scholarly journals Human adenosine deaminase expression in mice

Blood ◽  
1990 ◽  
Vol 75 (10) ◽  
pp. 2085-2092 ◽  
Author(s):  
KA Moore ◽  
FA Fletcher ◽  
DK Villalon ◽  
AE Utter ◽  
JW Belmont
Keyword(s):  
Stem Cells ◽  
Adenosine Deaminase ◽  
Bone Marrow Cells ◽  
High Titer ◽  
Experimental Period ◽  
Hematopoietic Stem ◽  
Provirus Integration

Abstract A replication defective retroviral vector containing a human adenosine deaminase (hADA) cDNA was produced by GP + E-86 packaging cells at high titer. We report long-term expression of hADA in the hematopoietic tissues of mice transplanted with bone marrow cells infected by in vitro co-cultivation with vector producing cells. Western analysis using an hADA-specific antibody allowed detection of the protein in the peripheral blood of all 37 transplanted mice for at least 9 weeks. Sixty-eight percent of the animals continued to express hADA in one or more of their hematopoietic tissues for the experimental period, and hADA was found in both spleen colonies and tissues of secondary recipients. There was provirus integration and expression in myeloid, erythroid, and lymphoid cell lineages, indicating extensive repopulation by the progeny of infected stem cells. The vector did not contain a selectable marker, and the infected stem cells did not have a competitive in vivo advantage. Nevertheless, we observed consistent gene transfer into hematopoietic stem cells and long-term expression of a human gene product in their progeny.

scholarly journals Human adenosine deaminase expression in mice

Blood ◽  
1990 ◽  
Vol 75 (10) ◽  
pp. 2085-2092
Author(s):  
KA Moore ◽  
FA Fletcher ◽  
DK Villalon ◽  
AE Utter ◽  
JW Belmont
Keyword(s):  
Stem Cells ◽  
Adenosine Deaminase ◽  
Bone Marrow Cells ◽  
High Titer ◽  
Experimental Period ◽  
Hematopoietic Stem ◽  
Provirus Integration

A replication defective retroviral vector containing a human adenosine deaminase (hADA) cDNA was produced by GP + E-86 packaging cells at high titer. We report long-term expression of hADA in the hematopoietic tissues of mice transplanted with bone marrow cells infected by in vitro co-cultivation with vector producing cells. Western analysis using an hADA-specific antibody allowed detection of the protein in the peripheral blood of all 37 transplanted mice for at least 9 weeks. Sixty-eight percent of the animals continued to express hADA in one or more of their hematopoietic tissues for the experimental period, and hADA was found in both spleen colonies and tissues of secondary recipients. There was provirus integration and expression in myeloid, erythroid, and lymphoid cell lineages, indicating extensive repopulation by the progeny of infected stem cells. The vector did not contain a selectable marker, and the infected stem cells did not have a competitive in vivo advantage. Nevertheless, we observed consistent gene transfer into hematopoietic stem cells and long-term expression of a human gene product in their progeny.

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

2020 ◽  
Vol 15 ◽  
Author(s):  
Fatima Aerts-Kaya
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Stress Conditions ◽  
Stress Factors ◽  
Hematopoietic Stem ◽  
Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

scholarly journals Hematopoietic stem/progenitor cells, generation of induced pluripotent stem cells, and isolation of endothelial progenitors from 21- to 23.5-year cryopreserved cord blood

Blood ◽  
2011 ◽  
Vol 117 (18) ◽  
pp. 4773-4777 ◽  
Author(s):  
Hal E. Broxmeyer ◽  
Man-Ryul Lee ◽  
Giao Hangoc ◽  
Scott Cooper ◽  
Nutan Prasain ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Proliferative Potential ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Induced Pluripotent

Abstract Cryopreservation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) is crucial for cord blood (CB) banking and transplantation. We evaluated recovery of functional HPC cryopreserved as mononuclear or unseparated cells for up to 23.5 years compared with prefreeze values of the same CB units. Highly efficient recovery (80%-100%) was apparent for granulocyte-macrophage and multipotential hematopoietic progenitors, although some collections had reproducible low recovery. Proliferative potential, response to multiple cytokines, and replating of HPC colonies was extensive. CD34+ cells isolated from CB cryopreserved for up to 21 years had long-term (≥ 6 month) engrafting capability in primary and secondary immunodeficient mice reflecting recovery of long-term repopulating, self-renewing HSCs. We recovered functionally responsive CD4+ and CD8+ T lymphocytes, generated induced pluripotent stem (iPS) cells with differentiation representing all 3 germ cell lineages in vitro and in vivo, and detected high proliferative endothelial colony forming cells, results of relevance to CB biology and banking.

scholarly journals Efficient retroviral gene transfer to purified long-term repopulating hematopoietic stem cells

