Gene Transfer to Hematopoietic Stem/Progenitor Cells As a Novel Approach for Immunotherapy Against B-Lineage Malignancies: In Vivo Xenograft Model,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4168-4168
Author(s):  
Satiro N. De Oliveira ◽  
Francesca Giannoni ◽  
Cinnamon Hardee ◽  
Arineh Sahaghian ◽  
Laurence J N Cooper ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Malignant Cells ◽  
Hematopoietic Stem ◽  
Effector Cells ◽  
Nsg Mice ◽  
Novel Approach ◽  
B Lineage

Abstract Abstract 4168 Chimeric Antigen Receptors (CAR) against CD19 have been shown to direct T cells to specifically target B-lineage malignant cells in animal models and clinical trials, with efficient tumor cell lysis. But, there has been insufficient persistence of effector cells, limiting the clinical efficacy. We propose gene transfer to hematopoietic stem/progenitor cells (HSPC) as a novel approach to ensure persistent production of effector cells targeting B-lineage malignant cells, exponentially increasing the number of effectors that may be generated against tumor cells. Experiments were performed using NOD-SCID-IL2 receptor gamma chain null (NSG) mice engrafted with human CD34+ HSPCs transduced with lentiviral vectors carrying first and second generations of CD19-specific CAR. There was efficient and stable transduction with 1–2 copies of CAR/cell as determined by qPCR. Differentiation of modified HSPC in vivo was not impaired by gene transfer, as observed in vitro. Results of in vivo studies showed that CAR-transduced human HSPC successfully differentiated into all lineages, with CAR-expressing T, NK and myeloid cells populating bone marrow, spleen and peripheral blood. The human CD19+ B cell populations normally formed in the xenografted NSG mice were significantly reduced when the transplanted HSPC were transduced with the anti-CD19 CAR, demonstrating in vivo biological activity. Cells harvested from bone marrow and spleen of mice engrafted with modified HSPC lysed CD19-positive cell targets ex vivo. Leukemic challenges of engrafted mice are in progress. Our results provide evidence for the feasibility and efficacy of the modification of HSPC with CAR as a protocol for generation of effector cells for immunotherapy against B-lineage malignancies. Disclosures: No relevant conflicts of interest to declare.

Fluorescence-Based Selection of Gene-Corrected Hematopoietic Stem and Progenitor Cells From Acid Sphingomyelinase-Deficient Mice: Implications for Niemann-Pick Disease Gene Therapy and the Development of Improved Stem Cell Gene Transfer Procedures

Blood ◽  
1999 ◽  
Vol 93 (1) ◽  
pp. 80-86 ◽  
Author(s):  
Shai Erlich ◽  
Silvia R.P. Miranda ◽  
Jan W.M. Visser ◽  
Arie Dagan ◽  
Shimon Gatt ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Fetal Liver ◽  
Acid Sphingomyelinase ◽  
Hematopoietic Stem

Abstract The general utility of a novel, fluorescence-based procedure for assessing gene transfer and expression has been demonstrated using hematopoietic stem and progenitor cells. Lineage-depleted hematopoietic cells were isolated from the bone marrow or fetal livers of acid sphingomyelinase–deficient mice, and retrovirally transduced with amphotropic or ecotropic vectors encoding a normal acid sphingomyelinase (ASM) cDNA. Anti–c-Kit antibodies were then used to label stem- and progenitor-enriched cell populations, and the Bodipy fluorescence was analyzed in each group after incubation with a Bodipy-conjugated sphingomyelin. Only cells expressing the functional ASM (ie, transduced) could degrade the sphingomyelin, thereby reducing their Bodipy fluorescence as compared with nontransduced cells. The usefulness of this procedure for the in vitro assessment of gene transfer into hematopoietic stem cells was evaluated, as well as its ability to provide an enrichment of transduced stem cells in vivo. To show the value of this method for in vitro analysis, the effects of retroviral transduction using ecotropic versus amphotropic vectors, various growth factor combinations, and adult bone marrow versus fetal liver stem cells were assessed. The results of these studies confirmed the fact that ecotropic vectors were much more efficient at transducing murine stem cells than amphotropic vectors, and that among the three most commonly used growth factors (stem cell factor [SCF] and interleukins 3 and 6 [IL-3 and IL-6]), SCF had the most significant effect on the transduction of stem cells, whereas IL-6 had the most significant effect on progenitor cells. In addition, it was determined that fetal liver stem cells were only approximately twofold more “transducible” than stem cells from adult bone marrow. Transplantation of Bodipy-selected bone marrow cells into lethally irradiated mice showed that the number of spleen colony-forming units that were positive for the retroviral vector (as determined by polymerase chain reaction) was 76%, as compared with 32% in animals that were transplanted with cells that were nonselected. The methods described within this manuscript are particularly useful for evaluating hematopoietic stem cell gene transfer in vivo because the marker gene used in the procedure (ASM) encodes a naturally occurring mammalian enzyme that has no known adverse effects, and the fluorescent compound used for selection (Bodipy sphingomyelin) is removed from the cells before transplantation.

