scholarly journals Generation of Red Blood Cells from Human Embryonic Stem Cells (ES) with Increased Efficacy

2012 ◽  
Vol 18 (2) ◽  
pp. S236
Author(s):  
A.S. Srivastava ◽  
B. Ismaeli-Azad ◽  
R. DenecocheaCampio ◽  
E. Carrier
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Red Blood Cells ◽  
Human Embryonic Stem Cells ◽  
Blood Cells ◽  
Embryonic Stem

Large-Scale Liquid Culture Production of Erythroid Cells from Human Embryonic Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3631-3631
Author(s):  
Emmanuel N. Olivier ◽  
Caihong Qiu ◽  
Eric E. Bouhassira
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Red Blood Cells ◽  
Human Embryonic Stem Cells ◽  
Blood Cells ◽  
Liquid Culture ◽  
Embryonic Stem ◽  
Erythroid Cells ◽  
Early Human

Abstract Early human erythropoiesis is difficult to study because the material is hard to access experimentally. Hence, relatively little is known about the gene expression profiles or the mechanism of globin gene expression in these early cells. We report here a system to produce large quantities in liquid culture of virtually pure erythroid cells starting from H1 human embryonic stem cells (hESCs). The system is adapted from methods to produce enucleated red blood cells from cord blood and consist of five steps. During the first step, hESCs are differentiated by co-culture on immortalized human fetal hepatocytes (FH-B-hTERT) for two weeks to produce hematopoietic cells. CD34 positive cells are then magnetically sorted and placed in step 2 for seven days in serum free medium in the presence of SCF, Epo, hydro-cortisone, flt-3 ligand, BMP-4 and IL3. In step 3, the cells are incubated for seven days in the same medium and cytokine cocktail but with IGF-1 and without flt-3-ligand. In step 4, the cells are incubated with Epo for 3 days, and in step 5 the cells are incubated without cytokine on a feeder layer of MS-5 cells. In a typical experiment, 2 millions hESCs (two 10cm2 wells) yield 50,000 sorted CD34 positive cells. Culture of these cells for about three weeks yields about 5 millions erythroid cells. This corresponds to a 5 to 10,000-fold amplification of the sorted hematopoietic cells since we estimate that only a few percent of the cells recovered with the CD34 magnetic beads are hematopoietic. Flow cytometry analysis revealed that at the beginning of the second step the CD34+ cells are CD45−, CD71low and CD235a−. After 7 days in liquid culture CD34 expression is less than 10%, CD45 and CD71 expressions are more than 95% and CD235a is less than 20%. Eight days later the cells are 95% CD34− CD45− CD71high and CD235a+. Finally at the end of the culture the cells become CD34−, CD45−, CD71− and CD235a+. Morphological analysis by Wright-Giemsa staining revealed that the differentiation process in the liquid culture is relatively synchronous and that at the end of the culture the majority of the cells are orthochromatic erythroblasts. In contrast to cord blood derived cells placed in similar differentiation conditions, very few enucleated red blood cells could be obtained from hESCs. Hemoglobin can first be detected spectrophotometrically after day 10 of liquid culture and reach a concentration of 20 pmol/106 cells at the end of the culture. Globin chain analysis by PCR and HPLC reveals that ξ, α, ε, and γ globin chains are synthesized by these cells but not β-globin could be detected. A detailed analysis of globin expression in early human erythroid cells will be presented in an accompanying abstract. This experimental system will be useful to study early erythropoiesis, to test gene therapy vectors, and to create genetically modified red blood cells.

Generation of Red Blood Cells From Human Embryonic Stem Cells (ES) with Increased Efficacy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4334-4334
Author(s):  
Anand S Srivastava ◽  
Babak Esmaeli Azad ◽  
Rosalia De Necochea Campion ◽  
Ewa Carrier
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Red Blood Cells ◽  
Human Embryonic Stem Cells ◽  
Blood Cells ◽  
Culture System ◽  
Embryonic Stem ◽  
Employment Equity ◽  
Equity Ownership

