Long-Term Engraftment and Self-Renewal of AML Stem Cells in the Newborn NOD-scid/IL2rgnull Immunodeficient Mouse Model.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1261-1261 ◽  
Author(s):  
Shuro Yoshida ◽  
Fumihiko Ishikawa ◽  
Masaki Yasukawa ◽  
Toshihiro Miyamoto ◽  
Goichi Yoshimoto ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Bone Marrow Cells ◽  
Scid Mice ◽  
Leukemic Cells ◽  
Self Renewal ◽  
Human T Cells

Abstract Transplantation of human leukemic cells into severe combined immunodeficiency (SCID) mice has been used to analyze developmental mechanisms of human leukemogenesis. Previous models, however, were limited in efficient or long-term engraftment of leukemia initiating cells. Here we report a new SCID model that supports highly efficient long-term engraftment of primary human acute myelogenous leukemia (AML) cells. We have established a novel immune-compromised mouse by backcrossing a complete null mutation of the common cytokine receptor g chain onto NOD-scid mice (NOD/SCID/IL2rgnull mice), and reported that normal human cord blood-derived hematopoietic stem cells efficiently engrafted in newborn NOD/SCID/IL2rgnull mice as compared to NOD/SCID/b2mnull mice (Ishikawa et al, Blood in press). Injection of 5x106 total bone marrow mononuclear cells from primary AML patients (FAB subtypes: M1, M2, M3, M4 and M7) into sublethally-irradiated newborn NOD/SCID/IL2rgnull mice, however, did not result in efficient engraftment of AML cells, while predominant proliferation of human CD4+ and CD8+ T cells was seen. These human T cells expressed CD45RO, and levels of human IFN-g in sera of the recipients significantly elevated, suggesting that human T cells were activated and inhibited the engraftment of human AML cells in the xenogeneic setting. We thus transplanted AML cells after T cell depletion. Strikingly, transplantation of 4x106 T cell-depleted AML bone marrow cells into neonatal NOD/SCID/IL2rgnull mice resulted in the efficient AML engraftment, whose levels were significantly higher than those in transplantation of the same number of T cell-depleted AML cells into NOD/SCID/b2mnull newborns or NOD/SCID/IL2rgnull adults. We also transplanted 103–104 hCD34+hCD38− bone marrow cells purified from AML patients. These low-doses of hCD34+hCD38− cells also successfully engrafted, progressively giving rise to hCD34+hCD38+ and hCD34− leukemic cells over 16 weeks. hCD34+hCD38− cells purified from the bone marrow of primary NOD/SCID/IL2rgnull recipients again reconstituted AML in secondary recipients, indicating that this system supports self-renewal capacity of AML stem cells within the hCD34+hCD38− fraction. Thus, the NOD/SCID/IL2rgnull newborn system provides a powerful model to study human leukemogenesis as well as the interaction between human T cells and AML cells in vivo.

scholarly journals Lymphoid reconstitution of the human fetal thymus in SCID mice with CD34+ precursor cells.

1991 ◽  
Vol 174 (5) ◽  
pp. 1283-1286 ◽  
Author(s):  
B Péault ◽  
I L Weissman ◽  
C Baum ◽  
J M McCune ◽  
A Tsukamoto
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Precursor Cell ◽  
Scid Mice ◽  
Hematopoietic Stem ◽  
Fetal Thymus

The search for human hematopoietic stem cells has been hampered by the lack of appropriate assay systems. Demonstration of the ability of precursor cell candidates to give rise to T cells is of significant difficulty since dissociated in vitro cultured thymus stroma cells lose their ability to sustain thymocyte maturation. To define further the differentiative capacities of the rare human fetal liver and bone marrow cells that express the CD34 surface antigen and exhibit in vitro myeloid and pre-B cell activities, we have microinjected them into HLA-mismatched fetal thymus fragments, partially depleted of hematopoietic cells by low temperature culture. In vitro colonized thymuses have then been allowed to develop upon engraftment into immunodeficient SCID mice. Using this modification of the SCID-hu system, we show that low numbers of fetal CD34+ progenitor cells can repopulate the lymphoid compartment in the human thymus.