scholarly journals In vivo gene transfer into rat bone marrow progenitor cells using rSV40 viral vectors

Blood ◽  
2005 ◽  
Vol 106 (8) ◽  
pp. 2655-2662 ◽  
Author(s):  
Bianling Liu ◽  
Judy Daviau ◽  
Carmen N. Nichols ◽  
David S. Strayer
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Transgene Expression ◽  
Viral Vectors ◽  
Ex Vivo ◽  
Simian Virus 40 ◽  
Hematopoietic Stem ◽  
Entire Study

AbstractHematopoietic stem cell (HSC) gene transfer has been attempted almost entirely ex vivo and has been limited by cytokine-induced loss of self-renewal capacity and transplantation-related defects in homing and engraftment. Here, we attempted to circumvent such limitations by injecting vectors directly into the bone marrow (BM) to transduce HSCs in their native environment. Simian virus 40 (SV40)–derived gene delivery vectors were used because they transduce resting CD34+ cells very efficiently. Rats received SV-(Nef-FLAG), carrying FLAG marker epitope—or a control recombinant SV40 (rSV40)—directly into both femoral marrow cavities. Intracellular transgene expression by peripheral blood (PB) or BM cells was detected by cytofluorimetry. An average of 5.3% PB leukocytes expressed FLAG for the entire study—56 weeks. Transgene expression was sustained in multiple cell lineages, including granulocytes (average, 3.3% of leukocytes, 20.4% of granulocytes), CD3+ T lymphocytes (average, 0.53% of leukocytes, 1% of total T cells), and CD45R+ B lymphocytes, indicating gene transfer to long-lived progenitor cells with multilineage capacity. An average of 15% of femoral marrow cells expressed FLAG up to 16.5 months after transduction. Thus, direct intramarrow administration of rSV40s yields efficient gene transfer to rat BM progenitor cells and may be worthy of further investigation.

scholarly journals Megakaryocytic TGFβ1 Partitions Hematopoiesis into Amplifying Stem and Progenitor Cells and Maturing Effector Cells

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 81-81
Author(s):  
Silvana Di Giandomenico ◽  
Pouneh Kermani ◽  
Nicole Molle ◽  
Mia Yabut ◽  
Fabienne Brenet ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Progenitor Cells ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Effector Cells ◽  
Erythroid Precursors ◽  
Stem And Progenitor Cells

