Bone Marrow Stem Cell Populations Research Articles

Apolipoprotein A1 deficiency in mice primes bone marrow stem cells for T cell lymphopoiesis.

ABSTRACTThe bone marrow has emerged as a potentially important target in cardiovascular disease as it generates all leukocytes involved in atherogenesis. In the current study, we evaluated whether a change in bone marrow functionality underlies the increased atherosclerosis susceptibility associated with high-density lipoprotein (HDL) deficiency. We found that HDL deficiency in mice due to the genetic lack of hepatocyte-derived apolipoprotein A1 (APOA1) was associated with an increase in the Lin−Sca-1+Kit+ (LSK) bone marrow stem cell population and lymphoid-primed multipotent progenitor numbers, which translated into a higher production and systemic flux of T cell subsets. In accordance with APOA1 deficiency-associated priming of stem cells to increase T lymphocyte production, atherogenic diet-fed low-density lipoprotein receptor knockout mice transplanted with bone marrow from APOA1-knockout mice displayed marked lymphocytosis as compared to wild-type bone marrow recipients. However, atherosclerotic lesion sizes and collagen contents were similar in the two groups of bone marrow recipients. In conclusion, systemic lack of APOA1 primes bone marrow stem cells for T cell lymphopoiesis. Our data provide novel evidence for a regulatory role of HDL in bone marrow functioning in normolipidemic mice.

Model for Studying the Effects of Chronic Metabolic Disease on Endogenous Bone Marrow Stem Cell Populations.

Disease-associated impairment/dysfunction of stem cell populations is prominent in chronic metabolic and inflammatory diseases, such as type 2 diabetes mellitus (DM) where the multifunctional properties (viability, proliferation, paracrine secretion, multilineage differentiation) of bone marrow resident mesenchymal stem cells (MSCs) can be affected. The growth and viability impairments make it difficult to study the underlying molecular mechanisms related to the dysfunction of these cells in vitro. We have consequently optimized the isolation and culture conditions for impaired/dysfunctional bone marrow MSCs from B6.Cg-Lepob/J obese prediabetic mice. The method described here permits ex vivo investigations into disease-associated functional impairments and the dysregulated molecular mechanisms in these primary MSCs through direct comparisons with their healthy wild-type C57BL6/J control mouse counterparts.

Carnosine Supplementation Mitigates the Deleterious Effects of Particulate Matter Exposure in Mice.

BackgroundExposure to fine airborne particulate matter (PM 2.5) induces quantitative and qualitative defects in bone marrow–derived endothelial progenitor cells of mice, and similar outcomes in humans may contribute to vascular dysfunction and the cardiovascular morbidity and mortality associated with PM 2.5 exposure. Nevertheless, mechanisms underlying the pervasive effects of PM 2.5 are unclear and effective interventional strategies to mitigate against PM 2.5 toxicity are lacking. Furthermore, whether PM 2.5 exposure affects other types of bone marrow stem cells leading to additional hematological or immunological dysfunction is not clear.Methods and ResultsMice given normal drinking water or that supplemented with carnosine, a naturally occurring, nucleophilic di‐peptide that binds reactive aldehydes, were exposed to filtered air or concentrated ambient particles. Mice drinking normal water and exposed to concentrated ambient particles demonstrated a depletion of bone marrow hematopoietic stem cells but no change in mesenchymal stem cells. However, HSC depletion was significantly attenuated when the mice were placed on drinking water containing carnosine. Carnosine supplementation also increased the levels of carnosine‐propanal conjugates in the urine of CAPs–exposed mice and prevented the concentrated ambient particles–induced dysfunction of endothelial progenitor cells as assessed by in vitro and in vivo assays.ConclusionsThese results suggest that exposure to PM 2.5 has pervasive effects on different bone marrow stem cell populations and that PM 2.5‐induced hematopoietic stem cells depletion, endothelial progenitor cell dysfunction, and defects in vascular repair can be mitigated by excess carnosine. Carnosine supplementation may be a viable approach for preventing PM 2.5‐induced immune dysfunction and cardiovascular injury in humans.

Microgrooved-surface topography enhances cellular division and proliferation of mouse bone marrow-derived mesenchymal stem cells.