Blood ◽  
1994 ◽  
Vol 84 (1) ◽  
pp. 74-83 ◽  
Author(s):  
SJ Szilvassy ◽  
S Cory
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Cell Biology ◽  
Bone Marrow Cells ◽  
High Titer ◽  
Marker Gene ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem

Abstract Efficient gene delivery to multipotential hematopoietic stem cells would greatly facilitate the development of effective gene therapy for certain hematopoietic disorders. We have recently described a rapid multiparameter sorting procedure for significantly enriching stem cells with competitive long-term lymphomyeloid repopulating ability (CRU) from 5-fluorouracil (5-FU)-treated mouse bone marrow. The sorted cells have now been tested as targets for retrovirus-mediated delivery of a marker gene, NeoR. They were cocultured for 4 days with fibroblasts producing a high titer of retrovirus in medium containing combinations of the hematopoietic growth factors interleukin-3 (IL-3), IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF) and then injected into lethally irradiated recipients, together with sufficient “compromised” bone marrow cells to provide short-term support. Over 80% of the transplanted mice displayed high levels (> or = 20%) of donor- derived leukocytes when analyzed 4 to 6 months later. Proviral DNA was detected in 87% of these animals and, in half of them, the majority of the hematopoietic cells were marked. Thus, infection of the stem cells was most effective. The tissue and cellular distribution of greater than 100 unique clones in 55 mice showed that most sorted stem cells had lymphoid as well as myeloid repopulating potential. Secondary transplantation provided strong evidence for infection of very primitive stem cells because, in several instances, different secondary recipients displayed in their marrow, spleen, thymus and day 14 spleen colony-forming cells the same proviral integration pattern as the primary recipient. Neither primary engraftment nor marking efficiency varied for stem cells cultured in IL-3 + IL-6, IL-3 + IL-6 + KL, IL-3 + IL-6 + LIF, or all four factors, but those cultured in IL-3 + IL-6 + LIF appeared to have lower secondary engraftment potential. Provirus expression was detected in 72% of the strongly marked mice, albeit often at low levels. Highly efficient retroviral marking of purified lymphomyeloid repopulating stem cells should enhance studies of stem cell biology and facilitate analysis of genes controlling hematopoietic differentiation and transformation.

Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1748-1755 ◽  
Author(s):  
David Bryder ◽  
Sten E. W. Jacobsen
Keyword(s):  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Calf Serum ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Divisions ◽  
Stem Cell Divisions

Abstract Although long-term repopulating hematopoietic stem cells (HSC) can self-renew and expand extensively in vivo, most efforts at expanding HSC in vitro have proved unsuccessful and have frequently resulted in compromised rather than improved HSC grafts. This has triggered the search for the optimal combination of cytokines for HSC expansion. Through such studies, c-kit ligand (KL), flt3 ligand (FL), thrombopoietin, and IL-11 have emerged as likely positive regulators of HSC self-renewal. In contrast, numerous studies have implicated a unique and potent negative regulatory role of IL-3, suggesting perhaps distinct regulation of HSC fate by different cytokines. However, the interpretations of these findings are complicated by the fact that different cytokines might target distinct subpopulations within the HSC compartment and by the lack of evidence for HSC undergoing self-renewal. Here, in the presence of KL+FL+megakaryocyte growth and development factor (MGDF), which recruits virtually all Lin−Sca-1+kit+ bone marrow cells into proliferation and promotes their self-renewal under serum-free conditions, IL-3 and IL-11 revealed an indistinguishable ability to further enhance proliferation. Surprisingly, and similar to IL-11, IL-3 supported KL+FL+MGDF-induced expansion of multilineage, long-term reconstituting activity in primary and secondary recipients. Furthermore, high-resolution cell division tracking demonstrated that all HSC underwent a minimum of 5 cell divisions, suggesting that long-term repopulating HSC are not compromised by IL-3 stimulation after multiple cell divisions. In striking contrast, the ex vivo expansion of murine HSC in fetal calf serum-containing medium resulted in extensive loss of reconstituting activity, an effect further facilitated by the presence of IL-3.