Bone Marrow Contains Virus-Specific Cytotoxic T Lymphocytes

Blood ◽  
1997 ◽  
Vol 90 (5) ◽  
pp. 2103-2108 ◽  
Author(s):  
Mark K. Slifka ◽  
Jason K. Whitmire ◽  
Rafi Ahmed
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Viral Infection ◽  
Cd8 T Cells ◽  
Acute Infection ◽  
Severe Combined Immunodeficiency ◽  
Scid Mice ◽  
Antiviral Immune Response

Abstract Immunizing bone marrow donors prior to bone marrow transplant (BMT) has the potential for adoptively transferring specific immunity against opportunistic pathogens. Studies have shown that long-term antibody production occurs in the bone marrow and that specific humoral immunity may be transferred from donor to recipient following BMT. However, the magnitude and duration of T-cell memory in the bone marrow compartment has not been adequately investigated. In this study, virus-specific CD8+ T-cell responses in the bone marrow were compared with those observed in the spleen of mice acutely infected with lymphocytic choriomeningitis virus (LCMV). During the acute stages of infection, most CD8+ T cells in the spleen and bone marrow showed upregulated surface expression of the activation/memory marker, LFA-1 (LFA-1hi). After clearing LCMV infection, the antiviral immune response subsided to homeostatic levels and the ratio of CD8+/LFA-1hi to CD8+/LFA-1lo T cells in the spleen and bone marrow of LCMV immune mice returned to the value observed in naive mice. Virus-specific ex vivo effector cytotoxic T-lymphocyte (CTL) responses could be identified in both spleen and bone marrow compartments at 8 days postinfection. LCMV-specific CTL precursor (CTLp) frequencies peaked in the bone marrow at 8 days postinfection and averaged one in 200 to one in 650 CD8+ T cells, a frequency similar to that observed in the spleen. After clearing the acute infection, potent LCMV-specific CTL memory responses could be demonstrated in the bone marrow for at least 325 days postinfection, indicating long-term persistence of antiviral T cells at this site. Adoptive transfer of LCMV-immune bone marrow into severe combined immunodeficiency (SCID) mice provided protection against viral challenge, whereas SCID mice that received naive bone marrow became chronically infected upon challenge with LCMV. These results indicate that after acute viral infection, virus-specific memory T cells can be found in the bone marrow compartment and are maintained for an extended period, and when adoptively transferred into an immunodeficient host, they are capable of conferring protection against chronic viral infection.

Immune-Mediated Hepatitis Drives Bone marrow to Hepatocyte Plasticity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3599-3599
Author(s):  
Marc H. Dahlke ◽  
Felix C. Popp ◽  
Pompiliu Piso ◽  
Hans J. Schlitt ◽  
Patrick Bertolino
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Liver Regeneration ◽  
T Cell Receptor ◽  
Bone Marrow Cells ◽  
Cell Receptor ◽  
Liver Pathology ◽  
Mhc Haplotype

Abstract Liver dysfunction is a major health burden world-wide. Future cell-based therapies for liver regeneration may benefit from the fact that bone marrow cells can fuse with or transdifferentiate into hepatocytes. All models demonstrating bone marrow to hepatocyte plasticity presented so far, however, have used highly artifical conditions of liver regeneration - applying toxins, genetic pressure models or liver resection. We have set up a model of transgenic T cell induced bystander hepatitis in bone marrow chimeras to assess the effect of hepatitis, a common liver pathology in humans, as an enhancer of bone marrow to hepatocyte plasticity events. MHC haplotype (Kb) transgenic bone marrow from 178.3 mice or control bone marrow from B10.BR (Kk) mice was transplanted into sublethally irradiated B10.BR (Kk) mice. Hepatitis was induced by repeated injections of Des Kk T cell receptor transgenic T cells against the Kb antigen. In additonal groups Retrorsine was used as an agent inhibiting endogenous hepatocyte proliferation and GCSF for mobilisation of bone marrow stem cells. Repeated injections of transgenic T cells induced subsequent waves of hepatitis in recipients of MHC haplotype transgenic bone marrow but not in control animals confirmed by serum ALT levels. Hepatocyte single cell suspensions from animals suffering from hepatitis revealed an increased expression of donor bone marrow derived antigen. This could be further enhanced by either increasing the number of circulating stem cells or by inhibiting the endogenous response of resident hepatocytes. FISH analysis showed fusion nuclei on a single cellular level. T cell receptor transgenic T cells induce bystander hepatitis in an antigen specific manner. This inflammatory response drives the plasticity of bone marrow cells to hepatocytes and their potential contribution to liver regeneration. Fusion between donor cells and resident hepatocytes is the underlying mechanism of liver regeneration in this model mimicking a common liver pathology.

