scholarly journals Mild preconditioning and low-level engraftment confer methotrexate resistance in mice transplanted with marrow expressing drug-resistant dihydrofolate reductase activity

Blood ◽  
2000 ◽  
Vol 96 (4) ◽  
pp. 1334-1341
Author(s):  
Rohaizah I. James ◽  
Christopher A. Warlick ◽  
Miechaleen D. Diers ◽  
Roland Gunther ◽  
R. Scott McIvor
Keyword(s):  
Stem Cells ◽  
Dihydrofolate Reductase ◽  
Reductase Activity ◽  
Lethal Dose ◽  
Hematopoietic Cells ◽  
High Dose ◽  
Hematopoietic Stem ◽  
Low Level ◽  
Marrow Cells

Effective engraftment of hematopoietic cells targeted for gene transfer is facilitated by cytoreductive preconditioning such as high-dose total body irradiation (TBI). To minimize the adverse side effects associated with TBI, experiments were conducted to determine whether sublethal doses of TBI would allow sufficient engraftment of MTX-resistant hematopoietic cells to confer survival on recipient mice administered MTX. FVB/N animals were administered 1, 2, or 4 Gy TBI (lethal dose, 8.5 Gy), transplanted with 107 FVB/N transgenic marrow cells expressing an MTX-resistant dihydrofolate reductase (DHFR) transgene, and then administered MTX daily for 60 days. Control mice administered 1 Gy with or without subsequent transplantation of normal marrow cells succumbed to MTX toxicity by day 45. In contrast, nearly all animals transplanted with transgenic marrow survived MTX administration, regardless of the TBI dose used for preconditioning. The donor DHFR transgenic marrow engraftment level was proportional to the preconditioning dose of TBI but was surprisingly reduced in animals given 2 or 4 Gy TBI and subsequently administered MTX when compared with control animals administered phosphate-buffered saline. Animals preconditioned with 1 Gy were also protected from MTX toxicity when transplanted with reduced amounts (5 × 106 and 1 × 106 cells) of DHFR transgenic donor marrow, resulting in low-level (approximately 1%) engraftment. In conclusion, very mild preconditioning allows sufficient low-level engraftment of genetically modified stem cells for in vivo manifestation of the modified phenotype, suggesting the usefulness of mild preconditioning regimens in human gene therapy trials targeting hematopoietic stem cells.

scholarly journals Mild preconditioning and low-level engraftment confer methotrexate resistance in mice transplanted with marrow expressing drug-resistant dihydrofolate reductase activity

Blood ◽  
2000 ◽  
Vol 96 (4) ◽  
pp. 1334-1341 ◽  
Author(s):  
Rohaizah I. James ◽  
Christopher A. Warlick ◽  
Miechaleen D. Diers ◽  
Roland Gunther ◽  
R. Scott McIvor
Keyword(s):  
Stem Cells ◽  
Dihydrofolate Reductase ◽  
Reductase Activity ◽  
Lethal Dose ◽  
Hematopoietic Cells ◽  
High Dose ◽  
Hematopoietic Stem ◽  
Low Level ◽  
Marrow Cells

Abstract Effective engraftment of hematopoietic cells targeted for gene transfer is facilitated by cytoreductive preconditioning such as high-dose total body irradiation (TBI). To minimize the adverse side effects associated with TBI, experiments were conducted to determine whether sublethal doses of TBI would allow sufficient engraftment of MTX-resistant hematopoietic cells to confer survival on recipient mice administered MTX. FVB/N animals were administered 1, 2, or 4 Gy TBI (lethal dose, 8.5 Gy), transplanted with 107 FVB/N transgenic marrow cells expressing an MTX-resistant dihydrofolate reductase (DHFR) transgene, and then administered MTX daily for 60 days. Control mice administered 1 Gy with or without subsequent transplantation of normal marrow cells succumbed to MTX toxicity by day 45. In contrast, nearly all animals transplanted with transgenic marrow survived MTX administration, regardless of the TBI dose used for preconditioning. The donor DHFR transgenic marrow engraftment level was proportional to the preconditioning dose of TBI but was surprisingly reduced in animals given 2 or 4 Gy TBI and subsequently administered MTX when compared with control animals administered phosphate-buffered saline. Animals preconditioned with 1 Gy were also protected from MTX toxicity when transplanted with reduced amounts (5 × 106 and 1 × 106 cells) of DHFR transgenic donor marrow, resulting in low-level (approximately 1%) engraftment. In conclusion, very mild preconditioning allows sufficient low-level engraftment of genetically modified stem cells for in vivo manifestation of the modified phenotype, suggesting the usefulness of mild preconditioning regimens in human gene therapy trials targeting hematopoietic stem cells.