Mesenchymal stem cells’ (MSCs) fate is largely determined by the various topographical features and a range of extracellular matrix (ECM) components present in their niches. Apart from maintaining structural stability, they regulate cell morphology, division, proliferation, migration and differentiation among others. Traditional MSC cultures, which are mainly based on two-dimensional smooth surfaces of culture dishes and plates, do not provide topographical cues similar to in vivo three-dimensional niches, impacting various cellular processes. Therefore, we culture the mouse bone marrow-derived MSCs on microgrooved bearing surface, partially mimicking in vivo reticulated niche, to study its effect on morphology, pluripotency factor-associated stemness, cell division and rate of proliferation. Following culture, morphological features, and MSC-specific marker gene expression, such as CD29, CD44, Sca-1 along with HSC (Haematopoietic stem cell)-specific markers like CD34, CD45, CD11b were evaluated by microscopy and immunophenotyping, respectively. HSC is another type of bone marrow stem cell population, which concertedly interacts with MSC during various functions, including haematopoiesis. In addition, mesenchymal stem cells were further analyzed for gene expression of pluripotency-associated transcription factors such as Oct3/4, Sox-2, Nanog and Myc, as well as differentiated into adipocytes, osteocytes and chondrocytes. Our results show that microgrooved surface-cultured mesenchymal stem cells (MMSCs) expressed higher levels of expected cell surface and pluripotency-associated markers and proliferated more rapidly (2–3×fold) with higher percentage of cells in S/G2-M-phase, consequently giving rise to higher cell yield compared to standard culture flask-grown cells (MSCs), taken as control. Furthermore, both MSCs and MMSCs showed considerable accumulation of intracellular lipid-droplets, higher alkaline phosphatase activity and secretion of extracellular matrix that are characteristics of adipogenesis, osteogenesis and chondrogenesis, respectively.

Mutant U2AF1 Expression Alters Hematopoiesis In Transgenic Mice

Mutations in 8 spliceosome genes have been identified in up to half of patients with myelodysplastic syndromes (MDS), suggesting that perturbations in pre-mRNA splicing play a role in MDS pathogenesis. Our group and others discovered recurrent heterozygous mutations in U2AF1, a gene encoding a splicing factor involved in intronic 3'-splice site recognition, in 11% of MDS patients. The most common U2AF1 mutation causes a serine to phenylalanine substitution at amino acid 34 (S34F). To study the effects of U2AF1(S34F) expression on hematopoiesis, we created site-specific, single-copy, doxycycline-inducible (controlled by the reverse tetracycline-transactivator [rtTA] transgene) U2AF1(WT) and U2AF1(S34F) transgenic mice. To examine the cell-autonomous effects of mutant U2AF1(S34F), we transplanted transgenic donor bone marrow into wild type mouse recipients prior to doxycycline induction of transgene expression.We observed a doxycycline dose-dependent increase in expression of U2AF1(S34F) and U2AF1(WT) transgenes by RT-PCR and pyrosequencing analysis, and we subsequently chose a dose that induces equivalent levels of exogenous U2AF1 transgene