Clonal Marking of Mouse Hematopoietic Stem and Lymphoid Progenitor Cell Populations.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5544-5544
Author(s):  
Mariluz P. Mojica-Henshaw ◽  
L. Jeanne Pierce ◽  
John D. Phillips ◽  
Vicente Planelles ◽  
Gerald J. Spangrude
Keyword(s):  
Bone Marrow ◽  
Transgene Expression ◽  
Progenitor Cell ◽  
Bone Marrow Cells ◽  
Random Sequence ◽  
Transduction Efficiency ◽  
Cell Populations ◽  
Lentivirus Vector ◽  
Cmv Promoter ◽  
Hematopoietic Stem

Abstract We have developed a method to clonally mark hematopoietic stem and lymphoid progenitor cell populations using a novel sequence tag approach. A library containing an 11-base random sequence tag is cloned into a lentivirus vector, packaged using the VSV-G glycoprotein and HIV-1 capsid, and transduced into freshly isolated mouse hematopoietic stem cell (Thy-1.1lowc-kithigh) or progenitor cell (Thy-1.1negc-kithigh) populations. To minimize artifacts introduced by prolonged culture, we have utilized a 3-hour spinoculation protocol performed in the absence of cytokines. Transduction efficiency was evaluated in vitro by methylcellulose colony assay and liquid cultures, and in vivo by transplanting the transduced cells into lethally irradiated mice. A bicistronic lentivirus vector with a CMV promoter driving expression of a transcript encoding Thy-1.2-IRES-GFP was used to optimize the transduction protocol. Liquid culture assays demonstrated 57% transduction efficiency after 5 days of growth, based on expression of the Thy-1.2 and GFP reporter proteins. Mice transplanted with transduced Thy-1.1negc-kithigh progenitor cells were sacrificed after 16 days, a time at which we have previously observed robust progenitor cell engraftment in the thymus while progeny of Thy-1.1lowc-kithigh HSC have not yet appeared. In 4 of 4 transplanted mice, we observed donor-derived cells in the bone marrow, lymph nodes and thymus. The percentage of total cells expressing the lentivirus-derived transgene ranged from 1.6% of bone marrow cells to 20% of thymocytes. Peripheral blood from mice transplanted with transduced HSC were analyzed and monitored every 4 weeks for transgene expression. We observed that although the Thy-1.2 marker was expressed and maintained up to 14 weeks after HSC transplant, GFP transgene expression was minimal. Based on these preliminary results, we have engineered a new lentivirus vector containing random sequence tags and the Thy-1.2 marker. This strategy provides a simple and efficient way of tracking the progeny of individual cells within a transplanted population, using PCR amplification of the random tags found within mature cell populations derived from the transduced cells. Sequence analysis of individual clones derived from different lineages of cells will enable us to better define the lineage potentials of specific progenitor cell subpopulations.

Kruppel Like Factor 7 Suppresses Hematopoietic Stem and Progenitor Cell Function

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

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

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

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1293-1293
Author(s):  
Hong Qian ◽  
Sten Eirik W. Jacobsen ◽  
Marja Ekblom
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Functional Blocking

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

Efficient Viral Transduction of Murine Hematopoietic Stem Cells Using HIV-1 Gag-Pol Cross Packaged Simian Immunodeficiency Viral Backbone.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5484-5484
Author(s):  
Yuan Lin ◽  
Stanton L. Gerson
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transgene Expression ◽  
Bone Marrow Cells ◽  
Lentiviral Vector ◽  
Bioluminescent Imaging ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Hiv 1 ◽  
Marrow Cells