scholarly journals Development of a Novel Selective Amplifier Gene for Controllable Expansion of Transduced Hematopoietic Cells

Blood ◽  
1997 ◽  
Vol 90 (10) ◽  
pp. 3884-3892 ◽  
Author(s):  
Keiko Ito ◽  
Yasuji Ueda ◽  
Masaki Kokubun ◽  
Masashi Urabe ◽  
Toshiya Inaba ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Selective Amplifier ◽  
Marrow Cells

Abstract To overcome the low efficiency of gene transfer into hematopoietic cells, we developed a novel system for selective expansion of transduced cells. To this end, we constructed a chimeric cDNA (GCRER) encoding the fusion protein between the granulocyte colony-stimulating factor receptor (G-CSFR) and the hormone-binding domain (HBD) of the estrogen receptor (ER) as a selective amplifier gene. Use of the intracellular signaling pathway of G-CSFR was considered to be appropriate, because G-CSF has the ability not only to stimulate the neutrophil production, but also to expand the hematopoietic stem/progenitor cell pool in vivo. To activate the exogenous G-CSFR signal domain selectively, the estrogen/ER-HBD system was used as a molecular switch in this study. When the GCRER gene was expressed in the interleukin-3 (IL-3)–dependent murine cell line, Ba/F3, the cells showed IL-3–independent growth in response to G-CSF or estrogen. Moreover, the Ba/F3 cells transfected with the Δ(5-195)GCRER, whose product lacks the extracellular G-CSF–binding domain, did not respond to G-CSF, but retained the ability for estrogen-dependent growth. Further, murine bone marrow cells transduced with the GCRER or Δ(5-195)GCRER gene with retroviral vectors formed a significant number of colonies in response to estrogen, as well as G-CSF, whereas estrogen did not stimulate colony formation by untransduced murine bone marrow cells. It is noteworthy that erythroid colonies were apparently formed by the bone marrow cells transduced with the GCRER gene in the presence of estrogen without the addition of erythropoietin, suggesting that the signals from the G-CSFR portion of the chimeric molecules do not preferentially induce neutrophilic differentiation, but just promote the differentiation depending on the nature of the target cells. We speculate that when the selective amplifier genes are expressed in the primitive hematopoietic stem cells, the growth signal predominates and that the population of transduced stem cells expands upon estrogen treatment, even if some of the cells enter the differentiation pathway. The present study suggests that this strategy is applicable to the in vivo selective expansion of transduced hematopoietic stem cells.

scholarly journals Isolation in a single step of a highly enriched murine hematopoietic stem cell population with competitive long-term repopulating ability