to endogenous mouse U2af1 transcripts. Following 4 weeks of doxycycline induction of transgene expression in vivo, U2AF1(S34F)-recipient mouse bone marrow cells showed increased alternative splicing of endogenous Fmr1 pre-mRNA, a transcript previously shown to be alternatively-spliced in U2AF1 mutant MDS patient samples, when compared to U2AF1(WT)- and rtTA only-recipient mouse bone marrow cells (p<0.02, n=9-11). This effect was seen as early as 5 days (p<0.001, n=3-7).Following 4 weeks of transgene induction, U2AF1(S34F)-recipient mice display a decrease in WBCs in the peripheral blood compared to U2AF1(WT)- and rtTA only-recipient controls (average 4.5 vs 6.7 and 7.1 K/µl, respectively; p≤0.01). However, there are no significant differences in bone marrow cellularity or spleen size in these mice (n=9-11). U2AF1(S34F)-recipient mice have reduced frequency of peripheral blood monocytes (average 1.2 vs 3.2 and 2.6%, p<0.05), as well as bone marrow monocytes (3.9 vs 7 and 6.1%, p<0.001) and B cells (average 10.1 vs 20.7 and 20.6%, p<0.001) when compared to U2AF1(WT)- and rtTA only-recipient mice, respectively (n=9-11). Moreover, bone marrow monocytes in U2AF1(S34F)-recipient mice have an increased frequency of Annexin V+ staining compared to controls (average 11.2 vs 6.9 and 7.5%, p<0.05, n=5-6), indicating that loss of these cells may be due to apoptosis. U2AF1(S34F)-recipient mice also have an increase in the frequency of bone marrow neutrophils compared to U2AF1(WT)- and rtTA only-recipient mice (average 65 vs 53 and 50%, respectively, p<0.001, n=9-11). Taken together, these data indicate U2AF1(S34F) expression alters hematopoietic lineage distribution in vivo.In addition, 4 weeks of U2AF1(S34F) expression results in alterations of progenitor and stem cell frequency. U2AF1(S34F)-recipient mice have approximately a 1.5-fold increase in the frequency of progenitor cells in their bone marrow measured by flow cytometry (linlow/c-kit+/Sca-1-) (average 4.1 vs 2.6 and 2.9%; p<0.001, n=5-6) and methylcellulose progenitor colony forming assays (average 85 vs 55 and 59 colonies/plate; p<0.02, n=9-11) when compared to U2AF1(WT)- and rtTA only-recipient mice, respectively. U2AF1(S34F)-recipient mice also display a 1.5-fold increase in the frequency of common myeloid progenitors compared to U2AF1(WT) and rtTA only controls in both bone marrow and spleen (p≤0.02), along with a 2-fold increase of the granulocyte-macrophage progenitors in the spleen (p≤0.03) (n=5-6). In addition to changes in progenitors, U2AF1(S34F)-recipient mice have a 1.4-fold increase in the frequency of bone marrow KLS cells, short-term HSCs, and dormant long-term HSCs (p≤0.05), as well as a 2-fold increase in spleen dormant long-term HSCs (p≤0.03) compared to U2AF1(WT)- and rtTA only-recipient controls (n=5-6). Competitive repopulation studies are ongoing.Collectively, these data indicate that mutant U2AF1(S34F) expression in mouse bone marrow cells results in altered pre-mRNA splicing and changes in hematopoietic cell lineage distribution, as well as increased bone marrow and spleen progenitor and stem cell populations. Furthermore, these results suggest that mutant U2AF1 may contribute to altered hematopoiesis in patients. Disclosures:No relevant conflicts of interest to declare.

