Identification of Cytogenetically Normal Human CD34+CD38+ Hematopoietic Stem/Progenitor Cells from Inv(16)+ Leukemic Bone Marrow

Abstract For many subtypes of AML including cases with the inv(16), mutations that give rise to the leukemic phenotype occur, at least in part, in the hematopoietic stem/progenitor (HSPC) cell subset, as suggested by studies showing that primitive CD34+ CD38- bone marrow cells can function as leukemia-initiating cells (LIC) when transferred into immunodeficient mice. A significant challenge has been that LIC share many of the same cell-surface markers as their normal HSPC counterparts, thus making it difficult to purify and functionally characterize either subset from the bulk bone marrow of leukemia patients. Here we report the FACS analysis of several previously reported human LIC markers on bone marrow samples from inv(16) AML patients and show that a combination of TIM3, CLL1, and CD33 can significantly enrich for a rare population of CD34+ CD38- cells that lack the inv(16) fusion mRNA when tested by nested RT-PCR. Heterogeneous expression of these markers among different patient samples often causes incomplete elimination of the fusion mRNA when FACS-sorting the CD34+ CD38- population as single TIM3-, CLL1-, or CD33- subsets. The combination of TIM3 with CLL1 and/or CD33 leads to a more consistent elimination of the fusion mRNA from the FACS-sorted CD34+ CD38- subsets. Results from methylcellulose assays showed that the TIM3- CLL1- CD33- subset of CD34+CD38- cells could form multiple colony types, including CFU-GEMM, that were all negative for the fusion mRNA by RT-PCR. In contrast, colonies derived from bulk bone marrow were all positive for the fusion mRNA. The TIM3- CLL1- CD33- subset of CD34+CD38- cells displayed greater than 600-fold enrichment for progenitor activity compared to bulk bone marrow but did not form additional colonies upon serial re-plating. These results have important implications for the therapeutic targeting of inv(16)+ hematopoietic stem/progenitor cells in patients with relapsed and refractory disease and for purification of normal HSPC from leukemic bone marrow samples. Disclosures No relevant conflicts of interest to declare.

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Abstract 577: Origin and Therapeutic Efficacy of Human Cardiac Progenitor Cells

Circulation ◽

10.1161/circ.116.suppl_16.ii_104-b ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Federico Mosna ◽

Claudia Bearzi ◽

Marcello Rota ◽

Toru Hosoda ◽

Jochen Tillmanns ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Connexin 43 ◽

Human Heart ◽

Bone Marrow Cells ◽

Ex Vivo ◽

Functional Integration ◽

Phenotypic Properties ◽

Hematopoietic Stem ◽

Immunodeficient Mice

To establish whether progenitor cells are present in the human heart (hCPCs) and have phenotypic properties distinct from human hematopoietic stem cells (hHSCs), samples of human myocardium were enzymatically dissociated and c-kit-positive-cells were sorted and plated immediately to obtain clones derived from single founder cells. By FACS analysis, freshly isolated hCPCs comprised 1.1±1.0% of the population and were negative for HSC markers, CD34 and CD133, and KDR. They were also negative for epitopes of monocytes, CD14 and CD16, mast cells, CD45, and lymphocytes, CD3 and CD20. The phenotype of hCPCs was completely different from that of human bone marrow cells which were positive for these surface antigens. Only small fractions of hCPCs expressed GATA4 and Nkx2.5. Of 1,530 seeded hCPCs, 11 clones were generated accounting for 0.7% cloning efficiency. Subsequently, these cells were injected in immunodeficient mice and rats at the time of coronary occlusion and 5 days after infarction. This was done to assess whether hCPCs differentiated into myocytes and coronary vessels immediately after ischemic injury and in the presence of a well-developed infarct. In both cases, hCPCs regenerated the infarcted myocardium. Connexin 43 and N-cadherin were detected between recipient rodent myocytes and newly formed human myocytes. The problem was then whether the structural integration of these two myocyte populations had a physiological counterpart. Studies were performed using an ex vivo preparation together with two-photon microscopy and laser line-scan imaging. EGFP-positive-hCPCs were injected in infarcted mice and the heart was studied 2-weeks later. The heart was perfused with an oxygenated Tyrode solution containing the calcium indicator Rhod-2 and stimulated at 1 Hz. Calcium transients was recorded in EGFP positive human myocytes and EGFP negative mouse myocytes. The synchronicity in calcium tracings between these two distinct cell pools was apparent, pointing to the functional integration of newly formed EGFP-positive human myocytes with the surrounding EGFP-negative mouse myocytes. Thus, the human heart contains hCPCs which are not of bone marrow origin and possess the ability to acquire the cardiomyocyte and coronary vascular cell lineages.