Blood ◽  
1989 ◽  
Vol 74 (3) ◽  
pp. 930-939 ◽  
Author(s):  
SJ Szilvassy ◽  
PM Lansdorp ◽  
RK Humphries ◽  
AC Eaves ◽  
CJ Eaves
Keyword(s):  
Simple Procedure ◽  
Hematopoietic Cells ◽  
Stem Cell Population ◽  
Proliferative Potential ◽  
Vitro Assay ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract A simple procedure is described for the quantitation and enrichment of murine hematopoietic cells with the capacity for long-term repopulation of lymphoid and myeloid tissues in lethally irradiated mice. To ensure detection of the most primitive marrow cells with this potential, we used a competitive assay in which female recipients were injected with male “test” cells and 1 to 2 x 10(5) “compromised” female marrow cells with normal short-term repopulating ability, but whose long-term repopulating ability had been reduced by serial transplantation. Primitive hematopoietic cells were purified by flow cytometry and sorting based on their forward and orthogonal light-scattering properties, and Thy-1 and H-2K antigen expression. Enrichment profiles for normal marrow, and marrow of mice injected with 5-fluorouracil (5- FU) four days previously, were established for each of these parameters using an in vitro assay for high proliferative potential, pluripotent colony-forming cells. When all four parameters were gated simultaneously, these clonogenic cells were enriched 100-fold. Both day 9 and day 12 CFU-S were copurified; however, the purity (23%) and enrichment (75-fold) of day 12 CFU-S in the sorted population was greater with 5-FU-treated cells. Five hundred of the sorted 5-FU marrow cells consistently repopulated recipient lymphoid and myeloid tissues (greater than 50% male, 1 to 3 months post-transplant) when co-injected with 1 to 2 x 10(5) compromised female marrow cells, and approximately 100 were sufficient to achieve the same result in 50% of recipients under the same conditions. This relatively simple purification and assay strategy should facilitate further analysis of the heterogeneity and regulation of stem cells that maintain hematopoiesis in vivo.

scholarly journals Short- and Long-Term Multilineage Repopulating Hematopoietic Stem Cells in Late Fetal and Newborn Mice: Models for Human Umbilical Cord Blood

Blood ◽  
1997 ◽  
Vol 90 (1) ◽  
pp. 174-181 ◽  
Author(s):  
David E. Harrison ◽  
Clinton M. Astle
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Umbilical Cord Blood ◽  
Functional Abilities ◽  
Newborn Mice ◽  
Hematopoietic Stem ◽  
Short And Long Term ◽  
Marrow Cells

Abstract Blood from late fetal and newborn mice is similar to umbilical cord blood obtained at birth in human beings, an important source of stem cells for clinical transplantation. The mouse model is useful because long-term functions can be readily assayed in vivo. To evaluate the functions of hematopoietic precursors in the blood and other tissues of late fetal and newborn mice, short- and long-term multilineage repopulating abilities were measured in vivo by competitive repopulation. Manipulations that might affect cell function, such as enrichment, tissue culture, or retroviral marking, were avoided. Hematopoietic stem cell functions of late fetal or newborn blood, liver, and spleen, were assayed as myeloid and lymphoid repopulating abilities relative to standard adult marrow cells. Donor cells from these tissues as well as adult control donor marrow cells were all of the same genotype. Cells from each donor tissue were mixed with portions from a pool of standard adult “competitor” marrow distinguished from the donors by genetic differences in hemoglobin and glucosephosphate isomerase. After 21 to 413 days, percentages of donor type myeloid and lymphoid cells in recipient blood were measured to assay the functional abilities of donor precursors relative to the standard. These relative measures are expressed as repopulating units, where each unit is equivalent to the repopulating ability found in 100,000 standard adult marrow cells. Thus, measures of repopulating units do not compare single cells but overall repopulating abilities of donor cell populations. Relative functional abilities in 1 million nucleated cells from late fetal or newborn blood were several times less than those found in adult marrow, but far more than in normal adult blood, and appeared to include long-term functional primitive hematopoietic stem cells (PHSC) similar to those in marrow. To estimate functional abilities of individual PHSC, variances among large groups of identical recipients were analyzed using both the binomial model and competitive dilution, a new model based on the Poisson distribution. The data best fit the hypothesis that individual PHSC from adult marrow, late fetal blood, or newborn blood each produce similar fractions of the total lymphoid and erythroid cells found in the recipient for many months.

scholarly journals Negative Regulation of the Differentiation of Flk2− CD34− LSK Hematopoietic Stem Cells by EKLF/KLF1

2020 ◽  
Vol 21 (22) ◽  
pp. 8448
Author(s):  
Chun-Hao Hung ◽  
Keh-Yang Wang ◽  
Yae-Huei Liou ◽  
Jing-Ping Wang ◽  
Anna Yu-Szu Huang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Gene Knockout ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Interesting Correlation ◽  
Regulatory Effects ◽  
High Level