Usefulness of Peripheral Blood Circulating DNAs As an Alternative to DNA From Bone Marrow Cells to Detect CpG Global Methylation Status and Genetic Mutations in Patients with Myelodysplastic Syndromes

Abstract 3854 Background:Several genetic/epigenetic abnormalities are associated with the pathogenesis of myelodysplastic syndromes (MDS). DNA methyltransferase inhibitors (DNMTi), azacitidine and decitabine, have recently come to be considered as standard therapeutics for patients with MDS. However, biomarkers that predict the effectiveness of DNMTi before and/or during treatment are still lacking. Although bone marrow (BM) aspiration is a common strategy to obtain MDS cells for evaluating their genetic/epigenetic status, repeated sampling is difficult because of pain and safety concerns. Therefore, alternatives are required. One possibility is to use circulating cell-free DNA in the plasma and serum of peripheral blood (PB); a technique previously used for the detection of genetic/epigenetic abnormalities in solid tumor cells. Aims:Assess the quality of PB circulating DNA from patients with MDS, and investigate its usefulness for analyzing serial genetic/epigenetic changes following treatment. Methods:DNA from BM, PB mononuclear cells (MNC), plasma and serum were obtained repeatedly from MDS patients and visualized via agarose gel electrophoresis. Bisulfite conversion of genomic or circulating DNA was performed and then pyrosequencing performed for the 4 CpG sites of long interspersed nuclear elements-1 (LINE-1) elements to detect global methylation. Bisulfite pyrosequencing for the 5 CpG sites of p15INK4B gene promoter was also performed. Genetic mutations were screened firstly by single nuclear polymorphism (SNP) array analysis and confirmed by DNA sequencing for the specific gene mutations. BM cells were sorted into CD34(+)/CD38(-), CD34(+)/CD38(+), and CD34(-) subpopulations, and the percentage of mutated DNA confirmed by pyrosequencing analysis. Results and Discussion:Circulating DNA from both healthy volunteer donors and MDS patients showed a ladder pattern of DNA fragments 160∼180 base pairs apart, suggesting their accumulation from mono-/di-nucleosomes. The plasma DNA concentration was relatively higher in patients with higher BM blast cell counts. Plasma DNA concentration had been changed even in the same patients according to the disease status. CpG methylation status of the LINE-1 promoter after treatment with azacitidine (days 1 to 28) was analyzed by pyrosequencing. Although the methylation status of 1 patient did not show any significant change, other 2 patients showed a serial decrease of the methylation percentage confirmed at days 3, 6, and 9 using DNA from plasma, serum, and PBMNC. Plasma DNA showed more rapid and significant changes at days 3 (p<0.001) and 6 (p<0.05) compared with serum DNA (not significant (N.S.) at day 3 and 6) and PBMNC (N.S. at day 3 and p<0.05 at day 6). These data suggest that circulating DNA from plasma can be used for analysis of the LINE-1 promoter as a measure of global methylation as an alternative strategy to using MNC in PB and/or BM. The changing ratio is different among patients and it may reflect the efficacy of DNMTi or the amount of MDS clones. p15INK4B gene promoter CpG methylation status was analyzed using BM cells, PBMNC, and circulating DNA. The methylation percentage was not significantly different among whole BM cells and 3 BM subpopulations, and plasma and serum DNA showed similar methylation pattern as whole BM DNA. Next, PB circulating DNA was utilized for the detection of genetic mutations in MDS cells. TET2 mutation (Y1245Term; TAC to TAG) was confirmed in a patient showing chromosome 4q uni-parental disomy (UPD), and the existence ratio of the mutation was significantly higher in plasma and serum DNA than in CD34(-) BM cells (p<0.05 and p<0.01, respectively) and almost equivalent to that in the CD34(+)/38(-) BM stem cell population. The high percentage of mutated genes in circulating DNA may result from abnormal DNA being released from fragile MDS clones, enriching the circulating DNA compared with DNA from BM or PBMNC but further molecular analyses and higher patient numbers are required to confirm this. Conclusions:PB circulating DNA can be reliably and sensitively used to detect epigenetic changes on genomic DNA after treatment with DNMTi. Genetic mutations in MDS clones can also be detected sensitively, at a level almost equivalent to that in BM CD34+/38- stem cells. These data suggest that genetic/epigenetic analyses using PB circulating DNA can be a better, less painful and safer alternative to using BM aspiration. Disclosures:No relevant conflicts of interest to declare.

Using peripheral blood circulating DNAs to detect CpG global methylation status and genetic mutations in patients with myelodysplastic syndrome

Myelodysplastic syndrome (MDS) is a hematopoietic stem cell disorder. Several genetic/epigenetic abnormalities are deeply associated with the pathogenesis of MDS. Although bone marrow (BM) aspiration is a common strategy to obtain MDS cells for evaluating their genetic/epigenetic abnormalities, BM aspiration is difficult to perform repeatedly to obtain serial samples because of pain and safety concerns. Here, we report that circulating cell-free DNAs from plasma and serum of patients with MDS can be used to detect genetic/epigenetic abnormalities. The plasma DNA concentration was found to be relatively high in patients with higher blast cell counts in BM, and accumulation of DNA fragments from mono-/di-nucleosomes was confirmed. Using serial peripheral blood (PB) samples from patients treated with hypomethylating agents, global methylation analysis using bisulfite pyrosequencing was performed at the specific CpG sites of the LINE-1 promoter. The results confirmed a decrease of the methylation percentage after treatment with azacitidine (days 3–9) using DNAs from plasma, serum, and PB mono-nuclear cells (PBMNC). Plasma DNA tends to show more rapid change at days 3 and 6 compared with serum DNA and PBMNC. Furthermore, the TET2 gene mutation in DNAs from plasma, serum, and BM cells was quantitated by pyrosequencing analysis. The existence ratio of mutated genes in plasma and serum DNA showed almost equivalent level with that in the CD34+/38- stem cell population in BM. These data suggest that genetic/epigenetic analyses using PB circulating DNA can be a safer and painless alternative to using BM cells.