Abstract Background: Chronic anemia is a significant problem affecting over 3 million Americans annually. Therapies are restricted to transfusion and Erythropoietin Stimulating Agents (ESA). There is a need for new approaches to treat chronic anemia. Immature erythroid progenitors are thought to be continuously produced and then permitted to survive and mature if there is sufficient erythropoietin (Epo) available. This model is elegant in that oxygen sensing within the kidney triggers Epo production so anemia can increase Epo and promote erythroid output. However, during homeostasis this model suggests that considerable energy is used to produce unneeded erythroid progenitors. We searched for independent control and compartmentalization of erythropoiesis that could couple early hematopoiesis to terminal erythroid commitment and maturation. Methods: We previously found the proportion of bone marrow megakaryocytes (MKs) staining for active, signaling-competent TGFβ transiently increases during bone marrow regeneration after chemotherapy. To assess the functional role of Mk-TGFβ, we crossed murine strains harboring a floxed allele of TGFβ1 (TGFβ1Flox/Flox) littermate with a Mk-specific Cre deleter to generate mice with Mk-specific deletion of TGFβ1 (TGFβ1ΔMk/ΔMk). We analyzed hematopoiesis of these mice using high-dimensional flow cytometry, confocal immunofluorescent microscopy and in vitro and in vivo assays of hematopoietic function (Colony forming assays, and in vivo transplantation). Results: Using validated, 9-color flow cytometry panels capable of quantifying hematopoietic stem cells (HSCs) and six other hematopoietic progenitor populations, we found that Mk-specific deletion of TGFβ1 leads to expansion of immature hematopoietic stem and progenitor cells (HSPCs) (Fig1A&B). Functional assays confirmed a more than three-fold increase in hematopoietic stem cells (HSCs) capable of serially-transplanting syngeneic recipients in the bone marrow (BM) of TGFβ1ΔMk/ΔMk mice compared to their TGFβ1Flox/Flox littermates. Expansion was associated with less quiescent (Go) HSCs implicating Mk-TGFβ in the control of HSC cell cycle entry. Similarly, in vitro colony forming cell assays and in vivo spleen colony forming assays confirmed expansion of functional progenitor cells in TGFβ1ΔMk/ΔMk mice. These results place Mk-TGFβ as a critical regulator of the size of the pool of immature HSPCs. We found that the blood counts and total BM cellularity of TGFβ1ΔMk/ΔMk mice was normal despite the dramatic expansion of immature HSPCs. Using a combination of confocal immunofluorescence microscopy (cleaved caspase 3) (Fig1C) and flow cytometry (Annexin V and cleaved caspase 3) (Fig1D), we found ~10-fold greater apoptosis of mature precursor cells in TGFβ1ΔMk/ΔMk BM and spleens. Coincident with this, we found the number of Epo receptor (EpoR) expressing erythroid precursors to be dramatically increased. Indeed, apoptosis of erythroid precursors peaked as they transitioned from dual positive Kit+EpoR+ precursors to single positive cells expressing EpoR alone. Epo levels were normal in the serum of these mice. We reasoned that the excess, unneeded EpoR+ cells were not supported physiologic Epo levels but might respond to even small doses of exogenous Epo. Indeed, we found that the excess erythroid apoptosis could be rescued by administration of very low doses of Epo (Fig1E). Whereas TGFβ1Flox/Flox mice showed minimal reticulocytosis and no change in blood counts, TGFβ1ΔMk/ΔMk mice responded with exuberant reticulocytosis and raised RBC counts almost 10% within 6 days (Fig. 1F). Low dose Epo also rescued survival of Epo receptor positive erythroid precursors in the bone marrow, spleen and blood of TGFβ1ΔMk/ΔMk mice. TGFβ1ΔMk/ΔMk mice showed a similarly brisk and robust erythropoietic response during recovery from phenylhydrazine-induced hemolysis (Fig.1G). Exogenous TGFβ worsened BM apoptosis and caused anemia in treated mice. Pre-treatment of wild-type mice with a TGFβ signaling inhibitor sensitized mice to low dose Epo. Conclusion: These results place megakaryocytic TGFβ1 as a gate-keeper that restricts the pool of immature HSPCs and couples immature hematopoiesis to the production of mature effector cells. This work promises new therapies for chronic anemias by combining TGFβ inhibitors to increase the outflow of immature progenitors with ESAs to support erythroid maturation. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Cloning and Functional Analysis of the Rhesus Macaque ABCG2 Gene: Forced Expression Confers an SP Phenotype among Hematopoietic Stem Cell Progeny in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3219-3219 ◽  
Author(s):  
Takahiro Ueda ◽  
Sebastian Brenner ◽  
Harry Malech ◽  
Saskia Langemeijer ◽  
Martha Kirby ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Rhesus Macaque ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Abcg2 Gene