Erythroid Krüppel-like factor (EKLF/KLF1) was identified initially as a critical erythroid-specific transcription factor and was later found to be also expressed in other types of hematopoietic cells, including megakaryocytes and several progenitors. In this study, we have examined the regulatory effects of EKLF on hematopoiesis by comparative analysis of E14.5 fetal livers from wild-type and Eklf gene knockout (KO) mouse embryos. Depletion of EKLF expression greatly changes the populations of different types of hematopoietic cells, including, unexpectedly, the long-term hematopoietic stem cells Flk2− CD34− Lin− Sca1+ c-Kit+ (LSK)-HSC. In an interesting correlation, Eklf is expressed at a relatively high level in multipotent progenitor (MPP). Furthermore, EKLF appears to repress the expression of the colony-stimulating factor 2 receptor β subunit (CSF2RB). As a result, Flk2− CD34− LSK-HSC gains increased differentiation capability upon depletion of EKLF, as demonstrated by the methylcellulose colony formation assay and by serial transplantation experiments in vivo. Together, these data demonstrate the regulation of hematopoiesis in vertebrates by EKLF through its negative regulatory effects on the differentiation of the hematopoietic stem and progenitor cells, including Flk2− CD34− LSK-HSCs.

Enforced Expression of the GATA-2 Transcription Factor Blocks Normal Hematopoiesis

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 488-499 ◽  
Author(s):  
Derek A. Persons ◽  
James A. Allay ◽  
Esther R. Allay ◽  
Richard A. Ashmun ◽  
Donald Orlic ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Blood Cell ◽  
Fluorescent Protein ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Immunoblot Analysis ◽  
Marrow Cells

Abstract The zinc finger transcription factor GATA-2 is highly expressed in immature hematopoietic cells and declines with blood cell maturation. To investigate its role in normal adult hematopoiesis, a bicistronic retroviral vector encoding GATA-2 and the green fluorescent protein (GFP) was used to maintain the high levels of GATA-2 that are normally present in primitive hematopoietic cells. Coexpression of the GFP marker facilitated identification and quantitation of vector-expressing cells. Bone marrow cells transduced with the GATA-2 vector expressed GFP as judged by flow cytometry and GATA-2 as assessed by immunoblot analysis. A 50% to 80% reduction in hematopoietic progenitor-derived colony formation was observed with GATA-2/GFP-transduced marrow, compared with marrow transduced with a GFP-containing vector lacking the GATA-2 cDNA. Culture of purified populations of GATA-2/GFP-expressing and nonexpressing cells confirmed a specific ablation of the colony-forming ability of GATA-2/GFP-expressing progenitor cells. Similarly, loss of spleen colony-forming ability was observed for GATA-2/GFP-expressing bone marrow cells. Despite enforced GATA-2 expression, marrow cells remained viable and were negative in assays to evaluate apoptosis. Although efficient transduction of primitive Sca-1+Lin- cells was observed with the GATA-2/GFP vector, GATA-2/GFP-expressing stem cells failed to substantially contribute to the multilineage hematopoietic reconstitution of transplanted mice. Additionally, mice transplanted with purified, GATA-2/GFP-expressing cells showed post-transplant cytopenias and decreased numbers of total and gene-modified bone marrow Sca-1+ Lin−cells. Although Sca-1+ Lin− bone marrow cells expressing the GATA-2/GFP vector were detected after transplantation, no appreciable expansion in their numbers occurred. In contrast, control GFP-expressing Sca-1+Lin− cells expanded at least 40-fold after transplantation. Thus, enforced expression of GATA-2 in pluripotent hematopoietic cells blocked both their amplification and differentiation. There appears to be a critical dose-dependent effect of GATA-2 on blood cell differentiation in that downregulation of GATA-2 expression is necessary for stem cells to contribute to hematopoiesis in vivo.