Effects of the Homeodomain Gene Pitx2 on Self-Renewal of Hematopoietic Stem Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 95-95 ◽  
Author(s):  
Hui Z. Zhang ◽  
Svetlana Rogulina ◽  
Wendy Chen ◽  
Barbara A. Degar ◽  
Bernard G. Forget
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Leukemic Cells ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Colony Forming Units ◽  
Marrow Cells

Abstract Pitx2, a homeodomain gene preferentially expressed in murine hematopoietic stem/progenitor cells, is also a downstream target of genes important for hematopoiesis such as MLL and Wnt/Dvl/β-Catenin. We have previously reported that Pitx2 null hematopoietic stem cells (HSCs) can contribute to multi-lineage hematopoiesis under physiologic conditions. We have now carried out serial bone marrow transplantation experiments and demonstrated that after the 3rd round of serial transplantation, Pitx2 null cells reconstituted only 28.6% of the recipient hematopoietic cells as compared to 60% in the case of wild type cells (P<0.001). There were no Pitx2 null donor-derived cells in recipient mice after the 4th round of transplantation, whereas donor-derived chimerism was 57% with wild type cells (P<0.001), and 26% with Pitx2 +/− cells (P<0.001). Therefore, Pitx2 null HSCs have decreased self renewal capacity. To further study the function of Pitx2 in HSC, we constitutively overexpressed the Pitx2 gene in murine bone marrow cells following transduction using a MSCV/IRES/GFP retroviral vector, and analyzed the effects on hematopoiesis in vitro and in vivo. Bone marrow cells overexpressing Pitx2 were isolated on the basis of their GFP expression and analyzed for their colony forming ability in vitro. Retrovirally transduced bone marrow cells were also transplanted into lethally irradiated mice, and the transplanted mice were observed for long-term reconstitution. Colony-forming unit assays showed that Pitx2 overexpressing bone marrow cells, compared to control cells transduced with vector only, had increased numbers of GM colony forming units and reduced numbers of megakaryocytic colony forming units. Pitx2-overexpressing cells continued to form GM colonies after more than eight serial replatings. When these cells were cultured in liquid medium containing SCF, IL-3 and IL-6, they gave rise to cells that stained positively either for alpha naphthyl butyrate, indicating monocytic differentiation, or for peroxidase, indicating neutrophilic differentiation. The ability of these GM-colony forming cells to cause leukemia is currently under investigation. Long-term reconstitution of hematopoiesis in mice by Pitx2 over-expressing HSCs was demonstrated by identifying GFP positive multi-lineage peripheral blood cells four months following transplantation. One of these mice manifested leukemia at this time, as evidenced by a markedly elevated WBC count and other hematologic abnormalities. The leukemic WBCs had very high levels of GFP and Pitx2 expression and were shown to contain two retroviral integration sites, neither of which involved a known oncogene or overexpression of the gene at the integration site. Immunophenotyping by flow cytometry demonstrated that the majority of the leukemic cells were c-kit positive and expressed the megakaryocytic marker CD41, as well as the common myeloid progenitor marker, CD16/32. Some of the cells expressed the erythroid marker Ter119. The leukemic cells did not express any lymphoid markers, including CD3ε, B220, CD19, and IL7R3. This Pitx2-overexpression-associated leukemia was transplantable. Experiments are under way to characterize the leukemia initiating cells. Taken together, our results provide evidence that the homeodomain gene Pitx2 plays a role in the self-renewal of hematopoietic stem/progenitor cells.

Kruppel Like Factor 7 Suppresses Hematopoietic Stem and Progenitor Cell Function

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2356-2356
Author(s):  
Laura Schuettpelz ◽  
Felipe Giuste ◽  
Priya Gopalan ◽  
Daniel Link
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Progenitor Cell ◽  
Bone Marrow Cells ◽  
Transduction Efficiency ◽  
Hematopoietic Stem ◽  
Short And Long Term ◽  
Marked Suppression

