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 In VitroLarge Scale Production of Human Mature Red Blood Cells from Hematopoietic Stem Cells by Coculturing with Human Fetal Liver Stromal Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Jiafei Xi ◽  
Yanhua Li ◽  
Ruoyong Wang ◽  
Yunfang Wang ◽  
Xue Nan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Red Blood Cells ◽  
Stromal Cells ◽  
Blood Cells ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Erythroid Cells ◽  
Hematopoietic Stem

In vitromodels of human erythropoiesis are useful in studying the mechanisms of erythroid differentiation in normal and pathological conditions. Here we describe an erythroid liquid culture system starting from cord blood derived hematopoietic stem cells (HSCs). HSCs were cultured for more than 50 days in erythroid differentiation conditions and resulted in a more than 109-fold expansion within 50 days under optimal conditions. Homogeneous erythroid cells were characterized by cell morphology, flow cytometry, and hematopoietic colony assays. Furthermore, terminal erythroid maturation was improved by cosculturing with human fetal liver stromal cells. Cocultured erythroid cells underwent multiple maturation events, including decrease in size, increase in glycophorin A expression, and nuclear condensation. This process resulted in extrusion of the pycnotic nuclei in up to 80% of the cells. Importantly, they possessed the capacity to express the adult definitiveβ-globin chain upon further maturation. We also show that the oxygen equilibrium curves of the cord blood-differentiated red blood cells (RBCs) are comparable to normal RBCs. The large number and purity of erythroid cells and RBCs produced from cord blood make this method useful for fundamental research in erythroid development, and they also provide a basis for future production of available RBCs for transfusion.

scholarly journals PSC-RED, an Albumin-Free Robust Erythroid Differentiation Method to Produce Enucleated Red Blood Cells from Human Pluripotent Stem Cells

2019 ◽  
Author(s):  
Emmanuel N Olivier ◽  
Shouping Zhang ◽  
Zi Yan ◽  
Sandra Suzuka ◽  
Karl Roberts ◽  
...  
Keyword(s):  
Stem Cells ◽  
Red Blood Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Blood Cells ◽  
Erythroid Differentiation ◽  
Economic Viability ◽  
List Type ◽  
Potential Applications ◽  
Induced Pluripotent

AbstractCultured red blood cells (cRBCs) have many potential applications in transfusion medicine and drug delivery. We report that we have developed chemically defined, albumin-free Robust Erythroid Differentiation (RED) methods to produce enucleated cRBCs from human induced pluripotent stem cells (iPSCs). Human iPSC-derived cRBCs produced with either the short or long variation of the RED protocol respectively express embryonic/fetal or a mixture of fetal and adult hemoglobins. The long version of the protocol produces up to 50% of enucleated cells at an unprecedented yield. RED is scalable and relies on inexpensive components and therefore dramatically increases the feasibility and economic viability of all translational applications of cRBCs.HighlightsPSC-RED: A chemically-defined, albumin-free Robust Erythroid Differentiation (RED) methods to produce cRBCs from human induced pluripotent stem cells.PSC-RED produces up to 50% enucleated cells at an unprecedented yield.PSC-RED is scalable and relies on inexpensive components and therefore increases the feasibility and economic viability of translational applications of cRBCs.

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 Human-induced pluripotent stem cells from blood cells of healthy donors and patients with acquired blood disorders

Blood ◽  
2009 ◽  
Vol 114 (27) ◽  
pp. 5473-5480 ◽  
Author(s):  
Zhaohui Ye ◽  
Huichun Zhan ◽  
Prashant Mali ◽  
Sarah Dowey ◽  
Donna M. Williams ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Blood Cells ◽  
Ips Cells ◽  
Patient Specific ◽  
Hematopoietic Differentiation ◽  
Ips Cell ◽  
Healthy Donors ◽  
Cd34 Cells ◽  
Induced Pluripotent