IGF-1Ea induces vessel formation after injury and mediates bone marrow and heart cross-talk through the expression of specific cytokines

The aim of this study was to investigate whether supplemental IGF-1Ea transgene expression induces activation of local cardiac and bone marrow stem cell population to mediate mammalian heart repair. In physiologic conditions, cardiac overexpression of the IGF-1Ea propeptide is associated with an enrichment of c-Kit/Sca-1 positive side population cells in the bone marrow and the occurrence of an endothelial-primed CD34 positive side population in the heart. This cellular profile is shown here to correlate with the expression of cytokines involved in stem cell mobilization and vessel formation. This molecular and cellular interplay favored IGF-1Ea-mediated vessel formation in injured hearts. The physiologic and pathologic connection between cytokines and stem cells in response to IGF-1Ea may represent an important model to understand how to elicit endogenous reparative signaling.

Substance P stimulates the recovery of bone marrow after the irradiation

The therapeutic use of ionizing radiation (e.g., X-rays and γ-rays) needs to inflict minimal damage on non-target tissue. Recent studies have shown that substance P (SP) mediates multiple activities in various cell types, including cell proliferation, anti-apoptotic responses, and inflammatory processes. The present study investigated the effects of SP on γ-irradiated bone marrow stem cells (BMSCs). In mouse bone marrow extracts, SP prolonged activation of Erk1/2 and enhanced Bcl-2 expression, but attenuated the activation of apoptotic molecules (e.g., p38 and cleaved caspase-3) and down-regulated Bax. We also observed that SP-decreased apoptotic cell death and stimulated cell proliferation in γ-irradiated mouse bone marrow tissues through TUNEL assay and PCNA analysis. To determine how SP affects bone marrow stem cell populations, mouse bone marrow cells were isolated and colony-forming unit (CFU) of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) was estimated. SP-pretreated ones showed higher CFUs of MSC and HSC than untreated ones. Furthermore, when SP was pretreated in cultured human MSC, it significantly decreased apoptotic cells at 48 and 72 h after γ-irradiation. Compared with untreated cells, SP-treated human MSCs showed reduced cleavage of apoptotic molecules such as caspase-8, -9, -3, and poly ADP-ribose polymerase (PARP). Thus, our results suggest that SP alleviates γ-radiation-induced damage to mouse BMSCs and human MSCs via regulation of the apoptotic pathway.

Rapidly mobilizable stem cells. Do they belong to a special subpopulation?

Some characteristics of rapidly mobilized stem cells as a possible distinct subset of the murine bone marrow stem cell population are overviewed. Some of the agents that rapidly mobilize stem cells are toxic and possibly act through disrupting anchorage to the microenvironment. The mobilization occurring days after cytostatics and/or colony-stimulating factors (CSF), however, is a consequence of increased production or differentiation. While stem cells (colony-forming units-spleen; CFU-S) circulating normally in blood have low self-renewal capacity (SRC), SRC of rapidly mobilized CFU-S is closer to that of bone marrow stem cells and is similar to that of the late mobilized stem cells. The survival rate of mice after transplantation of rapidly mobilized stem cells did not differ from that of bone marrow stem cells. One year after transplantation of rapidly mobilized stem cells, the SRC value of bone marrow did not differ from those transplanted with bone marrow cells. Replacement of a rapidly mobilizable stem cell pool requires 48 h under physiological conditions and a longer time after damage to hemopoiesis (irradiation, hydroxyurea injection). Possible physiological mechanisms in the anchorage of stem cells are discussed.

Mobilization of Bone Marrow Stem Cell Populations as a Result of Unopposed Estrogen: A Mechanism for Estrogen-Mediated Angiogenesis

We hypothesize that the mechanism by which estrogen promotes tumor progression is more complex than stimulation of the estrogen receptor (ER) on tumor cells that express the receptor. ERα knockout mice have been shown to be deficient in angiogenesis. Recent data have shown that bone marrow derived elements including stromal cells and endothelial cells are increased in the tumor microenvironment during states of unopposed estrogen. We hypothesize that mesenchymal stem cells, as the “stromal stem cells,” are the targets of estrogen-mediated angiogenesis.

Adult Human Blood Leukocytes as an Efficient Source for Tissue-Committed Neural Progenitors.

