Expansion of human cord blood CD34+CD38−cells in ex vivo culture during retroviral transduction without a corresponding increase in SCID repopulating cell (SRC) frequency: dissociation of SRC phenotype and function

Blood ◽  
2000 ◽  
Vol 95 (1) ◽  
pp. 102-110 ◽  
Author(s):  
Craig Dorrell ◽  
Olga I. Gan ◽  
Daniel S. Pereira ◽  
Robert G. Hawley ◽  
John E. Dick
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cord Blood ◽  
Cell Function ◽  
Cultured Cells ◽  
Genetic Manipulation ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Retroviral Transduction ◽  
Large Expansion

Abstract Current procedures for the genetic manipulation of hematopoietic stem cells are relatively inefficient due, in part, to a poor understanding of the conditions for ex vivo maintenance or expansion of stem cells. We report improvements in the retroviral transduction of human stem cells based on the SCID-repopulating cell (SRC) assay and analysis of Lin− CD34+CD38−cells as a surrogate measure of stem cell function. Based on our earlier study of the conditions required for ex vivo expansion of Lin−CD34+ CD38− cells and SRC, CD34+–enriched lineage–depleted umbilical cord blood cells were cultured for 2 to 6 days on fibronectin fragment in MGIN (MSCV-EGFP-Neo) retroviral supernatant (containing 1.5% fetal bovine serum) and IL-6, SCF, Flt-3 ligand, and G-CSF. Both CD34+CD38− cells (20.8%) and CFC (26.3%) were efficiently marked. When the bone marrow of engrafted NOD/SCID mice was examined, 75% (12/16) contained multilineage (myeloid and B lymphoid) EGFP+ human cells composing as much as 59% of the graft. Half of these mice received a limiting dose of SRC, suggesting that the marked cells were derived from a single transduced SRC. Surprisingly, these culture conditions produced a large expansion (166-fold) of cells with the CD34+CD38− phenotype (n = 20). However, there was no increase in SRC numbers, indicating dissociation between the CD34+CD38− phenotype and SRC function. The underlying mechanism involved apparent downregulation of CD38 expression within a population of cultured CD34+CD38+ cells that no longer contained any SRC function. These results suggest that the relationship between stem cell function and cell surface phenotype may not be reliable for cultured cells. (Blood. 2000;95:102-110)

Expansion of human cord blood CD34+CD38−cells in ex vivo culture during retroviral transduction without a corresponding increase in SCID repopulating cell (SRC) frequency: dissociation of SRC phenotype and function

Blood ◽  
2000 ◽  
Vol 95 (1) ◽  
pp. 102-110 ◽  
Author(s):  
Craig Dorrell ◽  
Olga I. Gan ◽  
Daniel S. Pereira ◽  
Robert G. Hawley ◽  
John E. Dick
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cord Blood ◽  
Cell Function ◽  
Cultured Cells ◽  
Genetic Manipulation ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Retroviral Transduction ◽  
Large Expansion

Current procedures for the genetic manipulation of hematopoietic stem cells are relatively inefficient due, in part, to a poor understanding of the conditions for ex vivo maintenance or expansion of stem cells. We report improvements in the retroviral transduction of human stem cells based on the SCID-repopulating cell (SRC) assay and analysis of Lin− CD34+CD38−cells as a surrogate measure of stem cell function. Based on our earlier study of the conditions required for ex vivo expansion of Lin−CD34+ CD38− cells and SRC, CD34+–enriched lineage–depleted umbilical cord blood cells were cultured for 2 to 6 days on fibronectin fragment in MGIN (MSCV-EGFP-Neo) retroviral supernatant (containing 1.5% fetal bovine serum) and IL-6, SCF, Flt-3 ligand, and G-CSF. Both CD34+CD38− cells (20.8%) and CFC (26.3%) were efficiently marked. When the bone marrow of engrafted NOD/SCID mice was examined, 75% (12/16) contained multilineage (myeloid and B lymphoid) EGFP+ human cells composing as much as 59% of the graft. Half of these mice received a limiting dose of SRC, suggesting that the marked cells were derived from a single transduced SRC. Surprisingly, these culture conditions produced a large expansion (166-fold) of cells with the CD34+CD38− phenotype (n = 20). However, there was no increase in SRC numbers, indicating dissociation between the CD34+CD38− phenotype and SRC function. The underlying mechanism involved apparent downregulation of CD38 expression within a population of cultured CD34+CD38+ cells that no longer contained any SRC function. These results suggest that the relationship between stem cell function and cell surface phenotype may not be reliable for cultured cells. (Blood. 2000;95:102-110)