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Differential Effects of TLR1/2 and TLR2/6 Signaling on Normal and Premalignant Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood-2018-99-119719 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1806-1806

Author(s):

Darlene A. Monlish ◽

Zev J. Greenberg ◽

Sima T. Bhatt ◽

Dagmar Ralphs ◽

John L. Keller ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Progenitor Cells ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

White Blood Cells ◽

Hematopoietic Stem ◽

Specific Effects ◽

Stem And Progenitor Cells ◽

Cell Cycling

Abstract Prior studies from our lab and others have demonstrated a role for Toll-like receptor 2 (TLR2) in regulating both normal and premalignant hematopoietic stem and progenitor cells (HSPCs), however the contributions of its binding partners, TLR1 and TLR6, remain unknown. In CD34+ bone marrow cells of patients with myelodysplastic syndrome (MDS), increased TLR2 was associated with lower-risk disease, elevated rates of apoptosis associated with improved prognosis, and enhanced survival. Conversely, increased levels of TLR6, but not TLR1, was associated with higher-risk disease and an increased percentage of bone marrow blasts (Zeng et al., Exp Cell Res 2016 and Wei et al., Leukemia 2013). These data suggest that there may be heterodimer-specific effects of TLR2 signaling on HSPCs influencing disease progression. To elucidate the unique contributions of the heterodimer pairs in MDS pathogenesis and leukemogenesis, we utilized a well-established mouse model of MDS that expresses the NUP98-HOXD13 fusion from the hematopoietic Vav-1 promoter. The "NHD13" mice recapitulate many of the salient features of human MDS and succumb to cytopenias or leukemia by 14 months of age (Lin et al., Blood 2005). Importantly, we observed significantly increased expression of TLRs 1, 2, and 6 on the c-Kit+, Sca-1+, Lineage- ("KSL") HSPCs of the NHD13 mice, similar to the increased expression of these TLRs on CD34+ cells of MDS patients. To begin to delineate the heterodimeric differences, NHD13 mice were treated chronically with either PAM2CSK4 (PAM2), a TLR2/6-specific agonist, or PAM3CSK4 (PAM3), a TLR1/2-specific agonist, to assess the effects on cytopenias and survival. After five months of treatment, a significant increase was observed in the total number of white blood cells in NHD13 mice treated with PAM2 (p=0.007), but not PAM3 (vs. vehicle (water)-treated controls), a finding that was not recapitulated in wild-type (WT) controls. On the contrary, a significant decrease in the total number of platelets in both NHD13 and WT mice treated with PAM3 was observed as compared to vehicle-treated controls (p=0.024 and p=0.011, respectively). Further supporting the existence of heterodimer-specific differences, death was expedited in NHD13 mice treated with PAM2 as compared to those treated with PAM3 (p=0.019), with a median survival of 243 days vs. 338 for the PAM3-treated cohort. The cause of death, as determined by a hematopathologist based on cytology and blast percentage, was most often due to leukemia. To investigate the potential mechanism through which enhanced TLR2/6 signaling accelerates leukemogenesis and death in NHD13 mice, the HSPCs of premalignant NHD13 mice treated with PAM2 or PAM3 were characterized by flow cytometry and evaluated for cell cycling and cell death. Both the total number and frequency of KSL cells were significantly increased in NHD13 mice treated with PAM2 (p=0.007 and p<0.0001, respectively), but not PAM3, vs. water-treated controls. No significant changes were noted in either cell cycling or apoptosis following agonist treatment. A microarray of bone marrow KSL cells revealed that stimulation of the TLR2/6 pathway is associated with an activated c-Myc signature, suggesting that enhanced signaling through this pathway, but not TLR1/2, may enhance leukemogenesis via Myc activation. Further, the expression levels of six downstream targets of c-Myc, including BAX, APEX1, ODC1, FKBP4, NCL, and HSPD1, were significantly increased in both WT and NHD13 mice following PAM2 treatment. Evaluation of serum cytokines also revealed heterodimer-specific alterations, including increased IL-6 levels in NHD13 mice treated with PAM2, but not PAM3. These data corroborate numerous previous reports linking IL-6 to MDS pathogenesis and transformation to acute myeloid leukemia. Ongoing studies involving mass cytometry, IL-6knockout mice, and pharmacological inhibitors of both IL-6 and c-Myc aim to further elucidate the mechanism through which TLR2/6-specific activation accelerates leukemogenesis and death in the NHD13 mouse model of MDS. These studies hope to inform more targeted therapeutics that could potentially delay MDS progression and reduce off-target effects. Disclosures No relevant conflicts of interest to declare.