Exhaustion of Donor Hematopoietic Stem Cells in Lethally-Irradiated Hosts Can Be Sbstantially Mitigated by Targeting p18INK4C.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1200-1200
Author(s):  
Hui Yu ◽  
Youzhong Yuan ◽  
Xianmin Song ◽  
Feng Xu ◽  
Hongmei Shen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Kinase Inhibitor ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Total Period ◽  
Over Expression

Abstract Hematopoietic stem cells (HSCs) are significantly restricted in their ability to regenerate themselves in the irradiated hosts and this exhausting effect appears to be accelerated in the absence of the cyclin-dependent kinase inhibitor (CKI), p21. Our recent study demonstrated that unlike p21 absence, deletion of the distinct CKI, p18 results in a strikingly positive effect on long-term engraftment owing to increased self-renewing divisions in vivo (Yuan et al, 2004). To test the extent to which enhanced self-renewal in the absence of p18 can persist over a prolonged period of time, we first performed the classical serial bone marrow transfer (sBMT). The activities of hematopoietic cells from p18−/− cell transplanted mice were significantly higher than those from p18+/+ cell transplanted mice during the serial transplantation. To our expectation, there was no detectable donor p18+/+ HSC progeny in the majority (4/6) of recipients after three rounds of sBMT. However, we observed significant engraftment levels (66.7% on average) of p18-null progeny in all recipients (7/7) within a total period of 22 months. In addition, in follow-up with our previous study involving the use of competitive bone marrow transplantation (cBMT), we found that p18−/− HSCs during the 3rd cycle of cBMT in an extended long-term period of 30 months were still comparable to the freshly isolated p18+/+ cells from 8 week-old young mice. Based on these two independent assays and the widely-held assumption of 1-10/105 HSC frequency in normal unmanipulated marrow, we estimated that p18−/− HSCs had more than 50–500 times more regenerative potential than p18+/+ HSCs, at the cellular age that is equal to a mouse life span. Interestingly, p18 absence was able to significantly loosen the accelerated exhaustion of hematopoietic repopulation caused by p21 deficiency as examined in the p18/p21 double mutant cells with the cBMT model. This data directly indicates the opposite effect of these two molecules on HSC durability. To define whether p18 absence may override the regulatory mechanisms that maintain the HSC pool size within the normal range, we performed the transplantation with 80 highly purified HSCs (CD34-KLS) and then determined how many competitive reconstitution units (CRUs) were regenerated in the primary recipients by conducting secondary transplantation with limiting dilution analysis. While 14 times more CRUs were regenerated in the primary recipients transplanted with p18−/−HSCs than those transplanted with p18+/+ HSCs, the level was not beyond that found in normal non-transplanted mice. Therefore, the expansion of HSCs in the absence of p18 is still subject to some inhibitory regulation, perhaps exerted by the HSC niches in vivo. Such a result was similar to the effect of over-expression of the transcription factor, HoxB4 in hematopoietic cells. However, to our surprise, the p18 mRNA level was not significantly altered by over-expression of HoxB4 in Lin-Sca-1+ cells as assessed by real time PCR (n=4), thereby suggesting a HoxB4-independent transcriptional regulation on p18 in HSCs. Taken together, our current results shed light on strategies aimed at sustaining the durability of therapeutically transplanted HSCs for a lifetime treatment. It also offers a rationale for the feasibility study intended to temporarily target p18 during the early engraftment for therapeutic purposes.

Telomere Length of Hematopoietic Cells in Long-Term Post-Autograft Survivors: No Evidence of Accelerated Replicative Cell Senescence Despite the Persistent Reduction of Both Committed and Immature Progenitor Cell Compartments.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4123-4123
Author(s):  
Alberto Rocci ◽  
Irene Ricca ◽  
Chiara Della Casa ◽  
Paolo Longoni ◽  
Mara Compagno ◽  
...  
Keyword(s):  
Stem Cells ◽  
Telomere Length ◽  
Progenitor Cell ◽  
Hematopoietic Cells ◽  
High Dose ◽  
Hematopoietic Stem ◽  
Replicative Stress