Abstract Hematopoietic cells can be highly enriched for repopulating ability based upon efflux of the fluorescent Hoechst 33342 dye by sorting for side population (SP) cells, a phenotype attributed to expression of ABCG2, a member of the ABC transporter superfamily. Intriguingly, murine studies suggest that forced ABCG2 expression prevents hematopoietic differentiation. We sought to determine the effects of forced expression of the ABCG2 gene in hematopoietic stem cells in the nonhuman primate model, a model with proven relevance to human hematopoiesis. We cloned the full-length rhesus ABCG2 (rh-ABCG2) cDNA using a series of primers spanning the entire sequence designed using the published human sequence. Sequence homology was greater than 96%. The rh-ABCG2 gene was then introduced into an MFGS based retroviral vector pseudotyped with the RD114 envelope. Mobilized human peripheral blood CD34-positive cells were transduced with either rh-ABCG2 or human GP91-phox vector with no other payload. All transductions were initiated with 4 x10e5 cells using X-VIVO10/1%HSA/4mM/L L-glutamine supplemented with 100ng/ml_FLT3L, 100ng/ml SCF, 100ng/ml TPO and polybrene (5 ug/ml). RD114 vector was concentrated by ultracentrifugation (83,000g 90minutes 4°C). Gene transfer rates to CFU of greater than 80% were achieved using both vectors with similar gene transfer rate estimated by flow cytometry. ABCG2-transduced human peripheral blood progenitor cells (PBPCs) acquired the SP phenotype, but showed significantly reduced growth compared to control (Day 8: cell counts 7.67+/− 2.54 vs. 17.83+/−6.64 x10e5 for ABCG2 and GP91-phox transduced cells, respectively p=0.0024, n=5). We then examined the engraftment of ABCG2-expressing stem and progenitor cells in the rhesus macaque autologous transplant model. GCSF/SCF mobilized PBPCs were collected from 2 animals and the CD34+ cells were divided and transduced with either vector and infused after lethal irradiation. In vivo marking levels post transplant measured in mononuclear cells and granulocytes from peripheral blood and bone marrow ranged initially from 0.5–4% by Realtime PCR, declined equally over time, and were similar between transduced fractions, with no discrepancy between bone marrow and peripheral blood marking. Furthermore, peripheral blood T cells, B cells and granulocytes expressed ABCG2 at levels predicted by vector copy number long term, and the differential of such cells within the SP gate matched that of the non-SP fraction demonstrating no block to differentiation in the large animal. In vitro studies showed selective protection against mitoxantrone among ABCG2-transduced rhesus PBPCs. Our results confirm the existence of rhesus-ABCG2, support its importance in conferring the SP phenotype, suggest no detrimental effect of its overexpression upon hematopoiesis, and imply a potential role for its overexpression as an in vivo selection strategy for gene therapy applications.

Fluorescence-Based Selection of Gene-Corrected Hematopoietic Stem and Progenitor Cells From Acid Sphingomyelinase-Deficient Mice: Implications for Niemann-Pick Disease Gene Therapy and the Development of Improved Stem Cell Gene Transfer Procedures

Blood ◽  
1999 ◽  
Vol 93 (1) ◽  
pp. 80-86 ◽  
Author(s):  
Shai Erlich ◽  
Silvia R.P. Miranda ◽  
Jan W.M. Visser ◽  
Arie Dagan ◽  
Shimon Gatt ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Fetal Liver ◽  
Acid Sphingomyelinase ◽  
Hematopoietic Stem