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R4 Rgs Subfamily Proteins Negatively Regulates SDF-1/CXCR4 Signaling in CD34+ Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v128.22.5048.5048 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5048-5048

Author(s):

Kam Tong Leung ◽

Yorky Tsin Sik Wong ◽

Karen Li ◽

Kathy Yuen Yee Chan ◽

Xiao-Bing Zhang ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Conflicts Of Interest ◽

Expression Patterns ◽

Rgs Proteins ◽

Hematopoietic Stem ◽

Cxcr4 Signaling ◽

Human Cd34 ◽

Cd34 Cells ◽

Stem And Progenitor Cells

Abstract RGS family proteins are known to negatively regulate G-protein-coupled receptor signaling through their GTPase-accelerating activity. In several types of hematopoietic cells (e.g., B lymphocytes and megakaryocytes), responses to stromal cell-derived factor-1 (SDF-1) are subjected to regulation by R4 subfamily RGS proteins. However, their expression patterns and functional roles in hematopoietic stem and progenitor cells (HSC) are poorly characterized. Here, we showed that human CD34+ HSC derived from cord blood (CB, n = 10) expressed 7 out of 10 R4 RGS proteins at mRNA level (RGS1-3, 5, 13, 16 and 18), whereas expressions of RGS4, 8 and 21 were undetectable. Exposure of CB CD34+ cells to SDF-1 significantly increased RGS1, 2, 13 and 16 expressions and decreased RGS3 and 18 expressions (P ≤ 0.0402, n = 5). Expressions of RGS1, 13 and 16 were significantly higher in bone marrow (BM, n = 10) CD34+ cells when compared to mobilized peripheral blood (MPB, n = 5) CD34+ cells (P ≤ 0.0160), while RGS3 and 18 expressions were lower in BM CD34+ cells (P ≤ 0.0471), suggesting a SDF-1- and niche-dependent regulation of RGS expressions. To investigate the potential involvement of RGS proteins in SDF-1-mediated homing-related functions, we introduced RGS overexpression constructs into CB CD34+ cells by lentiviral transduction. With >80% transduction efficiency, we showed that overexpression of RGS1, 13 and 16 but not RGS2 significantly inhibited migration of CD34+ cells to a SDF-1 gradient (P ≤ 0.0391, n = 4-5). Similarly, RGS1, 13 and 16 overexpression suppressed SDF-1-induced Akt phosphorylation (n = 2), but none of them affected SDF-1-mediated actin polymerization (n = 3). In the NOD/SCID mouse xenotransplantation model, preliminary results showed that bone marrow homing was impaired in RGS1- (16.3% reduction), RGS13- (12.7% reduction) or RGS16-overexpressing CD34+ cells (33.7% reduction). Taken together, we provided the first evidence that expressions of R4 RGS proteins are regulated by the SDF-1/CXCR4 axis in human CD34+ HSC. We also presented evidence that specific R4 RGS proteins (RGS1, 13 and 16) negatively regulate in vitro SDF-1-mediated responses and in vivo homing of CD34+ cells, suggesting that RGS proteins may serve as a feedback mechanism to regulate SDF-1/CXCR4 signaling. Strategies to inhibit RGS signaling could thus be a potential method for enhancing efficiency of HSC homing and long-term engraftment, which is particularly important in the setting of CB transplantation. Disclosures No relevant conflicts of interest to declare.