Highly Efficient Ex Vivo Expansion of Human Hematopoietic Stem Cells Using Delta1-Fc Chimeric Protein.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1332-1332
Author(s):  
Takahiro Suzuki ◽  
Yasuhisa Yokoyama ◽  
Keiki Kumano ◽  
Minoko Takanashi ◽  
Shiro Kozuma ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Chimeric Protein ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Clinical Transplantation

Abstract [Background and purposes] Ex vivo expansion of hematopoietic stem cells (HSCs) has been explored in the fields of stem cell biology, gene therapy and clinical transplantation. The use of Notch ligands or soluble IL-6 receptor combined with IL-6 has been a major technique that revealed several fold expansion of human cord blood SCID repopulating cells (SRCs). These studies, however, have been conducted in an independent manner, which hampered direct comparison and evaluation of the effect of combination of these methods. Our purpose of this study is to clarify these issues in a chemically defined serum-free medium that allows us to develop clinical usage of the culture condition. We also compared the efficiencies of SRC isolation with magnetic beads targeting CD34 and CD133. [Methods] Human cord blood CD133-sorted cells were cultured on immobilized Delta1 supplemented with stem cell factor, thrombopoietin, flt-3 ligand, IL-3 and IL-6/soluble IL-6 receptor chimeric protein (FP6) for three weeks, and cultured cells were transplanted into NOD/SCID mice after limiting dilution to calculate the number of SRCs. To confirm whether full multipotency and self-renewal capacity of SRCs were maintained during the culture, cells were transplanted serially into NOD/SCID/γcnull (NOG) mice, and hematopoietic reconstitution was examined. To compare the efficiencies of CD34- and CD133-sorting, we divided each sample into two aliquots and separated CD34+ and CD133+ cells, and calculated the SRC numbers recovered by both separation methods. [Results and discussion] The frequencies of SRCs in the culture-initiating CD133-sorted cells and cultured progeny were calculated as one out of 1,020 and one out of 175 (adjusted to culture-initiating cells), respectively, indicating 6-fold expansion of SRCs that was statistically significant. Delta1 significantly enhanced the expansion rate of SRCs, and addition of IL-3 to this condition further promoted the expansion. In the serial transplantation assays, we found human myeloid and lymphoid reconstitution both in the primary and secondary NOG recipients, verifying the SRC capacity in the cultured cells. Notably, the CD133-sorting was approximately 4.5 times more efficient in collecting SRCs than the CD34-sorting from the same number of mononuclear cells (MNCs) (308 and 254 SRCs by CD133-sorting vs. 67 and 59 SRCs by CD34-sorting from 108 MNCs). Our study provides a promising method to expand HSCs and encourages future trials on clinical transplantation.

InVitro Culture of Human Cord Blood Hematopoietic Stem/Progenitor Cells Interfered with Their Distribution to Other Bone Marrow Compartments after Intra-Bone Marrow Transplantation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4868-4868
Author(s):  
Kohshi Ohishi ◽  
Kentaro Yamamura ◽  
Masahiro Masuya ◽  
Naoyuki Katayama
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Transplantation ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Marrow Transplantation ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Left Tibia

Abstract Intra-bone marrow transplantation (IBMT) is a novel strategy for transplantation of hematopoietic stem cells because it can transfer various types of cells to bone marrow regardless of their homing capacity. However, reconstitution process of these cells after IBMT remains to be fully elucidated. Here, we investigated whether in vitro culture of cord blood hematopoietic stem/progenitor cells affects their reconstitution in bone marrow after IBMT. Freshly isolated AC133+ cells (5x104 cells/mouse) or all cells derived from AC133+ cells cultured with growth factors (stem cell factor, flt-3 ligand, and thrombopoietin) for 5 days were injected into the bone marrow of the left tibia in irradiated NOD/SCID mice. In the bone marrow of the injected left tibia, the engraftment levels of human CD45+ cells at 6 weeks after transplantation was not considerably different between transplantation of noncultured and cytokine-cultured cells (54±28% vs. 69±13%). However, the migration of transplanted cells to the bone marrow of other noninjected bones was extremely lower for cytokine-treated cells compared with noncultured cells (2±2% vs. 36±10%). Similar findings were observed for engraftment of CD34+ cells. To enhance the migration of cytokine-cultured cells after IBMT, we similarly transplanted cultured AC133+ cells into the bone marrow of the left tibia, assessed the engraftment in the injected and noninjected tibiae at 7 days after transplantation, and then subcutaneously administered G-CSF (250 μg/kg/d) for 5 days. Administration of G-CSF stimulated the migration of cytokine-cultured cells to the bone marrow of previously-aspirated right tibia but failed to induce their migration to intact bone marrow of femur. These data indicate that ex vivo manipulation of hematopoietic progenitor/stem cells adversely influences their migration properties to other bone marrow compartments after IBMT. Our data raise caution for future clinical applications of the IBMT method using ex vivo-manipulated hematopoietic stem cells.

