ◽  
Jing-E Song ◽  
Yu Han ◽  
Hong-Jiao Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Telomerase Activity ◽  
Human Pluripotent Stem Cells ◽  
Cell Counting ◽  
Cell Type ◽  
Alizarin Red

Abstract Background Derivation of osteoblast-like cells from human pluripotent stem cells (hPSCs) is a popular topic in bone tissue engineering. Although many improvements have been achieved, the low induction efficiency because of spontaneous differentiation hampers their applications. To solve this problem, a detailed understanding of the osteogenic differentiation process of hPSCs is urgently needed. Methods Monolayer cultured human embryonic stem cells and human-induced pluripotent stem cells were differentiated in commonly applied serum-containing osteogenic medium for 35 days. In addition to traditional assays such as cell viability detection, reverse transcription-polymerase chain reaction, immunofluorescence, and alizarin red staining, we also applied studies of cell counting, cell telomerase activity, and flow cytometry as essential indicators to analyse the cell type changes in each week. Results The population of differentiated cells was quite heterogeneous throughout the 35 days of induction. Then, cell telomerase activity and cell cycle analyses have value in evaluating the cell type and tumourigenicity of the obtained cells. Finally, a dynamic map was made to integrate the analysis of these results during osteogenic differentiation of hPSCs, and the cell types at defined stages were concluded. Conclusions Our results lay the foundation to improve the in vitro osteogenic differentiation efficiency of hPSCs by supplementing with functional compounds at the desired stage, and then establishing a stepwise induction system in the future.

scholarly journals Pluripotent stem cell SOX9 and INS reporters facilitate differentiation into insulin-producing cells

2021 ◽  
Author(s):  
Rabea Dettmer ◽  
Isabell Niwolik ◽  
Ilir Mehmeti ◽  
Anne Jörns ◽  
Ortwin Naujok
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Beta Cells ◽  
Pluripotent Stem Cells ◽  
Reporter Genes ◽  
Human Pluripotent Stem Cells ◽  
Endogenous Gene ◽  
Pancreatic Progenitors ◽  
Insulin Producing Cells ◽  
Endogenous Genes

AbstractDifferentiation of human pluripotent stem cells into insulin-producing stem cell-derived beta cells harbors great potential for research and therapy of diabetes. The SOX9 gene plays a crucial role during development of the pancreas and particularly in the development of insulin-producing cells as SOX9+ cells form the source for NEUROG3+ endocrine progenitor cells. For the purpose of easy monitoring of differentiation efficiencies into pancreatic progenitors and insulin-producing cells, we generated new reporter lines by knocking in a P2A-H-2Kk-F2A-GFP2 reporter genes into the SOX9 locus and a P2A-mCherry reporter gene into the INS locus mediated by CRISPR/CAS9-technology. The knock-ins enable co-expression of the endogenous genes and reporter genes, report the endogenous gene expression and enable the purification of pancreatic progenitors and insulin-producing cells using FACS or MACS. Using these cell lines we established a new differentiation protocol geared towards SOX9+ cells to efficiently drive human pluripotent stem cells into glucose-responsive beta cells.

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