The general utility of a novel, fluorescence-based procedure for assessing gene transfer and expression has been demonstrated using hematopoietic stem and progenitor cells. Lineage-depleted hematopoietic cells were isolated from the bone marrow or fetal livers of acid sphingomyelinase–deficient mice, and retrovirally transduced with amphotropic or ecotropic vectors encoding a normal acid sphingomyelinase (ASM) cDNA. Anti–c-Kit antibodies were then used to label stem- and progenitor-enriched cell populations, and the Bodipy fluorescence was analyzed in each group after incubation with a Bodipy-conjugated sphingomyelin. Only cells expressing the functional ASM (ie, transduced) could degrade the sphingomyelin, thereby reducing their Bodipy fluorescence as compared with nontransduced cells. The usefulness of this procedure for the in vitro assessment of gene transfer into hematopoietic stem cells was evaluated, as well as its ability to provide an enrichment of transduced stem cells in vivo. To show the value of this method for in vitro analysis, the effects of retroviral transduction using ecotropic versus amphotropic vectors, various growth factor combinations, and adult bone marrow versus fetal liver stem cells were assessed. The results of these studies confirmed the fact that ecotropic vectors were much more efficient at transducing murine stem cells than amphotropic vectors, and that among the three most commonly used growth factors (stem cell factor [SCF] and interleukins 3 and 6 [IL-3 and IL-6]), SCF had the most significant effect on the transduction of stem cells, whereas IL-6 had the most significant effect on progenitor cells. In addition, it was determined that fetal liver stem cells were only approximately twofold more “transducible” than stem cells from adult bone marrow. Transplantation of Bodipy-selected bone marrow cells into lethally irradiated mice showed that the number of spleen colony-forming units that were positive for the retroviral vector (as determined by polymerase chain reaction) was 76%, as compared with 32% in animals that were transplanted with cells that were nonselected. The methods described within this manuscript are particularly useful for evaluating hematopoietic stem cell gene transfer in vivo because the marker gene used in the procedure (ASM) encodes a naturally occurring mammalian enzyme that has no known adverse effects, and the fluorescent compound used for selection (Bodipy sphingomyelin) is removed from the cells before transplantation.

scholarly journals Hematopoietic stem and progenitor cells are present in healthy gingiva tissue

2021 ◽  
Vol 218 (4) ◽  
Author(s):  
Siddharth Krishnan ◽  
Kelly Wemyss ◽  
Ian E. Prise ◽  
Flora A. McClure ◽  
Conor O’Boyle ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Immune Responses ◽  
Progenitor Cells ◽  
Myeloid Cell ◽  
Extramedullary Hematopoiesis ◽  
Innate Immune ◽  
Innate Immune Cells ◽  
Hematopoietic Stem ◽  
Effector Cells ◽  
Stem And Progenitor Cells

Hematopoietic stem cells reside in the bone marrow, where they generate the effector cells that drive immune responses. However, in response to inflammation, some hematopoietic stem and progenitor cells (HSPCs) are recruited to tissue sites and undergo extramedullary hematopoiesis. Contrasting with this paradigm, here we show residence and differentiation of HSPCs in healthy gingiva, a key oral barrier in the absence of overt inflammation. We initially defined a population of gingiva monocytes that could be locally maintained; we subsequently identified not only monocyte progenitors but also diverse HSPCs within the gingiva that could give rise to multiple myeloid lineages. Gingiva HSPCs possessed similar differentiation potentials, reconstitution capabilities, and heterogeneity to bone marrow HSPCs. However, gingival HSPCs responded differently to inflammatory insults, responding to oral but not systemic inflammation. Combined, we highlight a novel pathway of myeloid cell development at a healthy barrier, defining a gingiva-specific HSPC network that supports generation of a proportion of the innate immune cells that police this barrier.

scholarly journals Dectin-1 Stimulation of Hematopoietic Stem and Progenitor Cells Occurs In Vivo and Promotes Differentiation Toward Trained Macrophages via an Indirect Cell-Autonomous Mechanism

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Cristina Bono ◽  
Alba Martínez ◽  
Javier Megías ◽  
Daniel Gozalbo ◽  
Alberto Yáñez ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Candida Albicans ◽  
Progenitor Cells ◽  
Recipient Mouse ◽  
Dependent Manner ◽  
Toll Like Receptor ◽  
Hematopoietic Stem ◽  
Content Type ◽  
Stem And Progenitor Cells