Abstract Telomere length is considered a valuable replicative capacity predictor of human hematopoietic stem cells. Indeed, a progressive telomere shortening affects hematopoietic cells upon in vitro expansion. However, less is known on the dynamics of telomere shortening in vivo following a non-physiological replicative stress. Aim of this study was to investigate markers for cellular senescence of hematopoietic cells exposed to replicative stress induced by bone marrow reconstitution following stem cell autograft. Thus, both telomere length and in vitro functional characteristics of bone marrow (BM) and peripheral blood (PB) were evaluated at long-term in subjects who had received intensive chemotherapy and autograft. Thirty-two adults with a previous diagnosis of lymphoma were examined, at a median time of 73 months (range 42–125) since autograft. They all had received a high-dose sequential chemotherapy treatment followed by peripheral blood progenitor cell (PBPC) autograft. There were 20 male and 12 female (median age at autograft: 40 yrs., range 21–60). A Southern blot procedure using a chemiluminescence-based assay was employed to determine telomere length on samples from grafted PBPC as well as on BM and PB samples obtained at long-term during follow-up. These latter samples were also studied for their in vitro growth characteristics, assessed by short and long-term culture assays. In all cases, autograft had been performed with large quantities of hematopoietic stem cells (median autografted CD34+ve cells/kg: 9.8 x 106, range 2–24), allowing a rapid and stable hematologic reconstitution. Telomere length was found slightly shorter in BM mononuclear cells from samples taken at follow-up compared to samples from grafted material (median telomere length: 6,895 bp vs 7,073 bp, respectively; p=ns). No marked differences were observed in telomere evaluation between BM and PB cells. No significant differences were observed as well when PB telomere length of follow-up samples was compared with telomere length of PB from age-related normal subjects. BM and PB samples were then assessed for their in vitro growth characteristics. Committed and stromal progenitors were grown from all samples in good though variable quantities. However, as compared to normal controls, a statistically significant reduction of marrow-derived hematopoietic progenitors (CFU-GM - BFU-E - CFU-Mix) as well as stromal progenitors (CFU-F) was observed. Additionally, the more immature LTC-IC progenitor cell compartment was dramatically reduced, both in BM and PB samples. The results indicate that: i. the proliferative stress induced by intensive chemotherapy and post-graft hematopoietic reconstitution does not imply marked telomere loss in BM and PB cells at long-term, provided that large quantities of PBPC are used for autograft; ii. stem cells present in the graft or surviving after high-dose therapy are capable of reconstituting a sufficiently adequate hematopoiesis although the committed progenitor cell compartment and even more the immature LTC-IC progenitors are persistently reduced even at up to 10 years since autograft.

Poly I:C Stimulates Sca-1 Expression in Murine Bone Marrow and Increases the Number of Phenotypic Hematopoietic Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2504-2504
Author(s):  
Russell Garrett ◽  
Gerd Bungartz ◽  
Alevtina Domashenko ◽  
Stephen G. Emerson
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Hematopoietic Cells ◽  
Poly I:C ◽  
Hematopoietic Stem ◽  
Marrow Cells ◽  
Myeloid Progenitors

