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 Human amniotic mesenchymal stromal cells support the ex vivo expansion of cord blood hematopoietic stem cells

2021 ◽  
Author(s):  
Valentina Orticelli ◽  
Andrea Papait ◽  
Elsa Vertua ◽  
Patrizia Bonassi Signoroni ◽  
Pietro Romele ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem

scholarly journals Merocyanine 540-sensitized photoinactivation of human erythrocytes parasitized by Plasmodium falciparum

Blood ◽  
1992 ◽  
Vol 80 (1) ◽  
pp. 21-24 ◽  
Author(s):  
OM Smith ◽  
SA Dolan ◽  
JA Dvorak ◽  
TE Wellems ◽  
F Sieber
Keyword(s):  
Stem Cells ◽  
Plasmodium Falciparum ◽  
Hematopoietic Stem Cells ◽  
Red Blood Cells ◽  
Human Blood ◽  
Blood Cells ◽  
Potential Usefulness ◽  
Hematopoietic Stem ◽  
Infected Blood ◽  
Merocyanine 540

The purpose of this study was to evaluate the photosensitizing dye merocyanine 540 (MC540) as a means for extracorporeal purging of Plasmodium falciparum-infected erythrocytes from human blood. Parasitized red blood cells bound more dye than nonparasitized cells, and exposure to MC540 and light under conditions that are relatively well tolerated by normal erythrocytes and normal pluripotent hematopoietic stem cells reduced the concentration of parasitized cells by as much as 1,000-fold. Cells parasitized by the chloroquine- sensitive HB3 clone and the chloroquine-resistant Dd2 clone of P falciparum were equally susceptible to MC540-sensitized photolysis. These data suggest the potential usefulness of MC540 in the purging of P falciparum-infected blood.

Induction of Adipocyte Differentiation and Reduction of Hematopoesis-Supporting Activity of Mouse Stromal Cells by Suppression of Transcription Factor GATA-2.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1392-1392
Author(s):  
Yoko Okitsu ◽  
Hideo Harigae ◽  
Masanori Seki ◽  
Toru Fujiwara ◽  
Shinichiro Takahashi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Hematopoietic Stem Cells ◽  
Stromal Cells ◽  
Colony Formation ◽  
Adipocyte Differentiation ◽  
Fetal Liver ◽  
Expression Level ◽  
Hematopoietic Stem

Abstract (Introduction) Aplastic anemia (AA) is characterized by peripheral pancytopenia and fatty bone marrow. An immunological attack to hematopoietic stem cells has been thought to be responsible for the development of the disease. Previously, we reported the expression of transcription factor GATA-2 is significantly decreased in CD34 positive cells in AA. Together with the phenotypes of hematopoietic stem cells in GATA-2 hetero-knockout mice, GATA-2 down-regulation may play a role in the reduction of a stem cell pool observed in AA. On the other hand, GATA-2 has been shown to be essential for the maintenance of immaturity of preadipocytes. If a pathological immune response in AA decreases the level of GATA-2 expression in not only hematopoietic stem cells but also stromal preadipocytes, it may accelerate the maturation of preadipocytes, leading to the formation of fatty bone marrow. To explore this possibility, the phenotypic change of stromal preadipocytes by suppression of GATA-2 was examined in this study. (Method) The GATA-2 expression level was suppressed by using siRNA for GATA-2 in mouse stromal preadipocyte cell lines, TBR9 and TBR343. After the treatment with siRNA, the adipocyte differentiation was induced by the incubation with insulin and dexamethasone for 7days. Then, the maturation level was examined by oil drops formation judged by oil red staining, and by the expression level of adipcin and PPAR-γ mRNA. Supporting activity of hematopoietic colony formation was also evaluated by using mouse fetal liver cells after siRNA treatment. (Results) By using designed siRNA, the GATA-2 expression was suppressed to 30% of control, whereas the expression level of GATA-3, which is co-expressed in preadipocytes, was unchanged. When GATA-2 was suppressed by siRNA, the oil drop formation and adipocyte-specific gene expression was significantly accelerated in both of stromal cells. Furthermore, the number of fetal liver hematopoetic colonies was significantly decreased by suppression of GATA-2, suggesting that GATA-2 down-regulation in stromal preadipocytes results in not only the acceleration of the maturation but also the reduced supporting activity of hematopoietic colony formation (Conclusion) These results suggest that suppression of GATA-2 in hematopoietic tissues induces the characteristic features of AA, i.e., decreased the number of hematopoietic stem cells and increased number of mature adipocytes.

