scholarly journals Assessment of the Hematopoietic Differentiation Potential of Human Pluripotent Stem Cells in 2D and 3D Culture Systems

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2858
Author(s):  
German Atzin Mora-Roldan ◽  
Dalia Ramirez-Ramirez ◽  
Rosana Pelayo ◽  
Karlen Gazarian
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Time Course ◽  
Hematopoietic Cells ◽  
3D Culture ◽  
Human Pluripotent Stem Cells ◽  
Hematopoietic Differentiation ◽  
Differentiation Potential ◽  
2D And 3D

Background. In vitro methods for hematopoietic differentiation of human pluripotent stem cells (hPSC) are a matter of priority for the in-depth research into the mechanisms of early embryogenesis. So-far, published results regarding the generation of hematopoietic cells come from studies using either 2D or 3D culture formats, hence, it is difficult to discern their particular contribution to the development of the concept of a unique in vitro model in close resemblance to in vivo hematopoiesis. Aim of the study. To assess using the same culture conditions and the same time course, the potential of each of these two formats to support differentiation of human pluripotent stem cells to primitive hematopoiesis without exogenous activation of Wnt signaling. Methods. We used in parallel 2D and 3D formats, the same culture environment and assay methods (flow cytometry, IF, qPCR) to investigate stages of commitment and specification of mesodermal, and hemogenic endothelial cells to CD34 hematopoietic cells and evaluated their clonogenic capacity in a CFU system. Results. We show an adequate formation of mesoderm, an efficient commitment to hemogenic endothelium, a higher number of CD34 hematopoietic cells, and colony-forming capacity potential only in the 3D format-supported differentiation. Conclusions. This study shows that the 3D but not the 2D format ensures the induction and realization by endogenous mechanisms of human pluripotent stem cells’ intrinsic differentiation program to primitive hematopoietic cells. We propose that the 3D format provides an adequate level of upregulation of the endogenous Wnt/β-catenin signaling.

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 Characterization of Phenotypic and Transcriptional Differences in Human Pluripotent Stem Cells under 2D and 3D Culture Conditions

2016 ◽  
Vol 5 (22) ◽  
pp. 2951-2958 ◽  
Author(s):  
Ken-ichiro Kamei ◽  
Yoshie Koyama ◽  
Yumie Tokunaga ◽  
Yasumasa Mashimo ◽  
Momoko Yoshioka ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
3D Culture ◽  
Culture Conditions ◽  
Human Pluripotent Stem Cells ◽  
2D And 3D

Hematopoietic Differentiation of Human Induced Pluripotent Stem Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 731-731
Author(s):  
Kyung-Dal Choi ◽  
Junying Yu ◽  
Kimberly Smuga-Otto ◽  
Jessica Dias ◽  
Giorgia Salvagiotto ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Differential Expression ◽  
Pluripotent Stem Cells ◽  
Blood Cells ◽  
Hematopoietic Cells ◽  
Patient Specific ◽  
Hematopoietic Differentiation ◽  
Differentiation Potential ◽  
Induced Pluripotent

Abstract Induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity for modeling of human diseases in vitro as well as for developing novel approaches for regenerative therapy based on immunologically compatible cells. In the present study, we employed an OP9 differentiation system to characterize the hematopoietic differentiation potential of seven human iPSC lines obtained from human fetal, neonatal, and adult fibroblasts through reprogramming with POU5F1, SOX2, NANOG, and LIN28 and compared it with the differentiation potential of five human embryonic stem cell lines (hESC; H1, H7, H9, H13, and H14). Similar to hESCs, all iPSCs in coculture with OP9 generated all types of colony forming cells (CFCs) as well as CD34+ cells that can be separated into distinct subsets based on differential expression of CD43 and CD31. CD34+CD31+CD43− cells obtained from all iPSCs expressed molecules present on endothelial cells and readily formed a monolayer when placed in endothelial conditions, while hematopoietic CFC potential was restricted to CD43+ cells. iPSC-derived CD43+ cells could be separated into three major subsets based on differential expression of CD235a/CD41a and CD45: CD235a+CD41a+/− (erythro-megakaryocytic progenitors), and lin-CD34+CD43+CD45− (multipotent), and lin-CD34+CD43+CD45+ (myeloid-skewed) primitive hematopoietic cells. Both subsets of primitive hematopoietic cells expressed genes associated with myeloid and lymphoid development, although myeloid genes were upregulated in CD45+ cells, which are skewed toward myeloid differentiation. Cytogenetic analysis demonstrated that iPSCs and derived from them CD43+ cells maintained normal karyotype. In addition short tandem repeat analysis of CFCs generated from IMR90-1 cells has been performed to confirm that blood cells are in fact derived from reprogrammed IMR90 cells, and not from contaminating hESCs. While we observed some variations in the efficiency of hematopoietic differentiation between different iPSCs, the pattern of differentiation was very similar in all seven tested iPSC and five hESC lines. Using different cytokine combinations and culture conditions we were able to expand iPSC-derived myeloid progenitors and induce their differentiation toward red blood cells, neutrophils, eosinophils, macrophages, ostoeclasts, dendritic and Langerhans cells. Although several issues remain to be resolved before iPSC-derived blood cells can be administered to humans for therapeutic purposes, patient-specific iPSCs can already be used for characterization of mechanisms of blood diseases and to identify molecules that can correct affected genetic networks.