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Inhibitory Receptor, gp49B1, Is Co-Expressed with c-Kit and Regulates Hematopoiesis during Development

Blood ◽

10.1182/blood.v126.23.4751.4751 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4751-4751

Author(s):

Mi Deng ◽

Zhigang Lu ◽

Jaehyup Kim ◽

Chengcheng Zhang

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Hematopoietic Cells ◽

Deficient Mouse ◽

Hematopoietic Progenitor ◽

Post Transplantation ◽

Hematopoietic Stem

Abstract During development, hematopoietic stem cells (HSCs) undergo dramatic expansion in fetal liver, and then migrate to spleen and bone marrow afterwards. Mouse HSCs are enriched in lineage- Sca-1+ c-Kit+ (LSK) cells and defined by their ability to reconstitute the hematopoietic system of lethally irradiated recipients. Although c-Kit is required for HSCs function, heterogeneous c-Kit expression represents functionally distinct subsets of HSCs and progenitor cells. Recently, we demonstrated the one of inhibitory leukocyte immunoglobulin-like receptors, LILRB2, and its mouse homolog, PIRB, are expressed in HSCs. Besides PIRB, the gp49B1 is the only other member of mouse LILRB family. The function of gp49B1 in hematopoiesis is not known. Here we demonstrated that gp49B1 is not expressed by LSK cells of adult mice but is expressed on neonatal bone marrow and spleen LSK cells. Two distinct populations of neonatal LSK cells can be identified based on c-Kit expression. In neonatal bone marrow, 96% of c-Kithi LSK cells are gp49B1+ whereas only 3% of c-Kitlo LSK cells express gp49B1. Similarly, 99% of c-Kithi but only 9% of c-Kitlo LSK cells are gp49B1+ in neonatal spleen. The gp49B1+ LSK cells showed 4.2-folds higher expression level of c-Kit than that of the gp49B1- LSK cells. Because c-Kit is required for hematopoietic progenitor or HSC (HSPC) function, we sought to test whether gp49B1 has regulatory effects on HSC activity. Neonatal splenic gp49B1- LSK cells produced 26-folds more colonies than gp49+ LSK cells after 7 day in methylcellulose media. To compare their reconstitution abilities, we injected 1,000 sorted neonatal splenic gp49B1+ or gp49B1- LSK cells into lethally irradiated 8 weeks-old C57BL6 mice. All mice transplanted by gp49B1+ LSK cells died within 2 weeks post-transplantation, whereas all gp49B1- LSK cells transplanted survived. These results suggest gp49B1- LSK cells, which have less c-Kit expression, are enriched for HSC activity. To further confirm it, 500 sorted gp49B1+ or gp49B1- LSK cells (CD45.2+) were transplanted with 100,000 competitor bone marrow cells (CD45.1+) into lethally irradiated congenic recipients (CD45.1+). Mice transplanted with gp49B1- LSK cells exhibited increasing peripheral blood donor CD45 chimerism levels from 3 to 18 weeks after transplant (14.7%~68.2%); but gp49B1+ LSK cells transplanted mice only have modest chimerism levels (<2%; exception of 1 out of 7 mice has 13% at 18 weeks). Interestingly, neonatal splenic gp49B1+ LSK cells exhibited a lineage bias compared to gp49B1- LSK cells after transplantation (B cell: 2% vs. 30.1%, p<0.01; T cell: 61.3% vs. 17.4%, p<0.05; and Myeloid cell: 42.8% vs. 60.2%, p=0.32). Consistently, c-Kithi LSK cells of which over 96% are gp49B1+ showed much less HSPC activities comparing with c-Kitlo LSK cells in colony formation (40-folds less), non-competitive transplantation (all died in 2 weeks vs. all survived after transplantation), and competitive transplantation (donor CD45 chimerism: 0.04~0.34% vs. 5.8~33.6%, from 6 to 20 weeks). We continued to study the function of gp49B1's function using gp49b1 deficient mice. We found that c-Kithi LSK cells were increased in gp49B1-deficient mouse (0.18% vs. 0.14%), whereas KO c-Kithi LSK% decreased (0.09% vs. 0.14%). The c-Kithi LSK cells of gp49B1-deficient mouse also exhibited low repopulation potential. While the same number of WT and KO LSK cells had comparable repopulation abilities, five hundred gp49b1-deficient c-Kitlo LSK cells exhibited a greater reconstitution capacity (65.3% vs. 33.6%) than wild-type c-Kitlo LSK cells. These results suggest that gp49B1 may regulate the repopulation of primitive neonatal hematopoietic cells. Together, our results demonstrate that the gp49B1 is co-expressed with high level of c-Kit in hematopoietic progenitor cells of neonatal mouse, and it regulates maturation, repopulation, and differentiation of hematopoietic cells during development. Disclosures No relevant conflicts of interest to declare.