In the last few years, significant progress has been made in the isolation and characterization of bone marrow stem cell populations and their potential to differentiate into a variety of cellular lineages. We hypothesized that peripheral blood can also be used as a source for precursor cells that can become committed progenitors for a variety of tissues. We report here the generation and characterization in vitro of neural progenitor cells from a newly discovered blood-derived multipotent cell population, named synergetic cell population (SCP). Human blood samples were obtained from the Israeli blood bank and SCP cells were purified based on cellular density. Neural progenitors were generated by culturing SCP cells in medium supplemented with autologous serum, followed by activation in a defined serum-free medium containing the specific differentiation-inducing factors F12, B27, bFGF, BDNF, and NGF.An average of 13.5x106 neural progenitor cells was generated from 450 ml blood. These cells developed irregular perikarya, from which filamentous extensions spread contacting neighboring cells and forming net-like structures. Immunostaining revealed that some of the cells express the early neuronal progenitor markers nestin and b-tubulin and Neu-N, a nuclear protein present in mature neurons. Other cells expressed glial-specific antigens, such as O4 (a marker of oligodendrocytes) and GFAP (a marker of astrocytes). Flow cytometry analysis showed that 44.4% and 34% of the cells were positive for nestin and b-tubulin, respectively. In addition to exhibiting phenotypic evidence of markers specific for the neural lineage, these progenitor cells also responded to the neurotransmitters glutamate and GABA, as detected by calcium influx through voltage-gated calcium channels, demonstrating functional differentiation.In this study we show that generation of neural progenitors from peripheral blood is feasible and efficient. Blood-derived angiogenic progenitors produced in our system are already safely and efficiently administrated to severe angina pectoris patients in a clinical trial we are conducting in Thailand (reported in a separate abstract by our group). The newly discovered source of these progenitors, the blood-derived multipotent population which we termed SCP, contains both hematopoietic stem cells as well as supportive cells that enable differentiation into various lineages. The therapeutic potential of these neural progenitors will be further characterized and evaluated in vivo using animal models.

CREB Transgenic Mice Develop Myeloproliferative Disease/Myelodysplastic Syndrome after a Prolonged Latency.

The cyclic AMP Response Element Binding Protein, CREB, is a basic leucine zipper transcription factor that induces genes that regulate cell proliferation and survival. CREB is a downstream target of GM-CSF signaling pathways in myeloid cells, resulting in transactivation of critical target genes. We previously demonstrated that both CREB protein and mRNA levels are increased in the bone marrow cells of patients with AML at diagnosis. In contrast, CREB is expressed at low levels in normal bone marrow cells. To examine the expression of CREB in specific bone marrow and leukemia stem cell populations, we performed quantitative real-time PCR. AML blast cells were sorted and were shown to have increased CREB mRNA expression in CD34+CD33+ and CD34-CD33+ population compared to normal bone marrow progenitor cells. To understand the role of CREB in normal hematopoiesis and leukemogenesis, we created transgenic mice in which the myeloid specific hMRP8 promoter controls CREB expression. Within months, these mice developed increased monocytes and neutrophils in the peripheral blood. The increased cells in the peripheral blood and bone marrow were Gr1+Mac1+. We did not observe increases in other cell lineages using FACS analysis and the markers, B220, CD3, and Ter119. Bone marrow cells from CREB transgenic mice displayed increased colony size, greater numbers of colonies, and immortalization in colony replating assays with methylcellulose containing IL-3, IL-6, and Stem Cell Factor, compared to age-matched littermate controls. Bone marrow from CREB transgenic mice grew in the absence of cytokines, demonstrating factor-independent growth. Increased numbers of CFU-M was observed with CREB transgenic mouse bone marrow in colony assays with methylcellulose containing M-CSF. Although the mice did not develop acute leukemia, 7 out of 8 CREB transgenic mice compared to 0 out of 13 control mice developed enlarged spleens and myeloproliferative disease (MPD) after 12 months of age. Histology of the spleens showed destruction of the normal architecture with aberrant myeloid cells, suggestive of a myeloproliferative disease/myelodysplastic syndrome. Both the indolent course and phenotype of our mice were similar to Chronic Myelomonocytic Leukemia (CMML) observed in humans. Interestingly, myeloid cells from patients with CML in chronic phase also express higher levels of CREB. Studies are underway to characterize CREB expression in peripheral blood or bone marrow cells from patients with MPD/MDS, including CML, CMML, and transient myeloproliferative disease of Down Syndrome. We are also analyzing the spleens from CREB transgenic mice that develop MPD/MDS. Our results demonstrate that overexpression of CREB is sufficient to induce myeloid cell transformation to a preleukemic state in vivo. Therefore, the CREB transgenic mouse provides a useful model to test novel therapies to treat MPD/MDS.