Abstract Abstract 4334 It is estimated that for every unit of donated blood, two units are required in North America. The current rate of blood donation is stagnant while the need increases by 6–8% annually. In order to overcome this difficulty, we have developed an improved method to generate red blood cells from human embryonic stem cells (H9) with increased efficacy. In addition to xeno-free conditions and standard cytokine cocktail used for hematopoietic differentiation of human embryonic stem cells (Carrier et al. J Transl Med. 2009; Vol 7: 27), we have introduced a new method of improved growth and differentiation of human ES cells with hypoxia-induced mesenchymal stem cells, obtained from allogeneic adult bone marrow donors. This technique increased efficacy of red blood cell production by 5–25 fold. We have developed a bioscaffold–> microsphere-based culture system with highly porous surface allowing culturing of a very large number of embryonic stem cells per one culture condition. This culture system avoids shear forces and damage to the cells, and facilitates removal and recycling of the microspheres. The in vitro obtained human ES-derived red blood cells are enucleated and do not produce tumors (efficacy of enucleation is 65–95%). The laser-based system is utilized to eliminate nucleated cells from the culture. The problem with hES-derived red blood cells is that they are produced in small numbers and process is very costly. We are developing a 3-phase bioreactor with computerized programming, which will increase every step of the differentiation process and allow recycling of feeder cells and cytokines. In this system we will utilize iron-loaded microspheres coated with hypoxia-processed mesenchymal stem cells as a main culture unit. The in vitro generated human ES-derived red blood cells upscaled in a bioreactor will be used for the off-shelf production of red blood cells for clinical use. Disclosures: Srivastava: Giostar: Employment, Equity Ownership. Azad:Dnamicroarray, Inc.: Employment, Equity Ownership. Carrier:Giostar: Consultancy; Samaritan Pharmaceuticals: Consultancy; Entest Biomedical: Consultancy, Equity Ownership; America Stem Cells: Consultancy, Equity Ownership; Millenium: Speakers Bureau; NovaRx: Employment, Equity Ownership.

scholarly journals Biologic properties and enucleation of red blood cells from human embryonic stem cells

Blood ◽  
2008 ◽  
Vol 112 (12) ◽  
pp. 4475-4484 ◽  
Author(s):  
Shi-Jiang Lu ◽  
Qiang Feng ◽  
Jennifer S. Park ◽  
Loyda Vida ◽  
Bao-Shiang Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Red Blood Cells ◽  
Human Embryonic Stem Cells ◽  
Blood Cells ◽  
Large Scale ◽  
Embryonic Stem ◽  
Globin Chain ◽  
Nuclear Condensation

Abstract Human erythropoiesis is a complex multistep process that involves the differentiation of early erythroid progenitors to mature erythrocytes. Here we show that it is feasible to differentiate and mature human embryonic stem cells (hESCs) into functional oxygen-carrying erythrocytes on a large scale (1010-1011 cells/6-well plate hESCs). We also show for the first time that the oxygen equilibrium curves of the hESC-derived cells are comparable with normal red blood cells and respond to changes in pH and 2,3-diphosphoglyerate. Although these cells mainly expressed fetal and embryonic globins, they also possessed the capacity to express the adult β-globin chain on further maturation in vitro. Polymerase chain reaction and globin chain specific immunofluorescent analysis showed that the cells increased expression of β-globin (from 0% to > 16%) after in vitro culture. Importantly, the cells underwent multiple maturation events, including a progressive decrease in size, increase in glycophorin A expression, and chromatin and nuclear condensation. This process resulted in extrusion of the pycnotic nuclei in up to more than 60% of the cells generating red blood cells with a diameter of approximately 6 to 8 μm. The results show that it is feasible to differentiate and mature hESCs into functional oxygen-carrying erythrocytes on a large scale.