Reversible Expression of CD34 by Murine Hematopoietic Stem Cells

Blood ◽  
1999 ◽  
Vol 94 (8) ◽  
pp. 2548-2554 ◽  
Author(s):  
Takashi Sato ◽  
Joseph H. Laver ◽  
Makio Ogawa
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Cell Populations ◽  
Hematopoietic Stem ◽  
Adult Mice ◽  
Cd34 Expression ◽  
Marrow Cells

We used a mouse transplantation model to address the recent controversy about CD34 expression by hematopoietic stem cells. Cells from Ly-5.1 C57BL/6 mice were used as donor cells and Ly-5.2 mice were the recipients. The test cells were transplanted together with compromised marrow cells of Ly-5.2 mice. First, we confirmed that the majority of the stem cells with long-term engraftment capabilities of normal adult mice are CD34−. We then observed that, after the injection of 150 mg/kg 5-fluorouracil (5-FU), stem cells may be found in both CD34− and CD34+ cell populations. These results indicated that activated stem cells express CD34. We tested this hypothesis also by using in vitro expansion with interleukin-11 and steel factor of lineage−c-kit+ Sca-1+ CD34− bone marrow cells of normal mice. When the cells expanded for 1 week were separated into CD34− and CD34+ cell populations and tested for their engraftment capabilities, only CD34+ cells were capable of 2 to 5 months of engraftment. Finally, we tested reversion of CD34+ stem cells to CD34− state. We transplanted Ly-5.1 CD34+post–5-FU marrow cells into Ly-5.2 primary recipients and, after the marrow achieved steady state, tested the Ly-5.1 cells of the primary recipients for their engraftment capabilities in Ly-5.2 secondary recipients. The majority of the Ly-5.1 stem cells with long-term engraftment capability were in the CD34− cell fraction, indicating the reversion of CD34+ to CD34−stem cells. These observations clearly demonstrated that CD34 expression reflects the activation state of hematopoietic stem cells and that this is reversible.

scholarly journals Expression of human adenosine deaminase in mice after transplantation of genetically-modified bone marrow

Blood ◽  
1990 ◽  
Vol 75 (8) ◽  
pp. 1733-1741 ◽  
Author(s):  
M Kaleko ◽  
JV Garcia ◽  
WR Osborne ◽  
AD Miller
Keyword(s):  
Bone Marrow ◽  
Adenosine Deaminase ◽  
Dna Sequences ◽  
Bone Marrow Cells ◽  
High Titer ◽  
Helper Virus ◽  
Lymphoid Tissues ◽  
Hematopoietic Stem ◽  
Wide Range

Abstract A high titer retroviral vector was used to transfer a human adenosine deaminase (h-ADA) cDNA into murine bone marrow cells in vitro. The h- ADA cDNA was linked to the retroviral promoter, and the vector also contained a neomycin phosphotransferase gene as a selectable marker. Infected marrow was transplanted into syngeneic W/Wv recipients, and h- ADA expression was monitored for 5.5 months. Several weeks after transplantation, h-ADA was detected in the erythrocytes of all nine recipients, eight of which expressed levels equal to the endogenous enzyme. This level of expression persisted in two of six surviving mice, while expression in three others stabilized at lower, but readily detectable, levels. Only one mouse had no detectable h-ADA after 5.5 months. Vector DNA sequences with common integration sites were found in hematopoietic and lymphoid tissues of the mice at 5.5 months, providing evidence that hematopoietic stem cells had been infected. Furthermore, all mice transplanted with marrow that had been selected in G418 before infusion had multiple vector copies per genome. While this category included the two highest h-ADA expressors, it also included the negative mouse. Thus, multiple copies of the vector were not sufficient to guarantee long-term h-ADA expression. Mice were monitored for “helper virus” infections with an assay designed to detect a wide range of replication-competent retroviruses, including those endogenous to the mouse genome. No helper virus was detected in the two highest h-ADA expressors, ruling out helper-assisted vector spread as a cause of the high h-ADA expression. These results help provide a foundation for the development of somatic gene therapy techniques to be used in the treatment of human disease.

scholarly journals The chemokine GROβ mobilizes early hematopoietic stem cells characterized by enhanced homing and engraftment

Blood ◽  
2007 ◽  
Vol 110 (3) ◽  
pp. 860-869 ◽  
Author(s):  
Seiji Fukuda ◽  
Huimin Bian ◽  
Andrew G. King ◽  
Louis M. Pelus
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem ◽  
Platelet Recovery ◽  
And Function ◽  
Stimulating Factor ◽  
Cxcr4 Axis