scholarly journals A chemical tool for improved culture of human pluripotent stem cells

2020 ◽  
Author(s):  
Laurence Silpa ◽  
Maximilian Schuessler ◽  
Gu Liu ◽  
Marcus Olivecrona ◽  
Lucia Groizard-Payeras ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Large Scale ◽  
3D Culture ◽  
Cost Effective ◽  
Human Pluripotent Stem Cells ◽  
Directed Differentiation ◽  
Kinase C ◽  
2D And 3D ◽  
Cell Cell

AbstractThe large-scale and cost-effective production of quality-controlled human pluripotent stem cells (hPSC) for use in cell therapy and drug discovery requires chemically-defined xenobiotic-free culture systems that enable easy and homogeneous expansion of pluripotent cells. Through phenotypic screening, we have identified a small molecule, OXS8360 (an optimized derivative of (-)-Indolactam V ((-)-ILV)), that stably disrupts hPSC cell-cell contacts. Proliferation of hPSC in OXS8360 is normal, as are pluripotency signatures, directed differentiation to hallmark lineages and karyotype over extended passaging. In 3D culture, OXS8360-treated hPSC form smaller, more uniform aggregates, that are easier to dissociate, greatly facilitating expansion. The mode of action of OXS8360 involves disruption of the localisation of the cell-cell adhesion molecule E-cadherin, via activation of unconventional Protein Kinase C isoforms. OXS8360 media supplementation is therefore able to yield more uniform, disaggregated 2D and 3D hPSC cultures, providing the hPSC field with an affordable tool to improve hPSC quality and scalability.

Norepinephrine Improves The Generation Of Hematopoietic Cells From Human Pluripotent Stem Cells With Increased Functional Properties

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1179-1179 ◽  
Author(s):  
Carolina Guibentif ◽  
Roger Emanuel Rönn ◽  
Roksana Moraghebi ◽  
Emanuela Monni ◽  
Marita Grönning Madsen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Progenitor Cell ◽  
Hematopoietic Cells ◽  
Fold Increase ◽  
Human Pluripotent Stem Cells ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Progenitors ◽  
Post Transplantation ◽  
Differentiation Potential

Abstract The possibility of differentiating human pluripotent stem cells (hPSCs) to hematopoietic stem cells (HSCs) could provide an unlimited source of donor cells for the treatment of hematological disorders and malignancies where HSC transplantation is required. Recently, signaling from the developing peripheral nervous system (PNS) has been implicated in the generation of HSCs in the aorta gonad mesonephros (AGM) of mouse embryos. Our own observations of AGM and urogenital ridge (UR) explants from a 6 weeks old human embryo show neurogenic potential. Since the AGM and UR are active sites of hematopoietic emergence, we hypothesize that migrating neural crest (NC) cells, precursors of the PNS, play an active role in HSC generation and maturation. Given that, at the time of HSC emergence, NC cells start expressing enzymes required for catecholamine production, we added norepinephrine (NE) to our optimized hPSC differentiation system and assessed for hematopoietic progenitor cell output. We observed approximately 50% increase of cells with an HSC immunophenotype (CD43+CD34+CD38-CD90+CD45RA-) compared to control settings (1.56 ± 0.25 fold increase, n=5 independent experiments, p=0.007). We identified this phenotype as an early progenitor cell with lymphoid and myeloid differentiation potential as well as highest colony forming unit (CFU) content. Importantly, the increase was specific to this cell fraction, since the proportion of more mature progenitors (CD43+CD34+ cells) and of total blood (CD43+) did not display a significant increase in the presence of NE (respectively, 1.053 ± 0.09 fold increase p=0.2104, and 0.97 ± 0.46 fold increase p=0.880, n=5 independent experiments). Continuing the differentiation culture for 5 additional days did not show additional increase in the frequency of these early progenitors. This indicates that higher proportion of early progenitors in the presence of NE is due to increased emergence from hemogenic endothelium rather than to maintenance of the early progenitors in culture. Improved generation of early progenitors was also consistent with an increase of colonies in the CFU assay (1.84 ± 0.29 fold increase in colony numbers n=4 independent experiments, p=0.010). The increased output of early hematopoietic progenitors was reversed when the Adrenergic Receptor β2 specific inhibitor ICI 118,551 was added together with NE, showing that the effect of NE is mediated by the activation of this receptor. Our preliminary results of transplantation in NSG mice show CD43+ human chimerism at week 6 post-transplantation 11 fold higher in mice transplanted with hematopoietic cells generated in the presence of NE compared to their counterparts transplanted with cells generated in standard conditions [2.33 ± 1.25% (n=3) vs 0.21 ± 0.02% (n=2), p=0.006]. However, since the detected human cells are predominantly of myeloid lineage (CD33+/CD15+) and the chimerism levels decrease back to levels comparable to PBS transplanted negative control at week 8-10 post-transplantation, this suggests that NE increases the homing and short term repopulation ability of pluripotent stem cell derived early hematopoietic progenitors. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Benchmark Side-by-Side Comparison of Two Well-Established Protocols for in vitro Hematopoietic Differentiation From Human Pluripotent Stem Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Francisco Gutierrez-Agüera ◽  
Virginia Rodriguez-Cortez ◽  
Paolo Petazzi ◽  
Clara Bueno ◽  
Pablo Menendez
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells ◽  
Embryoid Bodies ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Growth Factors ◽  
Hematopoietic Stem ◽  
Hesc Lines ◽  
Definitive Hematopoiesis