Abstract Abstract 2504 Poster Board II-481 Polyinosinic:polycytidlyic acid (poly I:C) is a synthetic double-stranded RNA used to mimic viral infections in order to study immune responses and to activate gene deletion in lox-p systems employing a Cre gene responsive to an Mx-1 promoter. Recent observations made by us and others have suggested hematopoietic stem cells, responding to either poly I:C administration or interferon directly, enter cell cycle. Twenty-two hours following a single 100mg intraperitoneal injection of poly I:C into 10-12 week old male C57Bl/6 mice, the mice were injected with a single pulse of BrdU. Two hours later, bone marrow was harvested from legs and stained for Lineage, Sca-1, ckit, CD48, IL7R, and BrdU. In two independent experiments, each with n = 4, 41 and 33% of Lin- Sca-1+ cKit+ (LSK) IL-7R- CD48- cells from poly I:C-treated mice had incorporated BrdU, compared to 7 and 10% in cells from PBS-treated mice. These data support recently published reports. Total bone marrow cellularity was reduced to 45 and 57% in the two experiments, indicating either a rapid death and/or mobilization of marrow cells. Despite this dramatic loss of hematopoietic cells from the bone marrow of poly I:C treated mice, the number of IL-7R- CD48- LSK cells increased 145 and 308% in the two independent experiments. Importantly, the level of Sca-1 expression increased dramatically in the bone marrow of poly I:C-treated mice. Both the percent of Sca-1+ cells and the expression level of Sca-1 on a per cell basis increased after twenty-four hours of poly I:C, with some cells acquiring levels of Sca-1 that are missing from control bone marrow. These data were duplicated in vitro. When total marrow cells were cultured overnight in media containing either PBS or 25mg/mL poly I:C, percent of Sca-1+ cells increased from 23.6 to 43.7%. Within the Sca-1+ fraction of poly I:C-treated cultures, 16.7% had acquired very high levels of Sca-1, compared to only 1.75% in control cultures. Quantitative RT-PCR was employed to measure a greater than 2-fold increase in the amount of Sca-1 mRNA in poly I:C-treated cultures. Whereas the numbers of LSK cells increased in vivo, CD150+/− CD48- IL-7R- Lin- Sca-1- cKit+ myeloid progenitors almost completely disappeared following poly I:C treatment, dropping to 18.59% of control marrow, a reduction that is disproportionately large compared to the overall loss of hematopoietic cells in the marrow. These cells are normally proliferative, with 77.1 and 70.53% accumulating BrdU during the 2-hour pulse in PBS and poly I:C-treated mice, respectively. Interestingly, when Sca-1 is excluded from the analysis, the percent of Lin- IL7R- CD48- cKit+ cells incorporating BrdU decreases following poly I:C treatment, in keeping with interferon's published role as a cell cycle repressor. One possible interpretation of these data is that the increased proliferation of LSK cells noted by us and others is actually the result of Sca-1 acquisition by normally proliferating Sca-1- myeloid progenitors. This new hypothesis is currently being investigated. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Loss of expression of the Hoxa-9 homeobox gene impairs the proliferation and repopulating ability of hematopoietic stem cells

Blood ◽  
2005 ◽  
Vol 106 (12) ◽  
pp. 3988-3994 ◽  
Author(s):  
H. Jeffrey Lawrence ◽  
Julie Christensen ◽  
Stephen Fong ◽  
Yu-Long Hu ◽  
Irving Weissman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cell Function ◽  
Bone Marrow Cells ◽  
Homeobox Gene ◽  
Normal Numbers ◽  
Hematopoietic Stem ◽  
Fold Reduction ◽  
Marrow Cells

The homeobox gene Hoxa-9 is normally expressed in primitive bone marrow cells, and overexpression of Hoxa-9 markedly expands hematopoietic stem cells, suggesting a function in early hematopoiesis. We present evidence for major functional defects in Hoxa-9-/- hematopoietic stem cells. Hoxa-9-/- marrow cells have normal numbers of immunophenotypic stem cells (Lin-c-kit+flk-2-Sca-1+ [KLFS] cells). However, sublethally irradiated Hoxa-9-/- mice develop persistent pancytopenia, indicating unusual sensitivity to ionizing irradiation. In competitive transplantation assays, Hoxa-9-/- cells showed an 8-fold reduction in multilineage long-term repopulating ability, a defect not seen in marrow cells deficient for the adjacent Hoxa-10 gene. Single-cell cultures of KLFS cells showed a 4-fold reduction in large high-proliferation potential colonies. In liquid cultures, Hoxa-9-deficient Lin-Sca-1+ cells showed slowed proliferation (a 5-fold reduction in cell numbers at day 8) and delayed emergence of committed progenitors (a 5-fold decrease in colony-forming cells). Slowing of proliferation was accompanied by a delay in myeloid maturation, with a decrease in Gr-1hiMac-1hi cells at the end of the culture. Retroviral transduction with a Hoxa-9 expression vector dramatically enhanced the cytokine-driven proliferation and in vivo engraftment of Hoxa-9-/- marrow cells. Hoxa-9 appears to be specifically required for normal hematopoietic stem cell function both in vitro and in vivo.

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