Abstract Mobilized peripheral blood hematopoietic stem cells (PBSCs) demonstrate accelerated engraftment compared with bone marrow; however, mechanisms responsible for enhanced engraftment remain unknown. PBSCs mobilized by GROβ (GROβΔ4/CXCL2Δ4) or the combination of GROβΔ4 plus granulocyte colony-stimulating factor (G-CSF) restore neutrophil and platelet recovery faster than G-CSF–mobilized PBSCs. To determine mechanisms responsible for faster hematopoietic recovery, we characterized immunophenotype and function of the GROβ-mobilized grafts. PBSCs mobilized by GROβΔ4 alone or with G-CSF contained significantly more Sca-1+-c-kit+-lineage− (SKL) cells and more primitive CD34−-SKL cells compared with cells mobilized by G-CSF and demonstrated superior competitive long-term repopulation activity, which continued to increase in secondary and tertiary recipients. GROβΔ4-mobilized SKL cells adhered better to VCAM-1+ endothelial cells compared with G-CSF–mobilized cells. GROβΔ4-mobilized PBSCs did not migrate well to the chemokine stromal derived factor (SDF)-1α in vitro that was associated with higher CD26 expression. However, GROβΔ4-mobilized SKL and c-Kit+ lineage− (KL) cells homed more efficiently to marrow in vivo, which was not affected by selective CXCR4 and CD26 antagonists. These data suggest that GROβΔ4-mobilized PBSCs are superior in reconstituting long-term hematopoiesis, which results from differential mobilization of early stem cells with enhanced homing and long-term repopulating capacity. In addition, homing and engraftment of GROβΔ4-mobilized cells is less dependent on the SDF-1α/CXCR4 axis.

Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1748-1755 ◽  
Author(s):  
David Bryder ◽  
Sten E. W. Jacobsen
Keyword(s):  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Calf Serum ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Divisions ◽  
Stem Cell Divisions

Although long-term repopulating hematopoietic stem cells (HSC) can self-renew and expand extensively in vivo, most efforts at expanding HSC in vitro have proved unsuccessful and have frequently resulted in compromised rather than improved HSC grafts. This has triggered the search for the optimal combination of cytokines for HSC expansion. Through such studies, c-kit ligand (KL), flt3 ligand (FL), thrombopoietin, and IL-11 have emerged as likely positive regulators of HSC self-renewal. In contrast, numerous studies have implicated a unique and potent negative regulatory role of IL-3, suggesting perhaps distinct regulation of HSC fate by different cytokines. However, the interpretations of these findings are complicated by the fact that different cytokines might target distinct subpopulations within the HSC compartment and by the lack of evidence for HSC undergoing self-renewal. Here, in the presence of KL+FL+megakaryocyte growth and development factor (MGDF), which recruits virtually all Lin−Sca-1+kit+ bone marrow cells into proliferation and promotes their self-renewal under serum-free conditions, IL-3 and IL-11 revealed an indistinguishable ability to further enhance proliferation. Surprisingly, and similar to IL-11, IL-3 supported KL+FL+MGDF-induced expansion of multilineage, long-term reconstituting activity in primary and secondary recipients. Furthermore, high-resolution cell division tracking demonstrated that all HSC underwent a minimum of 5 cell divisions, suggesting that long-term repopulating HSC are not compromised by IL-3 stimulation after multiple cell divisions. In striking contrast, the ex vivo expansion of murine HSC in fetal calf serum-containing medium resulted in extensive loss of reconstituting activity, an effect further facilitated by the presence of IL-3.

Hematopoietic stem cells express Tie-2 receptor in the murine fetal liver

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3757-3762 ◽  
Author(s):  
Hsiang-Chun Hsu ◽  
Hideo Ema ◽  
Mitsujiro Osawa ◽  
Yukio Nakamura ◽  
Toshio Suda ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Receptor Tyrosine Kinase ◽  
Fetal Liver ◽  
Newborn Mice ◽  
Hematopoietic Stem ◽  
Fetal Liver Cells

Tie-2 receptor tyrosine kinase expressed in endothelial and hematopoietic cells is believed to play a role in both angiogenesis and hematopoiesis during development of the mouse embryo. This article addressed whether Tie-2 is expressed on fetal liver hematopoietic stem cells (HSCs) at day 14 of gestation. With the use of anti–Tie-2 monoclonal antibody, its expression was detected in approximately 7% of an HSC population of Kit-positive, Sca-1–positive, lineage-negative or -low, and AA4.1-positive (KSLA) cells. These Tie-2–positive KSLA (T+ KSLA) cells represent 0.01% to 0.02% of fetal liver cells. In vitro colony and in vivo competitive repopulation assays were performed for T+ KSLA cells and Tie-2–negative KSLA (T− KSLA) cells. In the presence of stem cell factor, interleukin-3, and erythropoietin, 80% of T+ KSLA cells formed colonies in vitro, compared with 40% of T− KSLA cells. Long-term multilineage repopulating cells were detected in T+ KSLA cells, but not in T− KSLA cells. An in vivo limiting dilution analysis revealed that at least 1 of 8 T+ KSLA cells were such repopulating cells. The successful secondary transplantation initiated with a limited number of T+ KSLA cells suggests that these cells have self-renewal potential. In addition, engraftment of T+ KSLA cells in conditioned newborn mice indicates that these HSCs can be adapted equally by the adult and newborn hematopoietic environments. The data suggest that T+ KSLA cells represent HSCs in the murine fetal liver.

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