scholarly journals Robust generation of erythroid and multilineage hematopoietic progenitors from human iPSCs using a scalable monolayer culture system

2019 ◽  
Author(s):  
Juan Pablo Ruiz ◽  
Guibin Chen ◽  
Juan Jesus Haro Mora ◽  
Keyvan Keyvanfar ◽  
Chengyu Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Vascular Endothelium ◽  
Pluripotent Stem Cells ◽  
Culture System ◽  
Supernatant Fraction ◽  
Erythroid Cells ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Progenitors ◽  
Erythroid Progenitors ◽  
Human Ipscs

AbstractOne of the most promising objectives of clinical hematology is to derive engraftable autologous hematopoietic stem cells (HSCs) from human induced pluripotent stem cells (iPSCs). Progress in translating iPSC technologies to the clinic relies on the availability of scalable differentiation methodologies. In this study, human iPSCs were differentiated for 21 days using STEMdiff™, a monolayer-based approach for hematopoietic differentiation of human iPSCs that requires no replating, co-culture or embryoid body formation. Both monolayer and suspension cells were functionally characterized throughout differentiation. In the supernatant fraction, an early transient population of primitive CD235a+ erythroid cells first emerged, followed by hematopoietic progenitors with multilineage differentiation activity in vitro but no long-term engraftment potential in vivo. In later stages of differentiation, a nearly exclusive production of definitive erythroid progenitors was observed. In the adherent monolayer, we identified a prevalent population of mesenchymal stromal cells and limited arterial vascular endothelium (VE), suggesting that the cellular constitution of the monolayer may be inadequate to support the generation of HSCs with durable repopulating potential. Quantitative modulation of WNT/β-catenin and activin/nodal/TGFβ signaling pathways with CHIR/SB molecules during differentiation enhanced formation of arterial VE, definitive multilineage and erythroid progenitors, but was insufficient to orchestrate the generation of engrafting HSCs. Overall, STEMdiff™ provides a clinically-relevant and readily adaptable platform for the generation of erythroid and multilineage hematopoietic progenitors from human pluripotent stem cells.HighlightsRobust, scalable and clinically-relevant monolayer-based culture system for hematopoietic differentiation of human iPSCs.Successive emergence of primitive erythroid cells, definitive multilineage HSPCs and erythroid progenitors in the culture supernatant.Abundant mesenchymal cells and limited arterial vascular endothelium in the culture monolayer.CHIR/SB molecules increase arterial vascular endothelium formation, suppress primitive hematopoiesis and promote definitive multilineage and erythroid progenitors.

Induced Pluripotent Stem Cells and Erythrocyte Production

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. SCI-38-SCI-38
Author(s):  
Igor Slukvin
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Blood Cells ◽  
Embryonic Stem ◽  
Erythroid Cells ◽  
Hematopoietic Progenitors ◽  
Adult Hemoglobin ◽  
Equity Ownership ◽  
Induced Pluripotent

Abstract Abstract SCI-38 Induced pluripotent stem cells (iPSCs) are somatic cells that have been turned into embryonic-like stem cells by forced expression of factors critical for establishing pluripotency. Because iPSCs can be differentiated into any type of cell in the human body, including hematopoietic cells, they are seen as a logical alternative source of red blood cells (RBCs) for transfusion. In addition, the unlimited expansion potential of iPSCs makes it easy to adopt iPSC technology for RBC biomanufacturing. iPSCs can be generated from any type of donor, including O/Rh-negative universal donors and donors with very rare blood phenotypes, which makes it possible to generate blood products to accommodate virtually all patient groups. We have developed an approach for generating large quantities of RBCs from iPSCs by inducing them to differentiate into CD34+CD43+ hematopoietic progenitors in coculture with OP9 stromal cells, followed by selective expansion of erythroid cells in serum-free media with erythropoiesis-supporting cytokines. Erythroid cultures produced by this approach consist of leukocyte-free populations of CD235a+ RBCs with robust expansion potential and long (up to 90 days) life spans. In these cultures, up to 1.8×105 RBCs can be generated from a single iPSC. Similar to embryonic stem cells, iPSC-derived RBCs express predominantly embryonic and fetal hemoglobin, with very little adult hemoglobin. It is already feasible to adopt iPSC technologies for producing cGMP-grade RBCs using defined animal-product-free differentiation conditions. However, the induction of the complete switch from embryonic to fetal and adult hemoglobin, as well as the terminal maturation and enucleation of iPSC-derived erythroid cells, remains a significant challenge. We recently identified at least three distinct waves of hematopoietic progenitors with erythroid potential in iPSC differentiation cultures. The characterization of erythroid cells produced from these waves of hematopoiesis may help to define populations with definitive erythroid potential and facilitate the production of erythrocytes from iPSCs. Additional critical steps toward translating iPSC-based RBC technologies to the clinic include the development of bioreactor-based-technology for further scaling-up of cell production, and evaluation of the therapeutic potential and safety of human pluripotent stem cell-derived blood cells in animal models. Overall, the manufacturing of RBCs provides several advantages. It can improve the continuity of the blood supply, minimize/eliminate the risk of infection transmission, reduce the incidence of hemolytic and nonhemolytic transfusion reactions, and provide an opportunity to generate RBCs that fit specific clinical needs by using genetically engineered iPSCs or iPSCs with rare blood groups. Disclosures: Slukvin: CDI: Consultancy, Equity Ownership; Cynata: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