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The histone lysine acetyltransferase HBO1 (KAT7) regulates hematopoietic stem cell quiescence and self-renewal

Blood ◽

10.1182/blood.2021013954 ◽

2021 ◽

Author(s):

Yuqing Yang ◽

Andrew J Kueh ◽

Zoe Grant ◽

Waruni Abeysekera ◽

Alexandra L Garnham ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Hematopoietic Stem Cell ◽

Cell Function ◽

Bone Marrow Cells ◽

High Rate ◽

Embryonic Patterning ◽

Self Renewal ◽

Hematopoietic Stem

The histone acetyltransferase HBO1 (MYST2, KAT7) is indispensable for postgastrulation development, histone H3 lysine 14 acetylation (H3K14Ac) and the expression of embryonic patterning genes. In this study, we report the role of HBO1 in regulating hematopoietic stem cell function in adult hematopoiesis. We used two complementary cre-recombinase transgenes to conditionally delete Hbo1 (Mx1-Cre and Rosa26-CreERT2). Hbo1 null mice became moribund due to hematopoietic failure with pancytopenia in the blood and bone marrow two to six weeks after Hbo1 deletion. Hbo1 deleted bone marrow cells failed to repopulate hemoablated recipients in competitive transplantation experiments. Hbo1 deletion caused a rapid loss of hematopoietic progenitors (HPCs). The numbers of lineage-restricted progenitors for the erythroid, myeloid, B-and T-cell lineages were reduced. Loss of HBO1 resulted in an abnormally high rate of recruitment of quiescent hematopoietic stem cells (HSCs) into the cell cycle. Cycling HSCs produced progenitors at the expense of self-renewal, which led to the exhaustion of the HSC pool. Mechanistically, genes important for HSC functions were downregulated in HSC-enriched cell populations after Hbo1 deletion, including genes essential for HSC quiescence and self-renewal, such as Mpl, Tek(Tie-2), Gfi1b, Egr1, Tal1(Scl), Gata2, Erg, Pbx1, Meis1 and Hox9, as well as genes important for multipotent progenitor cells and lineage-specific progenitor cells, such as Gata1. HBO1 was required for H3K14Ac through the genome and particularly at gene loci required for HSC quiescence and self-renewal. Our data indicate that HBO1 promotes the expression of a transcription factor network essential for HSC maintenance and self-renewal in adult hematopoiesis.