Abstract Lentiviral vectors have been shown to infect non-dividing cells, including hematopoietic stem cell [HSC], and HIV lentiviral vector has been studied extensively in preclinical models. However low HIV lentiviral vector transduction efficiency compared to retroviral vectors, is seen in murine HSC, hampering transplantation and long-term expression of transgene in the recipients. Furthermore, concerns remain regarding the safety of HIV based vectors. Simian Immunodeficiency Viral [SIV] vectors could be safer since the parent virus does not cause disease in humans. However, to model this approach has been difficult because native SIV vectors do not transduce murine cells. We have generated a bicistronic SIV lentiviral SIN vector, containing MGMT and firefly luciferase genes linked by a self-cleavage FMDV 2A sequence. The SIV backbone was kindly provided by Dr. Donald Kohn (University of Southern California). The transgenes are controlled by the MND promoter, which has been shown to express well in murine hematopoietic stem cells. The vector was generated by cross-packaging SIV RNA with HIV-1 ΔR8.91 packaging plasmid and VSVG pseudotyped envelope (Ref. Retrovirology2005, 2:55). Unconcentrated viruses had an average titer of 1E+06 iu/ml, which was similar to HIV-1 lentiviral vectors. In vitro, HIV-1 cross-packaged SIV-mnd-MGMT-2A-Luc vector was able to transduce both human and murine cell lines with no reduction of expression for 10 weeks. In addition, this cross-packaged SIV vector was also able to transduce primary murine bone marrow cells from Balb/C mice with low MOI of 0.5 to 1. Transduced primary murine bone marrow cells maintained transgene expression during a 4 week culture. To analyze in vivo expression, Balb/C bone marrow cells were transduced for 48 hrs in cytokines with the HIV-1 packaged SIV vector and transplanted into irradiated recipients. We used bioluminescent imaging (BLI) to monitor the transgene expression and the dynamic engraftment of transduced murine bone marrow cells. At MOI of 0.5 or 5, transduction efficiencies in murine progenitor cells were 24.4% and 46.7% respectively by PCR of transgene from CFU colonies. Bioluminescent imaging indicated similar engraftment patterns of transduced bone marrow cells by HIV-1 lentiviral vector or cross-packaged SIV lentiviral vector, as early as day 5. Consistent BLI signals indicated sustained expression of transgene in SIV vector transduced bone marrow cells beyond 30 days. With this study, cross-packaged SIV SIN vector could be used as a potential gene transfer vector in both preclinical murine studies and perhaps in clinical trials.

scholarly journals Correlation of Expression of CD25 in Hematopoietic Stem/Progenitor Cell Fraction of Bone Marrow Cells with Response to Tyrosine Kinase Inhibitors in Chronic Myelogenous Leukemia Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3061-3061 ◽  
Author(s):  
Daiki Karigane ◽  
Hidenori Kasahara ◽  
Masatoshi Sakurai ◽  
Eri Matsuki ◽  
Keiichi Tozawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Quantitative Pcr ◽  
Research Funding ◽  
Progenitor Cell ◽  
Kinase Inhibitors ◽  
Bone Marrow Cells ◽  
Myelogenous Leukemia ◽  
Hematopoietic Stem ◽  
Copy Numbers ◽  
Tki Discontinuation

Abstract Introduction: Tyrosine kinase inhibitors (TKIs) have dramatically improved the prognosis of chronic myelogenous leukemia (CML). The treatment with TKIs continues to improve the depth of response and overall survival of CML patients, but the life-long use of TKI is known to be associated with late complications such as cardiovascular events as well as heavy financial burden, and thus impairs the quality of life. To overcome these issues, many studies evaluating the possibility of TKI discontinuation have been ongoing worldwide. In order to achieve durable treatment-free remission, it is crucial to understand the dynamics of CML-leukemia initiating cells (LICs). We previously reported that CD25 was highly expressed in murine and human CML-LICs (Kobayashi CI et al., Blood, 2014). The aim of this study was to assess whether the proportion of CD25 positive cells in hematopoietic stem/progenitor cell fraction of bone marrow cells in CML patients treated with TKIs is associated with their molecular response and could serve as a novel surrogate marker to stop TKI therapy. Methods: Bone marrow samples were obtained from patients with CML in chronic phase who were diagnosed and have been treated solely with TKIs at Keio University Hospital (Tokyo, Japan). This study was approved by the institutional ethical committee and informed consent was obtained from each patient. Both quantitative and qualitative PCR of BCR-ABL was performed using bone marrow mononuclear cells (BMMNCs). The proportion of CD25 positive cells in bone marrow hematopoietic stem/progenitor cell (HSPC; CD34+CD38-) fraction was evaluated by flow cytometry using FITC-labeled anti-CD34, PE- labeled anti-CD38 and APC-labeled anti-CD25 antibodies. The response to TKIs at the time of evaluation was determined according to the previous report (Yoshida C et al., Int J Clin Oncol, 2012): complete cytogenetic remission (CCyR) defined as Philadelphia chromosome undetectable and quantitative PCR copy numbers >731 among BMMNCs; major molecular remission (MMR) as quantitative PCR copy numbers ≤731, and complete molecular remission (CMR) as undetectable BCR-ABL by quantitative and qualitative PCR. Results: Bone marrow samples obtained from 95 patients were evaluated (median age 53 years old; male/female, 67/28). Analysis was performed prior to TKI exposure in nine patients and under TKI therapy including 2nd generation TKI in 64 patients (imatinib, 22; dasatinib, 33; nilotinib, 9). Remaining 22 patients were treatment free because they enrolled in a clinical trial of TKI discontinuation. The proportion of CD25 positive cells in HSPC fraction significantly decreased in patients with prior TKI exposure relative to patients at diagnosis (n=86; Mean 4.2%, SD 7.0% vs n=9; Mean 22.4%, SD 11.3%, P<0.01). In addition, the proportion of CD25 positive cells in HSPC fraction significantly correlated the level of quantitative PCR (Figure, P<0.0001), and MMR was also dividable from CMR by the proportion of CD25 positive cells (MMR; n=29, Mean 4.6%, SD 4.8% vs CMR; n=49, Mean 2.6%, SD 2.5%, P<0.05). The expression of CD25 was still detected in the majority of patients who achieved CMR, including those who sustained CMR after the discontinuation of TKIs. Conclusion: We confirmed that the expression of CD25 in HSPC fraction of CML patients was significantly correlated with the response to TKI therapy, and may serve as an asset to select patients who are likely to achieve durable treatment-free survival. Figure Figure. Disclosures Karigane: Celgene: Honoraria. Sakurai:Celgene: Honoraria. Matsuki:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Nippon Shinyaku: Honoraria. Kikuchi:Celgene: Honoraria; Takeda Pharmaceutical Company: Honoraria; Kyowa Hakko Kirin: Honoraria. Mitsuhashi:LSI Medience: Consultancy. Okamoto:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Asahi Kasei Pharma Corp.: Research Funding; Astellas Pharma Inc.: Research Funding; Shionogi & Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Alexion Pharmaceuticals, Inc.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Teijin Pharma Limited: Research Funding; Bristol-Myers Squibb K.K.: Honoraria, Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Honoraria, Research Funding; JCR Pharmaceuticals Co., Ltd.: Research Funding.