Umbilical Cord Blood Transplantation: A New Alternative Option

Hematology ◽  
2005 ◽  
Vol 2005 (1) ◽  
pp. 377-383 ◽  
Author(s):  
William Tse ◽  
Mary J. Laughlin
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cord Blood ◽  
Hematopoietic Stem Cell ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Ex Vivo ◽  
Cord Blood Transplantation ◽  
Alternative Source ◽  
Hematopoietic Stem

Abstract Allogeneic hematopoietic stem cell transplantation is a life-saving procedure for hematopoietic malignancies, marrow failure syndromes, and hereditary immunodeficiency disorders. However, wide application of this procedure is limited by availability of suitably HLA-matched adult donors. Umbilical cord blood (UCB) has being increasingly used as an alternative hematopoietic stem cell source for these patients. To date, over 6000 UCB transplant procedures in children and adults have been performed worldwide using UCB donors. Broader use of UCB for adult patients is however limited by the available infused cell dose. This has prompted intensive research on ex vivo expansion of UCB stem cells and UCB graft-engineering including accessory cells able to improve UCB engraftment and reconstitution and for tissue regenerative potential. Recently, two large European and North American retrospective studies demonstrated that UCB is an acceptable alternative source of hematopoietic stem cells for adult recipients who lack HLA-matched adult donors. UCB is anticipated to address needs in both transplantation and regenerative medicine fields. It has advantages of easy procurement, no risk to donors, low risk of transmitting infections, immediate availability and immune tolerance allowing successful transplantation despite HLA disparity.

Ex vivo targeting of p21Cip1/Waf1 permits relative expansion of human hematopoietic stem cells

Blood ◽  
2003 ◽  
Vol 102 (4) ◽  
pp. 1260-1266 ◽  
Author(s):  
Sebastian Stier ◽  
Tao Cheng ◽  
Randolf Forkert ◽  
Christoph Lutz ◽  
David M. Dombkowski ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Cell Function ◽  
Kinase Inhibitor ◽  
Ex Vivo ◽  
Cell Cycle Checkpoints ◽  
Hematopoietic Stem ◽  
Cell Cycling

Abstract Relative quiescence is a defining characteristic of hematopoietic stem cells. Reasoning that inhibitory tone dominates control of stem cell cycling, we previously showed that mice engineered to be deficient in the cyclin-dependent kinase inhibitor, p21Cip1/Waf1 (p21), have an increased stem cell pool under homeostatic conditions. Since p21 was necessary to maintain stem cell quiescence and its absence sufficient to permit increased murine stem cell cycling, we tested whether reduction of p21 alone in human adult–derived stem cells could affect stem cell proliferation. We demonstrate here that interrupting p21 expression ex vivo resulted in expanded stem cell number and in vivo stem cell function compared with control, manipulated cells. Further, we demonstrate full multilineage reconstitution capability in cells where p21 expression was knocked down. Therefore, lifting the brake on cell proliferation by altering cell cycle checkpoints provides an alternative paradigm for increasing hematopoietic stem cell numbers. This approach may be useful for relative ex vivo human stem cell expansion.

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 UMBILICAL CORD BLOOD BANKING IN THE WORLDWIDE HEMATOPOIETIC STEM CELL TRANSPLANTATION SYSTEM: PERSPECTIVES FOR UKRAINE

2017 ◽  
Vol 39 (3) ◽  
pp. 164-170 ◽  
Author(s):  
T O Kalynychenko
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Hematopoietic Stem ◽  
Blood Banks ◽  
Cord Blood Banks

Significant progress in the promotion of procedural technologies associated with the transplantation of hematopoietic stem cells caused a rapid increase in activity. The exchange of hematopoietic stem cells for unrelated donor transplantations is now much easier due to the relevant international professional structures and organizations established to support cooperation and standard setting, as well as rules for the functioning of both national donor registries and cord blood banks. These processes are increasing every year and are contributing to the outpacing rates of development in this area. Products within their country should be regulated by the competent government authorities. This study analyzes the work of international and national levels of support for transplantation activity in the field of unrelated hematopoietic stem cell transplantation, the standardization order of technologies, as well as data that justify the need to create a network of donated umbilical cord blood banks in Ukraine as a factor in the development of allogeneic transplantation. This will promote the accessibility of international standards for the treatment of serious diseases for Ukrainian citizens.

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