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Phenotypic correction of Fanconi anemia group C knockout mice

Blood ◽

10.1182/blood.v95.2.700 ◽

2000 ◽

Vol 95 (2) ◽

pp. 700-704 ◽

Cited By ~ 40

Author(s):

Kimberly A. Gush ◽

Kai-Ling Fu ◽

Markus Grompe ◽

Christopher E. Walsh

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Mitomycin C ◽

Fanconi Anemia ◽

Bone Marrow Cells ◽

Bone Marrow Failure ◽

Genetic Disorder ◽

Hematopoietic Stem ◽

Marrow Cells

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, congenital anomalies, and a predisposition to malignancy. FA cells demonstrate hypersensitivity to DNA cross-linking agents, such as mitomycin C (MMC). Mice with a targeted disruption of the FANCC gene (fancc −/− nullizygous mice) exhibit many of the characteristic features of FA and provide a valuable tool for testing novel therapeutic strategies. We have exploited the inherent hypersensitivity offancc −/− hematopoietic cells to assay for phenotypic correction following transfer of the FANCC complementary DNA (cDNA) into bone marrow cells. Murine fancc −/− bone marrow cells were transduced with the use of retrovirus carrying the humanfancc cDNA and injected into lethally irradiated recipients. Mitomycin C (MMC) dosing, known to induce pancytopenia, was used to challenge the transplanted animals. Phenotypic correction was determined by assessment of peripheral blood counts. Mice that received cells transduced with virus carrying the wild-type gene maintained normal blood counts following MMC administration. All nullizygous control animals receiving MMC exhibited pancytopenia shortly before death. Clonogenic assay and polymerase chain reaction analysis confirmed gene transfer of progenitor cells. These results indicate that selective pressure promotes in vivo enrichment offancc-transduced hematopoietic stem/progenitor cells. In addition, MMC resistance coupled with detection of the transgene in secondary recipients suggests transduction and phenotypic correction of long-term repopulating stem cells.

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Bone Marrow Oxidative Stress and Acquired Lineage-Specific Genotoxicity in Hematopoietic Stem/Progenitor Cells Exposed to 1,4-Benzoquinone

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph17165865 ◽

2020 ◽

Vol 17 (16) ◽

pp. 5865

Author(s):

Ramya Dewi Mathialagan ◽

Zariyantey Abd Hamid ◽

Qing Min Ng ◽

Nor Fadilah Rajab ◽

Salwati Shuib ◽

...

Keyword(s):