ABSTRACT Toll-like receptor (TLR) agonists drive hematopoietic stem and progenitor cells (HSPCs) to differentiate along the myeloid lineage. In this study, we used an HSPC transplantation model to investigate the possible direct interaction of β-glucan and its receptor (dectin-1) on HSPCs in vivo. Purified HSPCs from bone marrow of B6Ly5.1 mice (CD45.1 alloantigen) were transplanted into dectin-1−/− mice (CD45.2 alloantigen), which were then injected with β-glucan (depleted zymosan). As recipient mouse cells do not recognize the dectin-1 agonist injected, interference by soluble mediators secreted by recipient cells is negligible. Transplanted HSPCs differentiated into macrophages in response to depleted zymosan in the spleens and bone marrow of recipient mice. Functionally, macrophages derived from HSPCs exposed to depleted zymosan in vivo produced higher levels of inflammatory cytokines (tumor necrosis factor alpha [TNF-α] and interleukin 6 [IL-6]). These results demonstrate that trained immune responses, already described for monocytes and macrophages, also take place in HSPCs. Using a similar in vivo model of HSPC transplantation, we demonstrated that inactivated yeasts of Candida albicans induce differentiation of HSPCs through a dectin-1- and MyD88-dependent pathway. Soluble factors produced following exposure of HSPCs to dectin-1 agonists acted in a paracrine manner to induce myeloid differentiation and to influence the function of macrophages derived from dectin-1-unresponsive or β-glucan-unexposed HSPCs. Finally, we demonstrated that an in vitro transient exposure of HSPCs to live C. albicans cells, prior to differentiation, is sufficient to induce a trained phenotype of the macrophages they produce in a dectin-1- and Toll-like receptor 2 (TLR2)-dependent manner. IMPORTANCE Invasive candidiasis is an increasingly frequent cause of serious and often fatal infections. Understanding host defense is essential to design novel therapeutic strategies to boost immune protection against Candida albicans. In this article, we delve into two new concepts that have arisen over the last years: (i) the delivery of myelopoiesis-inducing signals by microbial components directly sensed by hematopoietic stem and progenitor cells (HSPCs) and (ii) the concept of “trained innate immunity” that may also apply to HSPCs. We demonstrate that dectin-1 ligation in vivo activates HSPCs and induces their differentiation to trained macrophages by a cell-autonomous indirect mechanism. This points to new mechanisms by which pathogen detection by HSPCs may modulate hematopoiesis in real time to generate myeloid cells better prepared to deal with the infection. Manipulation of this process may help to boost the innate immune response during candidiasis.

Phenotypic correction of Fanconi anemia group C knockout mice

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 700-704 ◽  
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.

STAT5 promotes multilineage hematolymphoid development in vivo through effects on early hematopoietic progenitor cells

Blood ◽  
2002 ◽  
Vol 99 (1) ◽  
pp. 95-101 ◽  
Author(s):  
Jonathan W. Snow ◽  
Ninan Abraham ◽  
Melissa C. Ma ◽  
Nancy W. Abbey ◽  
Brian Herndier ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Blood Cell ◽  
Progenitor Cells ◽  
Cell Types ◽  
Hematopoietic Stem ◽  
Signal Transducers ◽  
Adult Mice ◽  
Colony Forming Units ◽  
Proliferation Activity

The transcription factor signal transducers and activators of transcription 5 (STAT5) is activated by numerous cytokines that orchestrate blood cell development. Multilineage peripheral blood cytopenias were observed in adult mice lacking both isoforms of STAT5 (STAT5A and STAT5B) as well as accelerated rates of apoptosis in the bone marrow. Although the hematopoietic stem cell (HSC) population was preserved in a number of these mice, the post-HSC progenitor populations were diminished and a marked reduction in functional progenitors (spleen colony-forming units) was detected. Competitive bone marrow transplantation studies in vivo revealed a profound impairment of repopulation potential of STAT5-null HSCs, leading to complete lack of contribution to the myeloid, erythroid, and lymphoid lineages. These abnormalities were associated with heightened proliferation activity in the HSC fraction, suggesting the action of homeostatic mechanisms to maintain sufficient levels of diverse blood cell types for viability. Thus, STAT5 normally sustains the robust hematopoietic reserve that contributes to host viability through crucial survival effects on early progenitor cells.

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