Mesenchymal Stem Cells from the Wharton’s Jelly of Umbilical Cord Segments Support the Maintenance of Long Term Culture-Initiating Cells from Cord Blood.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3650-3650
Author(s):  
Kent W. Christopherson ◽  
Tiki Bakhshi ◽  
Shamanique Bodie ◽  
Shannon Kidd ◽  
Ryan Zabriskie ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Blood Cells ◽  
Hematopoietic Stem ◽  
Cord Blood Cells

Abstract Hematopoietic Stem Cells (HSC) are routinely obtained from bone marrow, mobilized peripheral blood, and umbilical Cord Blood. Traditionally, adult bone marrow has been utilized as a source of Mesenchymal Stem Cells (MSC). Bone marrow derived MSC (BM-MSC) have previously been shown to maintain the growth of HSC obtained from cord blood and have been utilized for cord blood expansion purposes. However, the use of a mismatched BM-MSC feeder stromal layer to support the long term culture of cord blood HSC is not ideal for transplant purposes. The isolation of MSC from a novel source, the Wharton’s Jelly of Umbilical Cord segments, was recently reported (Romanov Y, et al. Stem Cells.2003; 21: 105–110) (Lee O, et al. Blood.2004; 103: 1669–1675). We therefore hypothesized that Umbilical Cord derived MSC (UC-MSC) have the ability to support the long term growth of cord blood derived HSC similar to that previously reported for BM-MSC. To test this hypothesis, MSC were isolated from the Wharton’s Jelly of Umbilical Cord segments and defined morphologically and by cell surface markers. UC-MSC were then tested for their ability to support the growth of pooled CD34+ cord blood cells in long term culture - initiating cell (LTC-IC) assays as compared to BM-MSC. We observed that like BM-MSC, CB-MSC express a defined set of cell surface markers. By flow cytometry we determined that that both UC-MSC and BM-MSC are positive for CD29, CD44, CD73, CD90, CD105, CD166, HLA-A and negative for CD45, CD34, CD38, CD117, HLA-DR expression. Utilizing Mitomycin C treated (200 μM, 15 min.) UC-MSC from multiple donors as a feeder layer we observed that UC-MSC have the ability to support the maintenance of long term hematopoiesis during the LTC-IC assay. Specifically, UC-MSC isolated from separate umbilical cord donors support the growth of 69.6±11.9 (1A), 31.7±3.9 (2B), 67.0±13.5 (3A), and 38.5±13.7 (3B) colony forming cells (CFC) per 1×104 CD34+ cord blood cells as compared to 64.0±4.2 CFC per 1×104 CD34+ cord blood cells supported by BM-MSC (Mean±SEM, N=4 separate segments from three different donors). Thus, Umbilical Cord derived Mesenchymal Stem Cells, a recently described novel source of MSC, have the ability to support long term maintenance of Hematopoietic Stem Cells, as defined by the LTC-IC assay. These results may have potential therapeutic application with respect to ex vivo stem cell expansion of Cord Blood Hematopoietic Stem Cells utilizing a Mesenchymal Stem Cell stromal layer. In addition, these data suggest the possibility of co-transplantation of matched Mesenchymal and Hematopoietic Stem Cells from the same umbilical cord and cord blood donor respectively. Lastly, these results describe a novel model system for the future study of the interaction between Cord Blood Hematopoietic Stem Cells and the appropriate supportive microenvironment represented by the Umbilical Cord - Mesenchymal Stem Cells.

Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells

2005 ◽  
Vol 23 (1) ◽  
pp. 69-74 ◽  
Author(s):  
Marie-Catherine Giarratana ◽  
Ladan Kobari ◽  
Hélène Lapillonne ◽  
David Chalmers ◽  
Laurent Kiger ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Human Red Blood Cells

Podocalyxin is a CD34-related marker of murine hematopoietic stem cells and embryonic erythroid cells

Blood ◽  
2005 ◽  
Vol 105 (11) ◽  
pp. 4170-4178 ◽  
Author(s):  
Regis Doyonnas ◽  
Julie S. Nielsen ◽  
Shierley Chelliah ◽  
Erin Drew ◽  
Takahiko Hara ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Erythroid Cells ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Nucleated Red Blood Cells ◽  
Erythroid Precursors ◽  
Valuable Marker

Abstract Podocalyxin/podocalyxin-like protein 1 [PCLP1]/thrombomucin/MEP21 is a CD34-related sialomucin. We have performed a detailed analysis of its expression during murine development and assessed its utility as a marker of hematopoietic stem cells (HSCs) and their more differentiated progeny. We find that podocalyxin is highly expressed by the first primitive hematopoietic progenitors and nucleated red blood cells to form in the embryonic yolk sac. Likewise, podocalyxin is expressed by definitive multilineage hematopoietic progenitors and erythroid precursors in fetal liver. The level of podocalyxin expression gradually declines with further embryo maturation and reaches near-background levels at birth. This is followed by a postnatal burst of expression that correlates with the seeding of new hematopoietic progenitors to the spleen and bone marrow. Shortly thereafter, podocalyxin expression gradually declines, and by 4 weeks postpartum it is restricted to a rare population of Sca-1+, c-kit+, lineage marker- (Lin-) cells in the bone marrow. These rare podocalyxin-expressing cells are capable of serially reconstituting myeloid and lymphoid lineages in lethally irradiated recipients, suggesting they have HSC activity. In summary, we find that podocalyxin is a marker of embryonic HSCs and erythroid cells and of adult HSCs and that it may be a valuable marker for the purification of these cells for transplantation.