1104-191 Nicotine administration significantly alters peripheral, bone marrow and splenic stem cell populations in C57/B16 mice

1104-191 Nicotine administration significantly alters peripheral, bone marrow and splenic stem cell populations in C57/B16 mice

Antibody W7C5 defines a CD109 epitope expressed on CD34+ and CD34- hematopoietic and mesenchymal stem cell subsets.

The cell surface antigen recognized by monoclonal antibody W7C5 is expressed at low levels on human CD34(+) and CD34(-) bone marrow stem cell populations but at high levels on fetal liver CD34(+) cells. To identify the recognized antigen, we performed partial amino acid sequence and MALDI analysis of purified W7C5 antigens. The analyses revealed that MoAb W7C5 detects CD109, a recently cloned glycophosphatidylinositol-linked glycoprotein. The specificity of MoAb W7C5 was further confirmed by the selective recognition of CHO cells transfected with human CD109 cDNA.

Efficient oncoretroviral transduction of extended long-term culture-initiating cells and NOD/SCID repopulating cells: enhanced reconstitution with gene-marked cells through an ex vivo expansion approach.

Recent developments of surrogate assays for human hematopoietic stem cells (HSC) have facilitated efforts at improving HSC gene transfer efficiency. Through the use of xenograft transplantation models, such as nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, successful oncoretroviral gene transfer to transplantable hematopoietic cells has been achieved. However, because of the low frequency and/or homing efficiency of SCID repopulating cells (SRC) in bone marrow (BM), studies have primarily focused on cord blood (CB). The recently developed extended (> 60 days) long-term culture-initiating cell (ELTC-IC) assay detects an infrequent and highly quiescent candidate stem cell population in BM as well as CB of the CD34(+)CD38(-) phenotype. Although these characteristics suggest that ELTC-IC and SRC might be closely related, attempts to oncoretrovirally transduce ELTC-IC have been unsuccessful. Here, recently developed conditions (high concentrations of SCF + FL + Tpo in serum-free medium) supporting expansion of BM CD34(+)CD38(-) 12 week ELTC-IC promoted efficient oncoretroviral transduction of BM and CB ELTC-IC. Although SRC can be transduced with oncoretroviral vectors, this is frequently associated with loss of reconstituting activity, posing a problem for development of clinical HSC gene therapy. However, previous attempts at expanding transduced HSC posttransduction resulted in compromised rather than improved gene marking. Utilizing conditions promoting cell divisions and transduction of ELTC-IC we show that although 5 days of ex vivo culture is sufficient to obtain maximum gene transfer efficiency to SRC, extension of the expansion period to 12 days significantly enhances multilineage reconstitution activity of transduced SRC, supporting the feasibility of improving gene marking through ex vivo expansion.

ABC transporters as phenotypic markers and functional regulators of stem cells.

Characterization of molecules with tightly controlled expression patterns during differentiation represents an approach to understanding regulation of hematopoietic stem cell commitment. The multidrug resistance-1 (MDR1) gene product, P-glycoprotein, and the breast cancer resistance protein (BCRP) are expressed differentially during hematopoiesis, with the highest levels in primitive bone marrow stem cell populations that are CD34(low) and CD34(-), respectively. Roles for ATP-binding cassette (ABC) transporter superfamily members in conferring drug resistance have been extensively described. However, recent hematopoietic overexpression studies have begun to reveal previously unknown roles for ABC transporter function in normal and malignant hematopoiesis. Expression of MDR1 and BCRP transporters in the myeloid lineage has been reported in blasts from acute myeloid leukemia, but very low to undetectable in normal myelomonocytic cells. Retroviral-mediated dysregulated expression of the MDR1 transporter resulted in increased hematopoietic repopulating activity and myeloproliferative disease in mice. A distinct functional role for the BCRP transporter as a negative regulator of hematopoietic repopulating activity has recently been demonstrated using the same approach. Additionally, the presence of BCRP expression specifically on hematopoietic side-population stem cells and neural stem/progenitors, makes BCRP an attractive candidate marker for isolation of stem cells with the ability to respond to diverse environmental cues. Regulation of stem cell biology by ABC transporters has emerged as an important new field of investigation. In light of these findings, it will be critical to further characterize this family of proteins in hematopoietic lineage-restricted stem cells and in pluripotent stem cells capable of crossing lineage barriers.