Oxidative Stress ◽

Bone Marrow ◽

Dna Damage ◽

Progenitor Cells ◽

Bone Marrow Cells ◽

Protein Carbonyls ◽

Mouse Bone Marrow ◽

Tail Moment ◽

Erythroid Progenitor ◽

Hematopoietic Stem

Hematopoietic stem/progenitor cells (HSPCs) are susceptible to benzene-induced genotoxicity. However, little is known about the mechanism of DNA damage response affecting lineage-committed progenitors for myeloid, erythroid, and lymphoid. Here, we investigated the genotoxicity of a benzene metabolite, 1,4-benzoquinone (1,4-BQ), in HSPCs using oxidative stress and lineage-directed approaches. Mouse bone marrow cells (BMCs) were exposed to 1,4-BQ (1.25–12 μM) for 24 h, followed by oxidative stress and genotoxicity assessments. Then, the genotoxicity of 1,4-BQ in lineage-committed progenitors was evaluated using colony forming cell assay following 7–14 days of culture. 1,4-BQ exposure causes significant decreases (p < 0.05) in glutathione level and superoxide dismutase activity, along with significant increases (p < 0.05) in levels of malondialdehyde and protein carbonyls. 1,4-BQ exposure induces DNA damage in BMCs by significantly (p < 0.05) increased percentages of DNA in tail at 7 and 12 μM and tail moment at 12 μM. We found crucial differences in genotoxic susceptibility based on percentages of DNA in tail between lineage-committed progenitors. Myeloid and pre-B lymphoid progenitors appeared to acquire significant DNA damage as compared with the control starting from a low concentration of 1,4-BQ exposure (2.5 µM). In contrast, the erythroid progenitor showed significant damage as compared with the control starting at 5 µM 1,4-BQ. Meanwhile, a significant (p < 0.05) increase in tail moment was only notable at 7 µM and 12 µM 1,4-BQ exposure for all progenitors. Benzene could mediate hematological disorders by promoting bone marrow oxidative stress and lineage-specific genotoxicity targeting HSPCs.

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Sustained human hematopoiesis in immunodeficient mice by cotransplantation of marrow stroma expressing human interleukin-3: analysis of gene transduction of long-lived progenitors

Blood ◽

10.1182/blood.v83.10.3041.3041 ◽

1994 ◽

Vol 83 (10) ◽

pp. 3041-3051 ◽

Cited By ~ 204

Author(s):

JA Nolta ◽

MB Hanley ◽

DB Kohn

Keyword(s):

Stem Cells ◽

Progenitor Cells ◽

Blood Cells ◽

Gene Transduction ◽

Hematopoietic Stem ◽

Immunodeficient Mice ◽

Interleukin 3 ◽

Human Cd34 ◽

Time Period

Abstract We have developed a novel cotransplantation system in which gene- transduced human CD34+ progenitor cells are transplanted into immunodeficient (bnx) mice together with primary human bone marrow (BM) stromal cells engineered to produce human interleukin-3 (IL-3). The IL- 3-secreting stroma produced sustained circulating levels of human IL-3 for at least 4 months in the mice. The IL-3-secreting stroma, but not control stroma, supported human hematopoiesis from the cotransplanted human BM CD34+ progenitors for up to 9 months, such that an average of 6% of the hematopoietic cells removed from the mice were of human origin (human CD45+). Human multilineage progenitors were readily detected as colony-forming units from the mouse marrow over this time period. Retroviral-mediated transfer of the neomycin phosphotransferase gene or a human glucocerebrosidase cDNA into the human CD34+ progenitor cells was performed in vitro before cotransplantation. Human multilineage progenitors were recovered from the marrow of the mice 4 to 9 months later and were shown to contain the transduced genes. Mature human blood cells marked by vector DNA circulated in the murine peripheral blood throughout this time period. This xenograft system will be useful in the study of gene transduction of human hematopoietic stem cells, by tracing the development of individually marked BM stem cells into mature blood cells of different lineages.

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Involvement of the Integrin Alpha 6 Receptor in the Homing and Engraftment of Hematopoietic Stem and Progenitor Cells to Bone Marrow.

Blood ◽

10.1182/blood.v104.11.1293.1293 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1293-1293

Author(s):

Hong Qian ◽

Sten Eirik W. Jacobsen ◽

Marja Ekblom

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Progenitor Cell ◽

Bone Marrow Cells ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Functional Blocking