Abstract Abstract 2356 Kruppel like factor 7 (KLF7) expression is an independent predictor of poor outcome in pediatric acute lymphoblastic leukemia (Flotho, et al; Blood 2007). In addition, KLF7 overexpression is associated with Imatinib-resistant CML (Cammarata, et al; Clinical Leukemia 2007). The kruppel like factor (KLF) family of transcription factors are involved in regulating cellular growth and differentiation in multiple tissue types. KLF7 is important for neurogenesis, and mice lacking KLF7 die perinatally with severe neurologic defects (Laub, et al; Mol Cell Biol 2005). While no specific role for KLF7 in hematopoiesis has been previously reported, loss of the closely related family member KLF6 is associated with defective blood cell production (Matsumoto, et al; Blood 2006), and other KLF family members are involved in multiple aspects of hematopoiesis. Targets of KLF7 include known regulators of hematopoietic stem and progenitor cell (HSPC) function including TRKA, Cebp/a, and CDKN1A (p21). Normal HSPCs appear to have a low level of KLF7 expression based on RNA expression profiling of populations enriched for these cells. Given these findings, we hypothesized that KLF7 may play a role in regulating normal HSPC function, and may contribute to leukemogenesis or resistance to therapy. To test this hypothesis, we first analyzed the effect of the loss of KLF7 on hematopoiesis. Specifically, we generated Klf7−/− fetal liver chimeras and characterized their hematopoiesis. Long-term multilineage engraftment of Klf7−/− cells was comparable to control cells. Moreover, HSC self-renewal, as assessed by serial transplantation was not effected by the loss of KLF7. To model the effect of KLF7 overexpression on HSPC function, we generated retroviral and lentiviral vectors that express KLF7. KLF7 expression in wild type bone marrow cells transduced with KLF7 retrovirus was increased approximately 10-fold. Overexpression of KLF7 was associated with a marked suppression of myeloid progenitor cell growth, as assessed using colony-forming cell assays. Relative to the initial transduction efficiency, the number of myeloid colonies produced from KLF7-transduced cells compared to vector-alone transduced cells was reduced 5.7 ± 1.9 fold. We next assessed short- and long-term engraftment of KLF7-transduced cells by bone marrow transplantation. In experiments using bone marrow cells transduced with high efficiency (≥ 60% transduced cells), overexpression of KLF7 resulted in impaired radioprotection. Whereas all (12 of 12) recipients transplanted with control transduced cells survived, only 42% (5 out of 12) of recipients of KLF7 transduced cells survived more than two weeks after transplantation (P < 0.003). When these experiments were performed with a reduced multiplicity of infection to achieve a lower transduction efficiency, all recipient mice survived at least 3 months. Whereas control-transduced cells were readily detected at near input levels (on average, 40% of nucleated blood cells), minimal contribution of KLF7-transduced cells was observed in all lineages except T cells. Interestingly, KLF7-transduced T cells were present at near input levels. In summary, our show that KLF7 is not required for normal HSPC function. However, overexpression of KLF7 leads to a marked suppression of the short- and long-term repopulating activity of HSPC with the exception cells in the T cell lineage. Whether KLF7 expression contributes to T cell leukemogenesis through suppression of other hematopoietic lineages will require further study. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Class I PI3K Is Required for HSC Differentiation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2461-2461
Author(s):  
Kristina Ames ◽  
Imit Kaur ◽  
Meng Tong ◽  
Shayda Hemmati ◽  
Ellen Tein ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Growth Factor ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Transplant Recipients ◽  
Self Renewal ◽  
Gene Sets ◽  
Growth Factor Deprivation