The generation of transplantable hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) remains challenging. Current differentiation protocols from hPSCs generate mostly hematopoietic progenitors of the primitive HSC-independent program, and it remains unclear what is the best combination of cytokines and hematopoietic growth factors (HGFs) for obtaining functional hematopoietic cells in vitro. Here, we have used the AND1 and H9 hESC lines and the H9:dual-reporter RUNX1C-GFP-SOX17-Cherry to compare the hematopoietic differentiation in vitro based on the treatment of embryoid bodies (EBs) with the ventral mesoderm inducer BMP4 plus HGFs in the absence (protocol 1) or presence (protocol 2) of stage-specific activation of Wnt/β-catenin and inhibition of Activin/Nodal. Despite a slight trend in favor of protocol 1, no statistically significant differences were observed between protocols at any time point analyzed throughout EB development regarding the frequency of hemogenic endothelial (HE) precursors; CD43+ CD45−, CD45+, and CD45 + CD34 + hematopoietic derivatives; or the output of clonogenic progenitors. Similarly, the kinetics of emergence throughout EB development of both SOX17 + HE and RUNX1C + definitive hematopoiesis was very similar for both protocols. The expression of the early master mesendodermal transcription factors Brachyury, MIXL1, and KDR revealed similar gene expression kinetics prior to the emergence of RUNX1C + definitive hematopoiesis for both protocols. Collectively, the simpler protocol 1 is, at least, as efficient as protocol 2, suggesting that supplementation with additional morphogens/HGFs and modulation of Activin/Nodal and Wnt/β-catenin pathways seem dispensable for in vitro hematopoietic differentiation of hPSCs.

scholarly journals Enhanced HSC-like cell generation from mouse pluripotent stem cells in a 3D induction system cocultured with stromal cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Wei Shan ◽  
Qin Yu ◽  
Yan Long ◽  
Qian Luo ◽  
Honghu Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stromal Cells ◽  
Pluripotent Stem Cells ◽  
Hematopoietic Cells ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem ◽  
Hematopoietic Reconstitution ◽  
Induction System

Abstract Background Decades of efforts have attempted to differentiate the pluripotent stem cells (PSCs) into truly functional hematopoietic stem cells (HSCs), yet the problems of low differentiation efficiency in vitro and poor hematopoiesis reconstitution in vivo still exist, mainly attributing to the lack of solid, reproduced, or pursued differentiation system. Methods In this study, we established an in vitro differentiation system yielding in vivo hematopoietic reconstitution hematopoietic cells from mouse PSCs through a 3D induction system followed by coculture with OP9 stromal cells. The in vivo hematopoietic reconstitution potential of c-kit+ cells derived from the mouse PSCs was evaluated via m-NSG transplantation assay. Flow cytometry analysis, RNA-seq, and cell cycle analysis were used to detect the in vitro hematopoietic ability of endothelial protein C receptor (EPCR, CD201) cells generated in our induction system. Results The c-kit+ cells from 3D self-assembling peptide induction system followed by the OP9 coculture system possessed apparently superiority in terms of in vivo repopulating activity than that of 3D induction system followed by the 0.1% gelatin culture. We interestingly found that our 3D+OP9 system enriched a higher percentage of CD201+c-kit+cells that showed more similar HSC-like features such as transcriptome level and CFU formation ability than CD201-c-kit+cells, which have not been reported in the field of mouse PSCs hematopoietic differentiation. Moreover, CD201+ hematopoietic cells remained in a relatively slow cycling state, consistent with high expression levels of P57 and Ccng2. Further, we innovatively demonstrated that notch signaling pathway is responsible for in vitro CD201+ hematopoietic cell induction from mouse PSCs. Conclusions Altogether, our findings lay a foundation for improving the efficiency of hematopoietic differentiation and generating in vivo functional HSC-like cells from mouse PSCs for clinical application.

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.

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