scholarly journals A Reproducible and Simple Method to Generate Red Blood Cells from Human Pluripotent Stem Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1189-1189
Author(s):  
Marta A. Walasek ◽  
Crystal Chau ◽  
Christian Barborini ◽  
Matthew Richardson ◽  
Stephen J. Szilvassy ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Blood Cells ◽  
Culture System ◽  
Erythroid Differentiation ◽  
Average Frequency ◽  
Erythroid Cells ◽  
Ips Cell ◽  
High Yields

Erythroid cells generated from human pluripotent stem cells (hPSCs) can potentially offer an unlimited and safe supply of red blood cells (RBCs) for transfusion. Human PSC-derived erythroid cells at various stages of differentiation can also be used to model blood diseases, test new drug candidates, and develop cellular and genetic therapies. Although several protocols for deriving RBCs from hPSCs have been described, these are typically complex, involving multiple culture steps that may include co-culture with feeder cells, and exhibit large variability in erythroid cell yields between hPSC lines and replicate experiments. We have developed a straightforward, serum-free and feeder-free culture method to generate erythroid cells from hPSCs with high yields and high purity. The method has been validated on multiple human embryonic stem (ES) cell lines (H1, H7, H9) and induced pluripotent stem (iPS) cell lines (WLS-1C, STiPS-F016, STiPS-B004). The protocol involves two steps: hematopoietic specification of hPSCs, followed by differentiation of hPSC-derived hematopoietic stem and progenitor cells (HSPCs) into erythroid cells. Lineage specification and differentiation is driven by only three supplements that combine cytokines and other factors to support optimal differentiation efficiency and cell yield across cell lines. In the first step, small hPSC aggregates routinely maintained in feeder-free maintenance medium, are plated onto matrigel-coated microwells, and specification to mesoderm and subsequent hematoendothelial differentiation is induced by addition of successive expansion supplements. This phase promotes extensive hematopoietic progenitor cell generation, with a single hPSC producing on average 142 HSPCs (range: 50 - 360, n = 3 experiments) by day 10 across all six ES and iPS cell lines tested. The average frequency of cells expressing CD43, an embryonic pan-hematopoietic marker, is 92% (range: 85 - 95%), and the frequency of CD34+ cells ranges between 24-55%. In the second, erythroid differentiation step, hPSC-derived HSPCs expand on average 300-fold (range: 80 - 1000) within 10 - 14 days, and the average frequency of GlyA+ cells is 75% (range: 70 - 85%). Cumulatively, this results in the generation of on average 30,000 GlyA+ cells (range: 10,000 - 80,000) per initial hPSC after 20 - 24 days. Further maturation in 7-day cultures containing EPO and human serum resulted in a > 90% pure population of GlyA+ erythroid cells. Notably, no cell loss was observed during the maturation phase, resulting in an average yield of 50,000 GlyA+ cells (range: 10,000 - 220,000) per single initial hPSC on day 31. Differentiated cells were characterized by orthochromatic normoblast morphology and decreased CD71 expression, consistent with erythroid maturation. Erythroid cells generated in this differentiation culture system expressed a mix of 'primitive' and 'definitive' hemoglobin types, but with adult and fetal hemoglobin being expressed at higher levels than embryonic hemoglobin. The observed enucleation rates of hPSC-derived erythroid cells are consistent with current reports and are subject to further optimization. In summary, we have developed a two-step, serum- and feeder-free erythroid differentiation method to generate large numbers of erythroid cells from multiple hPSC lines. This culture system provides a simple, standardized and reproducible platform to generate RBCs from hPSCs with high yields and efficiency for basic and translational research. Disclosures Walasek: STEMCELL Technologies, Inc: Employment. Chau:STEMCELL Technologies, Inc: Employment. Barborini:STEMCELL Technologies, Inc: Employment. Richardson:STEMCELL Technologies, Inc: Employment. Szilvassy:STEMCELL Technologies, Inc: Employment. Louis:STEMCELL Technologies, Inc: Employment. Thomas:STEMCELL Technologies, Inc: Employment. Eaves:STEMCELL Technologies, Inc: Employment. Wognum:STEMCELL Technologies, Inc: Employment.