Large-scale production of embryonic red blood cells from human embryonic stem cells

2006 ◽  
Vol 34 (12) ◽  
pp. 1635-1642 ◽  
Author(s):  
Emmanuel N. Olivier ◽  
Caihong Qiu ◽  
Michelle Velho ◽  
Rhoda Elison Hirsch ◽  
Eric E. Bouhassira
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Red Blood Cells ◽  
Human Embryonic Stem Cells ◽  
Blood Cells ◽  
Large Scale ◽  
Embryonic Stem ◽  
Scale Production ◽  
Large Scale Production

A Novel Method for Efficient Production of Multipotential Hematopoietic Progenitors from Human Embryonic Stem Cells by Co-Culture with Murine Fetal Liver-Derived Stromal Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4214-4214
Author(s):  
Feng Ma ◽  
Dan Wang ◽  
Sachiyo Hanada ◽  
Hirohide Kawasaki ◽  
Yuji Zaike ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Stromal Cells ◽  
Blood Cells ◽  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Efficient Production ◽  
Hematopoietic Progenitors

Abstract Human embryonic stem cells provide a unique tool to study early events occurring in the development of human embryonic hematopoiesis, and their totipotent capability indicates a potent clinical application based on the cellular therapy and the evaluation of drug effects on hematopoietic and blood cells. To achieve efficient production of hematopoietic cells from human embryonic stem cells, we attempted to reproduce the circumstance surrounding embryonic hematopoietic cells in vitro. Since fetal liver is the predominant source of hematopoietic and blood cells in mammalian embryogenesis, we established stromal cells from mouse fetal liver at days 14 to 15 of gestation. In the co-culture of human embryonic stem cells with the established stromal cells, a number of hematopoietic progenitors were generated at around day 14 of co-culture, and this hematopoietic activity was highly enriched in the cobble stone-like cells under the stromal layer. Most of the cobble stone-like cells collected expressed CD34 and contained a variety of hematopoietic colony-forming cells, especially multilineage colony-forming cells, at a high frequency. The multipotential hematopoietic progenitors in the cobble stone-like cells produced all types of mature blood cells, including adult type hemoglobin-synthesizing erythrocytes and tryptase and chymase-bouble positive mast cells in the suspension cultiue with a cytokine cocktail. The developed co-culture system of human embryonic stem cells should offer a novel source for hematopoietic and blood cells applicable to cellular therapies and drug screening.

scholarly journals Colloidal Self-Assembled Patterns Maintain the Pluripotency and Promote the Hemopoietic Potential of Human Embryonic Stem Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Jiao Lin ◽  
Jiahui Zeng ◽  
Wencui Sun ◽  
Kun Liu ◽  
Myagmartsend Enkhbat ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Blood Cells ◽  
Early Stage ◽  
Embryonic Stem ◽  
Mapk Signaling ◽  
Sequencing Analysis ◽  
Erythroid Progenitors ◽  
Self Assembled

The generation of blood cells in a significant amount for clinical uses is still challenging. Human pluripotent stem cells-derived hemopoietic cells (hPSC-HCs) are a promising cell source to generate blood cells. Previously, it has been shown that the attached substrates are crucial in the maintenance or differentiation of hPSCs. In this study, a new family of artificial extracellular matrix (ECM) called colloidal self-assembled patterns (cSAPs: #1–#5) was used for the expansion of mouse and human PSCs. The optimized cSAP (i.e., #4 and #5) was selected for subsequent hemopoietic differentiation of human embryonic stem cells (hESCs). Results showed that the hematopoietic potential of hESCs was enhanced approx 3–4 folds on cSAP #5 compared to the flat control. The cell population of hematopoietic progenitors (i.e., CD34+CD43+ cells) and erythroid progenitors (i.e., CD71+GPA+ cells) were enhanced 4 folds at day 8 and 3 folds at day 14. RNA sequencing analysis of cSAP-derived hESCs showed that there were 300 genes up-regulated and 627 genes down-regulated compared to the flat control. The enriched signaling pathways, including up-regulation (i.e., Toll-like receptor, HIF-1a, and Notch) or down-regulation (i.e., FAs, MAPK, JAK/STAT, and TGF-β) were classic in the maintenance of hESC phenotype Real time PCR confirmed that the expression of focal adhesion (PTK2, VCL, and CXCL14) and MAPK signaling (CAV1) related genes was down-regulated 2-3 folds compared to the flat control. Altogether, cSAP enhances the pluripotency and the hematopoietic potential of hESCs that subsequently generates more blood-like cells. This study reveals the potential of cSAPs on the expansion and early-stage blood cell lineage differentiation of hPSCs.

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