Adult hematopoietic stem cells (HSCs) are a rare and unique population of stem cells that reside in the bone marrow, where they undergo self-renewal and differentiation to maintain the blood system. The maintenance of a proper balance between HSC self-renewal and differentiation requires growth factors, cytokines, and chemokines, most of which activate the phosphoinositide 3-kinase/Protein Kinase B (PI3K/AKT) signaling pathway. Pathologic activation of the AKT pathway is frequently observed in tumors, making it a desirable target for cancer treatment. Since several PI3K inhibitors are now in clinical use, it is critical to determine the roles of PI3K in adult HSCs. However, the specific roles of PI3K in HSC function are poorly understood. Hematopoietic cells express three Class IA catalytic PI3K isoforms (P110α, β, and δ), which can all transduce growth factor and cytokine signals, and can compensate for one another in some cell types. Individual Class 1A PI3K isoforms have unique functions in mature hematopoietic lineages, but are dispensable for HSC function. To uncover the potentially redundant roles of PI3K isoforms in HSCs, we have generated a triple knockout (TKO) mouse model with conditional deletion of p110α and p110β in hematopoietic cells using MX1-Cre, and germline deletion of p110δ. TKO mice develop pancytopenia, which is also observed upon transplantation of TKO bone marrow. Competitive repopulation assays reveal a defect in long-term multi-lineage chimerism. Surprisingly, loss of Class 1A PI3K causes significant expansion of donor-derived long-term (Lin-cKit+Flk2-CD150+CD48-) and short-term (Lin-cKit+Flk2-CD150-CD48-) HSCs in the bone marrow, but not committed progenitors. This phenotype could not be explained by alterations in HSC cell cycling or apoptosis in TKO HSCs. TKO transplant recipients also have dysplastic features in the bone marrow. Methylcellulose plating assays of TKO bone marrow revealed a relative increase in granulocyte erythroid macrophage megakaryocyte (GEMM) colonies and extended serial replating, suggesting increased self-renewal. Thus, our data are consistent with impaired HSC differentiation upon deletion of all Class IA PI3K isoforms, which leads to dysplastic changes. RNA sequencing of sorted long-term HSCs from the bone marrow of TKO transplant recipients revealed the enrichment of human and mouse HSC signatures, and the downregulation of DNA repair gene sets and RNA splicing gene sets in TKO HSCs. Interestingly, we also observed downregulation of autophagy gene sets in TKO HSCs. Macroautophagy has been shown to be essential for the maintenance of HSC metabolism and self-renewal. Analysis of the autophagosomal marker LC3-II in TKO HSCs revealed a decrease in autophagy upon growth factor deprivation. Surprisingly, we observed an increase in MTOR activation in TKO cKit+ bone marrow cells via compensatory signaling through the MAPK pathway. Given that MTOR is a known negative regulator of autophagy, this is consistent with the observed autophagy decrease in TKO HSCs. Additionally, we found that autophagy can still be induced in TKO HSCs with the MTOR inhibitor rapamycin. Furthermore, rapamycin treatment impairs serial replating of TKO bone marrow cells. In conclusion, we found that inactivation of all Class 1A PI3 kinases leads to impaired HSC differentiation, likely due to a defect in autophagy induction in response to growth factor deprivation. Disclosures No relevant conflicts of interest to declare.

A comparison of murine T-cell–depleted adult bone marrow and full-term fetal blood cells in hematopoietic engraftment and immune reconstitution

Blood ◽  
2002 ◽  
Vol 99 (1) ◽  
pp. 364-371 ◽  
Author(s):  
Benny J. Chen ◽  
Xiuyu Cui ◽  
Gregory D. Sempowski ◽  
Maria E. Gooding ◽  
Congxiao Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Blood Cells ◽  
Immune Reconstitution ◽  
Full Term ◽  
Fetal Blood ◽  
Adult Bone

Umbilical cord blood has been increasingly used as a source of hematopoietic stem cells. A major area of concern for the use of cord blood transplantation is the delay in myeloid and lymphoid recovery. To directly compare myeloid and lymphoid recovery using an animal model of bone marrow and cord blood as sources of stem cells, hematopoietic engraftment and immune recovery were studied following infusion of T-cell–depleted adult bone marrow or full-term fetal blood cells, as a model of cord blood in a murine allogeneic transplantation model (C57BL/6 [H-2b] → BALB/c [H-2d]). Allogeneic full-term fetal blood has poorer radioprotective capacity but greater long-term engraftment potential on a cell-to-cell basis compared with T-cell–depleted bone marrow. Allogeneic full-term fetal blood recipients had decreased absolute numbers of T, B, and dendritic cells compared with bone marrow recipients. Splenic T cells in allogeneic full-term fetal blood recipients proliferated poorly, were unable to generate cytotoxic effectors against third-party alloantigens in vitro, and failed to generate alloantigen-specific cytotoxic antibodies in vivo. In addition, reconstituting T cells in fetal blood recipients had decreased mouse T-cell receptorδ single-joint excision circles compared with bone marrow recipients. At a per-cell level, B cells from fetal blood recipients did not proliferate as well as those found in bone marrow recipients. These results suggest that full-term fetal blood can engraft allogeneic hosts across the major histocompatibility barrier with slower hematopoietic engraftment and impaired immune reconstitution.