scholarly journals The Roles of RUNX1 in Human Hematopoiesis and Megakaryopoiesis Revealed By Genome-Targeted Human iPSCs and an Improved Hematopoietic Differentiation Model

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1167-1167
Author(s):  
Bin-Kuan Chou ◽  
Hao Bai ◽  
Yongxing Gao ◽  
Ying Wang ◽  
Zhaohui Ye ◽  
...  
Keyword(s):  
Stem Cells ◽  
Culture System ◽  
Cell Formation ◽  
Hematopoietic Cells ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem ◽  
Runx1 Gene ◽  
Human Ipscs ◽  
Cd34 Cells ◽  
Definitive Hematopoiesis

Abstract The RUNX1 gene (also called AML1), one of the most mutated genes in acute myeloid leukemia (AML), was first identified decades ago that encodes a key regulatory transcriptional factor. Numerous studies using mouse and zebrafish models show that RUNX1 is essential for definitive hematopoiesis. In mice, its homozygous knock-out (KO) in hematopoietic stem/progenitor cells also causes defects in lymphoid and megakaryocytic (MK) development. However, heterozygous Runx1 gene mutations in laboratory mouse and zebrafish had little effects on development of hematopoietic stem/progenitor cells (HSPCs) or the MK cell lineage. In contrast, heterozygous germline mutations in RUNX1 were found in patients with familial platelet disorder (FPD) with predisposition to AML and MDS. The mechanisms underlying the observed differences between humans and small animal models remain unclear. In the past decade, we and others have utilized human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to investigate human hematopoiesis including the roles of the RUNX1 gene. Using a feeder-free culture system, we generated human CD34+CD45+ HSPCs cells from human ESCs and iPSCs around 11-14 days after embryoid body (EB) formation. The CD34+CD45+ HSPCs were capable to form multiple types of hematopoietic cells such as myeloid, erythroid, and polyploid MK cells (Connelly et al., 2014; Liu et al., 2015). We also reported that human iPSCs derived from FPD patients containing a heterozygous RUNX1 mutation were defective in MK formation, and targeted correction of the mutated RUNX1 allele by genome editing restored the MK potential (Connelly et al., 2014). Since then, we have extended our studies by precise genomic targeting in human wildtype iPSCs to ablate exon 5 that is common in all 3 isoforms of the RUNX1 gene, or exon 1B that is unique to the RUNX1c isoform. Bi-allelic KO of RUNX1 at exon 5 completely abolished the formation of hematopoietic cells at days 11-14 after EB formation. Complete disruption of exon 1B showed little effect, indicating that the RUNX1c isoform is dispensable for definitive hematopoiesis in the presence of the RUNX1a and RUNX1b isoforms transcribed from the downstream P2 promoter. Detailed analysis of EBs at days 6-8 revealed that bi-allelic RUNX1 KO at exon 5 (ablating all 3 isoforms) did not affect the formation of CD34+/CD31+/CD144+ endothelial-like cells. However, the endothelial-to-hematopoietic transition (EHT) was completely blocked and no CD45+ hematopoietic cells emerged from the EHT culture supplemented with hematopoietic cytokines. To elucidate the functions of different RUNX1 isoforms in early steps of human hematopoiesis, we adapted the EHT culture system that uses CD34+ cells isolated from earlier stages of EB formation (day 6-8), before definite hematopoiesis was observable (after day 8, Bai et al., 2015). Two different iPSCs clones with homozygous or bi-allelic RUNX1 KO at exon 5 both failed to form CD45+ hematopoietic cells after EHT culture. Because constitutive transgene expression of RUNX1b or RUNX1c (but not RUNX1a) cDNA in human iPSCs inhibits hematopoietic differentiation, we transduced the day 6 EB cells at the beginning of the EHT culture with RUNX1-expressing vectors. Lentiviral vectors constitutively express RUNX1b or RUNX1c (but not RUNX1a) cDNA partially restored the EHT and hematopoietic (CD45+) cell formation after 4-5 days in EHT culture. We further used a lentiviral vector in which RUNX1c (as an ER fusion protein) can be conditionally activated by 4-HT induction. Induction starting at day 4 and lasting for 3 days rendered the maximal effect of hematopoietic cell formation in the EHT culture using CD34+ cells isolated at day 6 of EB formation. Our data corroborate with limited in vivo data using human fetal tissues on the possible roles of RUNX1 in definitive hematopoiesis. At present we are analyzing iPSC clones with mono-allelic disruption of exon 5 in a wild type iPSC line, comparing to iPSCs derived from FPD patients. The current study of using isogenic human iPSCs will help to understand the roles of RUNX1 in human hematopoiesis and megakaryopoiesis, and offer an amenable system to study the RUNX1 gene functions and downstream target genes. Disclosures No relevant conflicts of interest to declare.