Abstract Human induced pluripotent stem (iPS) cells derived from somatic cells hold promise to develop novel patient-specific cell therapies and research models for inherited and acquired diseases. We and others previously reprogrammed human adherent cells, such as postnatal fibroblasts to iPS cells, which resemble adherent embryonic stem cells. Here we report derivation of iPS cells from postnatal human blood cells and the potential of these pluripotent cells for disease modeling. Multiple human iPS cell lines were generated from previously frozen cord blood or adult CD34+ cells of healthy donors, and could be redirected to hematopoietic differentiation. Multiple iPS cell lines were also generated from peripheral blood CD34+ cells of 2 patients with myeloproliferative disorders (MPDs) who acquired the JAK2-V617F somatic mutation in their blood cells. The MPD-derived iPS cells containing the mutation appeared normal in phenotypes, karyotype, and pluripotency. After directed hematopoietic differentiation, the MPD-iPS cell-derived hematopoietic progenitor (CD34+CD45+) cells showed the increased erythropoiesis and gene expression of specific genes, recapitulating features of the primary CD34+ cells of the corresponding patient from whom the iPS cells were derived. These iPS cells provide a renewable cell source and a prospective hematopoiesis model for investigating MPD pathogenesis.

scholarly journals Olfactomedin 4 Inhibits Erythroid Differentiation of Leukemic Cell Lines Induced By TGF-β: A Model of Preferential Commitment of Del(13q) Hematopoietic Stem Cells in Immune-Mediated Bone Marrow Failure

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5000-5000
Author(s):  
Kohei Thi Hosokawa ◽  
Kohei Hosokawa ◽  
Tanabe Mikoto ◽  
Md Mohiuddin ◽  
Takeshi Yoroidaka ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Lines ◽  
Western Blotting ◽  
Erythroid Differentiation ◽  
Erythroid Cells ◽  
Hematopoietic Stem ◽  
Protein Levels ◽  
Company Limited ◽  
Immune Mediated

[Background] Olfactomedin 4 (OLFM4), a member of the olfactomedin-related protein family, is constitutively expressed in bone marrow (BM) cells and many gastrointestinal organs and is involved in a variety of cellular functions, including proliferation, differentiation, apoptosis, and cell adhesion in tumor cells. Previous studies have suggested that OLFM4 supports the survival of leukemic stem cells derived from iPS cells of patients with chronic myeloid leukemia. Our recent study revealed a somatic mutation of OLFM4 in HLA allele-lacking granulocytes of a patients with acquired aplastic anemia (AA) in long-term remission (Blood advances, 2(9):1000-1012, 2018). The OLFM4 gene is located at chromosome 13q14.3, which is a commonly deleted region in AA patients with del(13q) who show a good response to immunosuppressive therapy and a high prevalence of increased glycosylphosphatidylinositol-anchored protein(GPI-AP)-deficient cells (Haematologica, 97(12):1845-9, 2012). There may be common mechanisms underlying the preferential commitment between GPI-AP- and del(13q) hematopoietic stem and progenitor cells (HSPCs), like insensitivity to inhibitory cytokines such as TGF-β, in immune-mediated BM failure. To confirm this hypothesis, we studied the effect of OLFM4 knockout on the erythroid differentiation of erythroid leukemia cell lines induced by TGF-β. [Methods] We established OLFM4 knockout (KO) K562 and TF-1 cells using a CRISPR-Cas9 system. OLFM4-knockdown (KD) cells were also prepared using siRNA to validate the results of OLFM4-KO cells. The OLFM4 mRNA and protein levels were determined using quantitative polymerase chain reaction, flow cytometry (FCM), Western blotting, immunocytochemistry, and immunofluorescence methods. The erythroid differentiation was assessed by measuring the expression of glycophorin A (GPA) with FCM, GATA-1 protein expression using Western blotting, and iron staining of the cells. [Results] The OLFM4 KO cells showed slower proliferation than wild-type (WT) cells. Both OLFM4-KO cells and OLFM4-KD cells showed a higher GPA expression than WT cells (median fluorescence intensity [MFI] of K562: 2924 and 2143 vs. 1469 and TF-1: 950 and 870 vs. 694, respectively). OLFM4-KO cells showed erythroid morphology, an elevated expression of GATA-1, and positivity for iron granules, suggesting that OLFM4 KO promoted the erythroid differentiation of K562 and TF-1 cells in RPMI1640 containing 10% fetal bovine serum (Figure 1). When WT cells were cultured in a serum-free culture medium (Steampro34) with or without TGF-β (6 ng/ml) for 8 days, the GPA expression was induced in both TF-1 (MFI: 4358 vs. 883), and K562 cell lines (33440 vs. 25655). The OLFM4 protein levels in these cell lines were significantly decreased by the TGF-β treatment in a dose-dependent manner, suggesting that TGF-β directly downregulated the OLFM4 expression in WT cells; the relative expression of OLFM4 was 1, 0.64, and 0.12 while that of TF-1 was 1, 0.12, and 0.05 at 0, 2, and 6 ng/ml of TGF-β, respectively (Figure 2). [Conclusion] OLFM4 prevents K562 and TF-1 cells from differentiating into erythroid cells in response to TGF-β. The erythroid differentiation of these leukemic cells may be mediated by the downregulation of OLFM4 induced by TGF-β. Haploinsufficiency of OLFM4 due to either a loss of function mutation or del(13q) may be related to the mechanisms underlying the preferential commitment of the mutant HSPCs to erythroid cells in patients with immune-mediated BM failure where TGF-β is abundantly present. Disclosures Yoroidaka: Ono Pharmaceutical: Honoraria. Nakao:Takeda Pharmaceutical Company Limited: Honoraria; Novartis Pharma K.K: Honoraria; Kyowa Kirin: Honoraria; Bristol-Myers Squibb: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ono Pharmaceutical: Honoraria; Celgene: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; SynBio Pharmaceuticals: Consultancy.