The Endothelial Antigen ESAM Marks Hematopoietic Stem Cells throughout Life

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 727-727 ◽  
Author(s):  
Takafumi Yokota ◽  
Kenji Oritani ◽  
Stefan Butz ◽  
Koichi Kokame ◽  
Paul W Kincade ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Endothelial Cell ◽  
Hematopoietic Stem Cells ◽  
Blood Cells ◽  
Fetal Liver ◽  
Side Population ◽  
Expression Profiles ◽  
Hematopoietic Stem

Abstract Hematopoietic stem cells (HSC) are an important cell type with the capacity for self-renewal as well as differentiation into multi-lineage blood cells, maintaining the immune system throughout life. Many studies have attempted to identify unique markers associated with these extremely rare cells. In bone marrow of adult mice, the Lin-c-kitHi Sca1+ CD34−/Lo Thy1.1Lo subset is known to include HSC with long-term repopulating capacity. However, several of these parameters differ between strains of mice, change dramatically during developmental age and/or are expressed on many non-HSC during inflammation. Efficient HSC-based therapies and the emerging field of regenerative medicine will benefit from learning more about what defines stem cells. We previously determined that the most primitive cells with lymphopoietic potential first develop in the paraaortic splanchnopleura/aorta-gonad-mesonephros (AGM) region of embryos using Rag1/GFP knock-in mice. We also reported that Rag1/GFP-c-kitHi Sca1+ cells derived from E14.5 fetal liver (FL) reconstituted lympho-hematopoiesis in lethally irradiated adults, while Rag1/GFPLo c-kitHi Sca1+ cells transiently contributed to T and B lymphopoiesis. To extend those findings, microarray analyses were conducted to search for genes that characterize the initial transition of fetal HSC to primitive lymphopoietic cells. The comparisons involved mRNA from Rag1Lo ckitHi Sca1+, early lymphoid progenitors (ELP) and the HSC-enriched Rag1-ckitHi Sca1+ fraction isolated from E14.5 FL. While genes potentially related to early lymphopoiesis were discovered, our screen also identified genes whose expression seemed to correlate with HSC. Among those, endothelial cell-selective adhesion molecule (ESAM) attracted attention because of its conspicuous expression in the HSC fraction and sharp down-regulation on differentiation to ELP. ESAM was originally identified as an endothelial cell-specific protein, but expression on megakaryocytes and platelets was also reported (J. Biol. Chem., 2001, 2002). Flow cytometry analyses with anti-ESAM antibodies showed that the HSC-enriched Rag1-c-kitHi Sca1+ fraction could be subdivided into two on the basis of ESAM levels. The subpopulation with the high density of ESAM was enriched for c-kitHi Sca1Hi cells, while ones with negative or low levels of ESAM were found in the c-kitHi Sca1Lo subset. Among endothelial-related antigens on HSC, CD34 and CD31/PECAM1 were uniformly present on Rag1-c-kitHi Sca1+ cells in E14.5 FL and neither resolved into ESAMHi and ESAM−/Lo fractions. Expression profiles of Endoglin and Tie2 partially correlate with ESAM. The primitive ESAMHi fraction uniformly expressed high levels of Endoglin and Tie2, but many of the more differentiated ESAM−/Lo cells still retained the two markers. ESAM expression correlated well with HSC activity. Cells in the ESAMHi Rag1-ckitHi Sca1+ fraction formed more and larger colonies than those in the ESAM-/Lo Rag1-ckitHi Sca1+ fraction. Particularly, most CFU-Mix, primitive progenitors with both myeloid and erythroid potential, were found in the ESAMHi fraction. In limiting dilution stromal cell co-cultures, we found that 1 in 2.1 ESAMHi Rag1-ckitHi Sca1+ cells and 1 in 3.5 ESAM−/Lo Rag1-ckitHi Sca1+ cells gave rise to blood cells. However, while only 1 in 125 ESAM−/Lo Rag1-ckitHi Sca1+ cells were lymphopoietic under these conditions, 1 in 8 ESAMHi Rag1-ckitHi Sca1+ cells produced CD19+ B lineage cells. In long-term reconstituting assays, ESAMHi Rag1-ckitHi Sca1+ cells contributed highly to the multi-lineage recovery of lympho-hematopoiesis in recipients, but no chimerism was detected in mice transplanted with ESAM−/Lo Rag1-ckitHi Sca1+ cells. These results suggested that HSC in E14.5 FL are exclusively present in the ESAMHi fraction. Tie2+ c-kit+ lympho-hematopoietic cells of E10.5 AGM also expressed high levels of ESAM. Furthermore, ESAM expression in adult bone marrow was detected on primitive progenitors and cells in the side population within the Lin-ckitHi Sca1+ fraction. Interestingly, the expression was up-regulated in aged mice. Based on these observations, we conclude that ESAM marks HSC throughout life in mice. We also observed that many of human cord blood CD34+ CD38− cells express ESAM, suggesting potential application for the purification of human HSC.

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