Efficient Production of Human Hematopoietic Progenitors from Human Pluripotent Stem Cells Using Chemically Defined Media without Serum or Feeder Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2463-2463
Author(s):  
Zhaohui Ye ◽  
Xiaobing Yu ◽  
Linzhao Cheng
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Es Cells ◽  
Ips Cells ◽  
Feeder Cells ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Progenitors ◽  
Serum Factors ◽  
Defined Media ◽  
Human Es Cells

Abstract As established cell lines, human pluripotent stem cells such as embryonic stem (ES) or induced pluripotent stem (iPS) cells can divide indefinitely while retaining their potential to differentiate into many cell types in culture. The induction of mesoderm formation and hematopoietic differentiation was achieved either via embryoid body (EB) formation by culturing ES cell aggregation in suspension or by co-cultures with mouse stromal cell lines. Traditionally serum factors are also added for mesoderm induction and hematopoietic differentiation. Although hematopoietic progenitors are obtained from multiple lines of human ES cells, the low efficiency and high variability have hindered the progress of using human ES cells as a model for studying human hematopoiesis. Normally <15% of cells obtained from a primary culture expressed CD34 (a marker for endothelial cells and hematopoietic progenitors) and even less for CD45 (a pan-leukocyte marker). To generate maximal output of CD34+CD45+ hematopoietic progenitors, we decided to adopt the serum-free and spin-EB formation method (Ng, Blood, 2005) and systematically improved culture conditions. 3,000 human ES cells were added into each well in 96-well plates and formed an aggregate after centrifugation. BMP4 and bFGF were added at day 1, and VEGF and hematopoietic cytokines was added at day 3–9. VEGF was then withdrawn after day 9. Single EB (occasionally 2) grew in each well. By day 8, small blast (or lymphocyte-) like cells were observed on the edge of EBs. By day 12–14, we observed the outgrowth of blast cells (in hundreds to thousands) surrounding each EB (Panel A). By FACS analysis (Panel B), we observed nearly 50% of the total cells express CD45 at day 12, and many co-express CD34. The lymphocytelike cells can be easily separately from EBs by passing through a 40-micron strainer, and nearly all the isolated cells express CD45 (and 50–75% of them co-express CD34). We obtained 6 million CD45+ cells from 0.9 million human ES cells 14 days after EB formation. We also observed that the conditional HES1-ER transgene expression further increased the frequency of CD34+CD45+ cells as we observed under a different culture condition (Yu. Cell Stem Cells, 2008). The isolated CD45+ cells formed efficiently hematopoietic colonies in the methylcellulose medium with a frequency of ~58+/− 4 colonies per 3,000 cells, with or without the HES1-ER transgene. We are currently testing in vivo activities of isolated CD45+ cell populations (+/− HES1-ER) in the NOD/SCID/γC−/− mice. We are also testing if this improved and defined method would also be applicable to hematopoietic differentiation of human iPS cells we recently derived (Mali, Stem Cells, 2008). The significantly improved method using defined media in the absence of serum factors or feeder cells warrants further investigation whether it is better and more reproducible to elucidate mechanisms that regulate early human hematopoiesis, and to generate a large quantity of CD34+CD45+ human hematopoietic progenitor cells for various applications. Figure Figure

scholarly journals Utilising Induced Pluripotent Stem Cells in Neurodegenerative Disease Research: Focus on Glia