scholarly journals Generating Large Numbers of Human Megakaryocytes from Pluripotent Stem Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4289-4289
Author(s):  
Jaspreet Gill ◽  
Leon Lin ◽  
Marta A. Walasek ◽  
Stephen J. Szilvassy ◽  
Wing Chang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Culture System ◽  
Method Development ◽  
Immunofluorescence Microscopy ◽  
Dna Ploidy ◽  
Scale Up ◽  
Embryonic Stem ◽  
Hematopoietic Stem

Abstract The process of hemostasis and blood coagulation relies heavily on a sufficient supply of platelets (PLTs, also known as thrombocytes) within a person's bloodstream. Platelet transfusion is an effective treatment for thrombocytopenia-related diseases, yet paucity of supply and limited shelf-life (5 - 7 days) remain challenging. PLTs are generated by the proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs) into megakaryocytes (MKs), a rare subset of large polyploid bone marrow cells. Methods to produce MKs in vitro by inducing mesodermal specification and hematopoietic differentiation of human pluripotent stem cells (hPSCs) could provide a reliable and safe supply of PLTs for transfusion and would also be amenable to gene editing for generation of HLA-null universal PLTs. Culture methods to generate hPSC-derived MKs and PLTs have been described, yet published protocols lack standardization, are PSC line dependent and/or promote differentiation of other lineages, resulting in low MK cell yields and purity. The use of feeder cells and/or viral vectors also limits the clinical and scale-up applicability. Here, we describe an efficient feeder cell-free and serum-free culture system that promotes the selective differentiation of hPSCs from multiple ES and iPS lines into polyploid MKs with high purity and yields and ability to generate platelets. The 17-day protocol includes two stages: a 12-day stage to differentiate hPSCs into megakaryocytic-biased HSPCs through endothelial-to-hematopoietic transition (H-phase), and a 5-day stage to further differentiate HSPCs into mature MKs (MK-phase). at the start of the H-phase, hPSC aggregates were plated in mTeSR TM media on Matrigel ®-coated plates at 16 aggregates (100 - 200 µm in diameter, ~100 cells per aggregate) per cm 2 to allow attachment overnight (Day -1). The cells were then cultured in mesoderm-induction medium for 3 days (Day 0 - 3), and subsequently in hematopoietic specification medium for 9 days (Day 3 - 12). During this phase, PSC-derived HSPCs emerged from an adherent layer of endothelial cells and were released into suspension. On day 12 these nonadherent cells were harvested and seeded at 1 - 3.5 × 10 5 cells/mL in MK maturation medium containing thrombopoietin (TPO) and other hematopoietic growth factors and cultured for 5 additional days (MK-phase, Day 12 - 17). At the end of H-phase (day 12) and MK-phase (day 17) the cells were counted and assessed for HSPC markers (CD34/CD45), the erythroid marker glycophorin A (GlyA), MK markers (CD41a/CD42b), DNA ploidy profile and PLT production by flow cytometry and immunofluorescence microscopy. Two embryonic stem (ES) cell lines (H1 and H9), and two induced pluripotent stem (iPS) cell lines (WLS-1C and STiPS-R038) were used in this study. At the end of H-phase (Day 12), on average 48% (range: 34 - 72%) of cells released into suspension co-expressed CD41a and CD42b markers, with an average yield of 93 CD41a +CD42b + cells per seeded hPSC (range: 30 - 200, n = 4 for H9/1C, n = 3 for H1/R038). The cells also expressed CD34 (average of 78% CD34 + cells) and GlyA (average of 71% GlyA + cells), indicating that the H-phase may support differentiation of PSCs to megakaryocyte-erythroid progenitors. At the end of MK-phase (Day 17), on average 82% of the cells expressed CD41a (range: 70 - 99%), 62% of the cells co-expressed CD41a and CD42b (range: 40 - 85%), and an average of 253 CD41a +CD42b + cells were generated per seeded hPSC (range: 70 - 700 MKs, n = 11 for H1/H9/R038, n = 7 for 1C). Of note, less than 5% of cells expressed GlyA, showing that the culture system is specific for megakaryocytic differentiation. The DNA ploidy profile of the CD41a +CD42b + cells generated on Day 17 showed that on average 26% and 9% of cells had 4N and 8N+ DNA ploidy, respectively (n = 11). Multinucleated MKs could also be readily observed by immunofluorescence microscopy. These PSC-derived MKs produced an average of 3.5 PLTs (range: 1 - 10 PLTs, n = 11) based on viable CD41a +CD45 -GlyA - PLT-like particles with a similar size and CD41 expression as control PLTs prepared from fresh blood. In conclusion, we have developed a simple two-step, yet robust serum- and feeder-free culture system for generating high numbers of hPSC-MKs that are large, polyploid, and capable of shedding PLTs. This culture method provides a platform to study thrombopoiesis and is amenable to scale-up method development. Disclosures No relevant conflicts of interest to declare.