Analysis of Hematopoietic Stem and Progenitor Cell Populations in Cytomegalovirus-Infected Mice

We have studied the effects of murine cytomegalovirus (MCMV) infection on bone marrow stem and progenitor cell populations to find an explanation for the defects in hematopoiesis that accompany CMV infections in patients. Sublethal MCMV infection of BALB/c mice resulted in a 5- to 10-fold decrease in the numbers of myeloid (colony-forming unit-granulocyte-macrophage [CFU-GM]) and erythroid (burst-forming unit-erythroid [BFU-E]) progenitor cells in the marrow, but not in primitive myeloerythroid progenitor cell (colony-forming unit-spleen [CFU-S]) numbers. In contrast, we observed a 10- to 20-fold reduction in CFU-S as well as CFU-GM and BFU-E in lethally infected mice. Depletion of marrow CFU-GM was less severe in C57BL/10 and C3H/HeJ mice, which are more resistant to the effects of MCMV infection. Treatment of bone marrow cells with MCMV preparations in vitro did not reduce the numbers of CFU-GM, although up to 10% of the cells were productively infected. This finding suggests that CFU-GM were not susceptible to lytic MCMV infection in vitro and are probably not eliminated by lytic infection in vivo. Increases in the frequencies of Sca-1+Lin- marrow cells, a population that includes cells with the characteristics of pluripotential stem cells, were observed in MCMV-infected BALB/c, C57BL/10, and DBA/2J mice. Increases in the frequencies of c-kit+Lin- marrow cells were only seen in DBA/2J mice. MCMV infection did not impair the function of pluripotential stem cells because transplantation of marrow from MCMV-infected donors into irradiated recipient mice resulted in successful reconstitution of the T, B, and myeloid cell lineages.

Proliferation inhibition of murine pluripotent haemopoietic stem cells by interferon or poly I:C.

The effect of mouse serum interferon (IF) in vitro and an inducer in vivo on the proliferation of a pluripotent stem cell population with high turnover rate was studied. Proliferation rate was characterized by the number of CFUs in the S phase of the cell cycle. Increased proliferation of bone marrow stem cell populations was produced either by irradiating the donor mice with 3.36 Gy (336 rad) 60Co-gamma rays 7 days before the experiment or by incubating normal bone marrow cells with 10(-11) M concentration of isoproterenol. IF considerably reduced the number of CFUs in S phase in both cases without reducing the CFUs content of the samples. Injection of IF inducer (4 mg/kg poly I:C) into regenerating mice also inhibited the proliferation of CFUs without decreasing the femoral CFUs level. Regeneration kinetics of CFUs from irradiated poly I:C-treated mice ran parallel with that of irradiated untreated animals but showed a characteristic delay corresponding to approximately one CFUs doubling. A transient, non-cytotoxic proliferation inhibitory effect of IF or IF inducer is, therefore, proposed.

Some Properties of the Circulating Hemopoietic Stem Cells

Abstract Some properties of hemopoietic stem cells (CFU) circulating in the peripheral blood of the normal mouse (PCFU) were studied. Those parameters were chosen that are well-known characteristics of bone marrow stem cell populations. Leukocytes separated from the peripheral blood were the source of PCFU. The radiosensitivity (D0 value), growth curve, seeding efficiency (f number), and the turnover state (number of PCFU in S phase measured by 3H-thymidine killing technique in vitro) of PCFU were compared to the same parameters for bone marrow CFU. Several characteristic changes in these parameters were found for PCFU: (1) Lower radiosensitivity (D0 = 140 R), (2) Higher seeding efficiency (f number 20.9%), (3) Higher sensitivity to 3H-thymidine in vitro (killing effect 32%). The doubling time was the same both for CFU and PCFU (21 hr), but the logarithmic growth of the PCFU population started by approximately 36 hr later than that of the marrow-derived CFU population. All these findings support the idea that PCFU represent a subpopulation of the heterogeneous bone marrow CFU population.