2021 ◽  
Vol 22 (9) ◽  
pp. 4334
Author(s):  
Katrina Albert ◽  
Jonna Niskanen ◽  
Sara Kälvälä ◽  
Šárka Lehtonen
Keyword(s):  
Stem Cells ◽  
Neurodegenerative Diseases ◽  
Neurodegenerative Disease ◽  
Induced Pluripotent Stem Cells ◽  
Glial Cells ◽  
Pluripotent Stem Cells ◽  
Cell Types ◽  
Human Ipscs ◽  
Induced Pluripotent

Induced pluripotent stem cells (iPSCs) are a self-renewable pool of cells derived from an organism’s somatic cells. These can then be programmed to other cell types, including neurons. Use of iPSCs in research has been two-fold as they have been used for human disease modelling as well as for the possibility to generate new therapies. Particularly in complex human diseases, such as neurodegenerative diseases, iPSCs can give advantages over traditional animal models in that they more accurately represent the human genome. Additionally, patient-derived cells can be modified using gene editing technology and further transplanted to the brain. Glial cells have recently become important avenues of research in the field of neurodegenerative diseases, for example, in Alzheimer’s disease and Parkinson’s disease. This review focuses on using glial cells (astrocytes, microglia, and oligodendrocytes) derived from human iPSCs in order to give a better understanding of how these cells contribute to neurodegenerative disease pathology. Using glia iPSCs in in vitro cell culture, cerebral organoids, and intracranial transplantation may give us future insight into both more accurate models and disease-modifying therapies.

scholarly journals A non-invasive method to generate induced pluripotent stem cells from primate urine

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Johanna Geuder ◽  
Lucas E. Wange ◽  
Aleksandar Janjic ◽  
Jessica Radmer ◽  
Philipp Janssen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Sendai Virus ◽  
Cell Types ◽  
Urine Samples ◽  
Comparative Approach ◽  
Non Invasive ◽  
Human Ipscs ◽  
Induced Pluripotent

AbstractComparing the molecular and cellular properties among primates is crucial to better understand human evolution and biology. However, it is difficult or ethically impossible to collect matched tissues from many primates, especially during development. An alternative is to model different cell types and their development using induced pluripotent stem cells (iPSCs). These can be generated from many tissue sources, but non-invasive sampling would decisively broaden the spectrum of non-human primates that can be investigated. Here, we report the generation of primate iPSCs from urine samples. We first validate and optimize the procedure using human urine samples and show that suspension- Sendai Virus transduction of reprogramming factors into urinary cells efficiently generates integration-free iPSCs, which maintain their pluripotency under feeder-free culture conditions. We demonstrate that this method is also applicable to gorilla and orangutan urinary cells isolated from a non-sterile zoo floor. We characterize the urinary cells, iPSCs and derived neural progenitor cells using karyotyping, immunohistochemistry, differentiation assays and RNA-sequencing. We show that the urine-derived human iPSCs are indistinguishable from well characterized PBMC-derived human iPSCs and that the gorilla and orangutan iPSCs are well comparable to the human iPSCs. In summary, this study introduces a novel and efficient approach to non-invasively generate iPSCs from primate urine. This will extend the zoo of species available for a comparative approach to molecular and cellular phenotypes.

scholarly journals Chemically defined and xeno-free culture condition for human extended pluripotent stem cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bei Liu ◽  
Shi Chen ◽  
Yaxing Xu ◽  
Yulin Lyu ◽  
Jinlin Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Culture Condition ◽  
Human Fibroblast ◽  
Culture System ◽  
Disease Modeling ◽  
Directed Differentiation

AbstractExtended pluripotent stem (EPS) cells have shown great applicative potentials in generating synthetic embryos, directed differentiation and disease modeling. However, the lack of a xeno-free culture condition has significantly limited their applications. Here, we report a chemically defined and xeno-free culture system for culturing and deriving human EPS cells in vitro. Xeno-free human EPS cells can be long-term and genetically stably maintained in vitro, as well as preserve their embryonic and extraembryonic developmental potentials. Furthermore, the xeno-free culturing system also permits efficient derivation of human EPS cells from human fibroblast through reprogramming. Our study could have broad utility in future applications of human EPS cells in biomedicine.

scholarly journals Reprogramming mechanisms influence the maturation of hematopoietic progenitors from human pluripotent stem cells

2018 ◽  
Vol 9 (11) ◽  
Author(s):  
Hye-Ryeon Heo ◽  
Haengseok Song ◽  
Hye-Ryun Kim ◽  
Jeong Eun Lee ◽  
Young Gie Chung ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells ◽  
Hematopoietic Progenitors
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