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.

330 DERIVATION OF PORCINE INDUCED PLURIPOTENT STEM CELLS FROM FIBROBLASTS OF A TRANSLOCATED AZOOSPERMIC BOAR

2015 ◽  
Vol 27 (1) ◽  
pp. 254
Author(s):  
A. Congras ◽  
H. Barasc ◽  
C. Delcros ◽  
F. Vignoles ◽  
A. Pinton ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cell Lines ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Genomic Stability ◽  
Comparative Genomic ◽  
Differentiation Potential ◽  
Ips Cell ◽  
The Impact

Chromosomal rearrangements have a crucial impact on the proper proceedings of meiosis and can lead by several mechanisms to the production of unbalanced gametes or to the complete arrest of gametes production. To assess the impact of these rearrangements in the early development of pig germ cells, we proposed to generate a library of stem cells from infertile boars that are carriers of chromosomal abnormalities as a new tool for the development of an in vitro differentiation system from pluripotent stem cells to germ cells. We report here the reprogramming of fibroblasts from an azoospermic boar carrying a reciprocal translocation t(Y:14) by integrative or nonintegrative viral overexpression of Oct4, Sox2, Klf4, and c-Myc. The iPS cell lines were characterised for pluripotency, cell cycle, and differentiation potential by conventional methods. Genomic stability was analysed by G-banding karyotype, comparative genomic hybridization, and FISH. The porcine iPS-like cell lines harbored characteristics of ground and naïve pluripotency when cultured in specific media. They expressed several pluripotency genes and harbored an ES-like cell cycle. Nevertheless, contrary to mouse and human iPS, they did not silence the integrated exogenes, leading to a poor differentiation potential. Moreover, cytogenetic analysis revealed a high genomic instability upon passaging, which suggests the development of a population with an increased selective advantage. We characterised the selected duplications and compared them to those previously described in other species. In contrast, the nonintegrative reprogrammation system gives us promising results regarding differentiation potential and genomic stability and will bring new insights into the molecular factors controlling and maintaining pluripotency in the pig species.

scholarly journals Nonhuman Primate Induced Pluripotent Stem Cells in Regenerative Medicine

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Yuehong Wu ◽  
Anuja Mishra ◽  
Zhifang Qiu ◽  
Steven Farnsworth ◽  
Suzette D. Tardif ◽  
...  
Keyword(s):  
Stem Cells ◽  
Regenerative Medicine ◽  
Induced Pluripotent Stem Cells ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Patient Specific ◽  
Ips Cell ◽  
Disease States ◽  
Autologous Cell Therapy ◽  
Induced Pluripotent

Among the various species from which induced pluripotent stem cells have been derived, nonhuman primates (NHPs) have a unique role as preclinical models. Their relatedness to humans and similar physiology, including central nervous system, make them ideal for translational studies. We review here the progress made in deriving and characterizing iPS cell lines from different NHP species. We focus on iPS cell lines from the marmoset, a small NHP in which several human disease states can be modeled. The marmoset can serve as a model for the implementation of patient-specific autologous cell therapy in regenerative medicine.

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