Generation of primary antigen-specific human cytotoxic T lymphocytes in human/mouse radiation chimera

Blood ◽  
1996 ◽  
Vol 88 (2) ◽  
pp. 721-730 ◽  
Author(s):  
H Segall ◽  
I Lubin ◽  
H Marcus ◽  
A Canaan ◽  
Y Reisner
Keyword(s):  
T Cells ◽  
T Cell ◽  
Human Lymphocytes ◽  
Scid Mice ◽  
Human Antibody ◽  
Adoptive Therapy ◽  
Activation Markers ◽  
Human T Cell ◽  
Human T Cells

Severe combined immunodeficient (SCID) mice are increasingly used as hosts for the adoptive transfer of human lymphocytes. Human antibody responses can be obtained in these xenogeneic chimeras, but information about the functionality of the human T cells in SCID mice is limited and controversial. Studies using human peripheral blood lymphocytes (PBL) injected intraperitoneally (IP) into SCID mice (hu-PBL-SCID mice) have shown that human T cells from these chimeras are anergic and have a defective signaling via the T-cell receptor. In addition, their antigenic repertoire is limited to xenoreactive clones. In the present study, we tested the functionality of human T cell in a recently described chimeric model. In this system, BALB/c mice are conditioned by irradiation and then transplanted with SCID bone marrow, followed by IP injection of human PBL. Our experiments demonstrated that human T cells, recovered from these hu-PBL-BALB mice within 1 month posttransplant, proliferated and expressed activation markers upon stimulation with anti-CD3 monoclonal antibody. A vigorous antiallogeneic human cytotoxic T-lymphocyte (CTL) response could be generated in these mice by immunizing them with irradiated allogeneic cells. Moreover, anti-human immunodeficiency virus type 1 (HIV-1) Net- specific human CTLs could be generated in vivo from naive lymphocytes by immunization of mouse-human chimeras with a recombinant vaccinia-nef virus. This model may be used to evaluate potential immunomodulatory drugs or cytokines, and could provide a relevant model for testing HIV vaccines, for production of antiviral T-cell clones for adoptive therapy, and for studying human T-cell responses in vivo.

scholarly journals Myelosuppressive conditioning is required to achieve engraftment of pluripotent stem cells contained in moderate doses of syngeneic bone marrow

Blood ◽  
1994 ◽  
Vol 83 (4) ◽  
pp. 939-948 ◽  
Author(s):  
Y Tomita ◽  
DH Sachs ◽  
M Sykes
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Whole Body ◽  
Mixed Chimerism ◽  
Hematopoietic Stem ◽  
Donor Cells ◽  
Donor Type ◽  
Low Levels

Abstract We have investigated the requirement for whole body irradiation (WBI) to achieve engraftment of syngeneic pluripotent hematopoietic stem cells (HSCs). Recipient B6 (H-2b; Ly-5.2) mice received various doses of WBI (0 to 3.0 Gy) and were reconstituted with 1.5 x 10(7) T-cell-depleted (TCD) bone marrow cells (BMCs) from congenic Ly-5.1 donors. Using anti-Ly-5.1 and anti-Ly-5.2 monoclonal antibodies and flow cytometry, the origins of lymphoid and myeloid cells reconstituting the animals were observed over time. Chimerism was at least initially detectable in all groups. However, between 1.5 and 3 Gy WBI was the minimum irradiation dose required to permit induction of long-term (at least 30 weeks), multilineage mixed chimerism in 100% of recipient mice. In these mice, stable reconstitution with approximately 70% to 90% donor-type lymphocytes, granulocytes, and monocytes was observed, suggesting that pluripotent HSC engraftment was achieved. About 50% of animals conditioned with 1.5 Gy WBI showed evidence for donor pluripotent HSC engraftment. Although low levels of chimerism were detected in untreated and 0.5-Gy-irradiated recipients in the early post-BM transplantation (BMT) period, donor cells disappeared completely by 12 to 20 weeks post-BMT. BM colony assays and adoptive transfers into secondary lethally irradiated recipients confirmed the absence of donor progenitors and HSCs, respectively, in the marrow of animals originally conditioned with only 0.5 Gy WBI. These results suggest that syngeneic pluripotent HSCs cannot readily engraft unless host HSCs sustain a significant level of injury, as is induced by 1.5 to 3.0 Gy WBI. We also attempted to determine the duration of the permissive period for syngeneic marrow engraftment in animals conditioned with 3 Gy WBI. Stable multilineage chimerism was uniformly established in 3-Gy-irradiated Ly-5.2 mice only when Ly-5.1 BMC were injected within 7 days of irradiation, suggesting that repair of damaged host stem cells or loss of factors stimulating engraftment may prevent syngeneic marrow engraftment after day 7.

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.

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