Abstract Within the bone marrow environment, adhesive interactions between stromal cells and extracellular matrix molecules are required for stem and progenitor cell survival, proliferation and differentiation as well as their transmigration between bone marrow (BM) and the circulation. This regulation is mediated by cell surface adhesion receptors. In experimental mouse stem cell transplantation models, several classes of cell adhesion receptors have been shown to be involved in the homing and engraftment of stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Using FACS analysis, the integrin a6 chain was now found to be ubiquitously (>95%) expressed in mouse hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, lin−Sca-1+c-Kit+CD34+) both in adult bone marrow and in fetal liver. In vitro, about 70% of mouse BM lin−Sca-1+c-Kit+ cells adhered to laminin-10/11 and 40% adhered to laminin-8. This adhesion was mediated by integrin a6b1 receptor, as shown by functional blocking monoclonal antibodies. We also used a functional blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of hematopoietic stem and progenitor cells. We found that the integrin a6 antibody inhibited the homing of bone marrow progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C was reduced by about 40% as compared to cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells (LTR), antibody treated bone marrow cells were first injected intravenously into lethally irradiated primary recipients. After three hours, bone marrow cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis 16 weeks after transplantation revealed an 80% reduction of stem cell activity of integrin a6 antibody treated cells as compared to cells treated with control antibody. These results suggest that integrin a6 plays an important role for hematopoietic stem and progenitor cell homing in vivo.

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3′ Distal Regulatory Elements Required for Human CD34 Expression in Transgenic Mice.

Blood ◽

10.1182/blood.v106.11.125.125 ◽

2005 ◽

Vol 106 (11) ◽

pp. 125-125

Author(s):

Elena Levantini ◽

Yutaka Okuno ◽

Pu Zhang ◽

Steffen Koschmieder ◽

Hanna S. Radomska ◽

...

Keyword(s):

Bone Marrow ◽

Transgenic Mice ◽

Progenitor Cells ◽

Regulatory Element ◽

Restriction Enzymes ◽

Regulatory Elements ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Expression

Abstract CD34 is the best-defined human hematopoietic stem cell (HSC) marker, however the regulation of its gene expression is still largely unknown. Therefore, unraveling the elements that regulate human CD34 expression would be an invaluable tool for a broad range of studies, including the establishment of models of leukemia in mice, which require targeting of the transgene to stem and/or early progenitor cells. Moreover, identification of such regulatory elements will provide important insights into the transcriptional agenda of stem and progenitor cells and most importantly will prove useful for gene therapy protocols. Studies from our laboratory demonstrated that human CD34 transgenes are expressed in murine repopulating HSCs, which resembles the expression of the CD34 gene in human hematopoiesis, thus indicating the mouse model as an excellent way to study the expression of human CD34. Using P1 derived artificial chromosome (PAC) clones encompassing the human CD34 gene to generate transgenic mice, we showed that 90kb of upstream and 26kb of downstream flanking sequences were capable of regulating human CD34 expression in murine transgenic lines. Successive deletions of this larger construct were then performed to identify the important control regions. Deletion of the 5′ region from −90kb to −18kb did not result in any loss of activity. PAC54A19, a clone extending from −18kb to +26kb, expressed RNA in various tissues in a manner similar to that of larger fragments. In contrast, deletions creating a construct spanning from −10kb to +17kb led to complete loss of expression in transgenic animals, indicating that critical distal elements are located between −18kb to −10kb and/or +17kb to +26kb. In order to facilitate identification of important regulatory elements present in the upstream (−18kb to −10 kb) and/or downstream (+17kb to +26kb) regions of human CD34, we created further deletions of PAC54A19 using rare-cutting restriction enzymes, and studied the effects of the deletions on human CD34 expression in transgenic mice. Interestingly, we did not detect any human CD34 mRNA and protein expression in bone marrow and HSCs from transgenic mice carrying a construct spanning from −18kb to +17.4kb. In contrast, we observed expression of human CD34 transcripts in the bone marrow of transgenic mice containing a PAC spanning from −12.8kb to +26kb. Furthermore, HSCs from this latter group of mice presented the human CD34 antigen on their surface, as detected by FACS. Taken together, these data are highly suggestive that critical cis regulatory element(s) required to drive human CD34 in vivo expression are located in a 8.6kb fragment placed between +17.4kb and +26kb downstream of the human CD34 gene. Our current efforts focus on identifying the element(s) within the 8.6kb 3′ region that might be required to achieve human CD34 expression in HSCs.

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