scholarly journals Genetic deletion of Cdc42GAP reveals a role of Cdc42 in erythropoiesis and hematopoietic stem/progenitor cell survival, adhesion, and engraftment

Blood ◽  
2006 ◽  
Vol 107 (1) ◽  
pp. 98-105 ◽  
Author(s):  
Lei Wang ◽  
Linda Yang ◽  
Marie-Dominique Filippi ◽  
David A. Williams ◽  
Yi Zheng
Keyword(s):  
Bone Marrow ◽  
Progenitor Cell ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Negative Regulator ◽  
Null Alleles ◽  
Hematopoietic Stem ◽  
Cell Functions ◽  
Rho Family

Abstract Rho family GTPases are key signal transducers in cell regulation. Although a body of literature has implicated the Rho family members Rac1 and Rac2 in multiple hematopoietic-cell functions, the role of Cdc42 in hematopoiesis remains unclear. Here we have examined the hematopoietic properties and the hematopoietic stem/progenitor cell (HSP) functions of gene-targeted mice carrying null alleles of cdc42gap, a negative regulator of Cdc42. The Cdc42GAP-/- fetal liver and bone marrow cells showed a 3-fold increase in Cdc42 activity but normal Rac and RhoA activities, indicating that Cdc42GAP knockout resulted in a gain of Cdc42 activity in the hematopoietic tissues. Cdc42GAP-/- mice were anemic. The cellularity of fetal liver and bone marrow, the number and composition percentage of HSPs, and the erythroid blast-forming unit and colony-forming unit (BFU-E/CFU-E) activities were significantly reduced in the homozygous mice. The decrease in HSP number was associated with increased apoptosis of the Cdc42GAP-/- HSPs and the activation of JNK-mediated apoptotic machinery. Moreover, homozygous HSPs showed impaired cortical F-actin assembly, deficiency in adhesion and migration, and defective engraftment. These results provide evidence that Cdc42 activity is important for erythropoiesis and for multiple HSP functions, including survival, adhesion, and engraftment.

High Level Expression of a Membrane-Anchored Inhibitor of HIV Infection in Murine Hematopoietic Stem and Progenitor Cells Does Not Pertubate Serial Multilineage Repopulation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1758-1758
Author(s):  
Axel Schambach ◽  
Bernhard Schiedlmeier ◽  
Jens Bohne ◽  
Dorothee von Laer ◽  
Geoff Margison ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transgene Expression ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Hiv 1 ◽  
Marrow Cells

Abstract T20 is a 36-amino-acid peptide that binds to HIV-1 gp41 and thereby acts as a fusion inhibitor, thus mediating potent and selective inhibition of HIV-1 entry in vitro and in vivo. An extended peptide expressed as an artificial, membrane-bound molecule (mbC46) efficiently inhibits HIV infection of primary human T-cells following retroviral vector mediated gene transfer (Egelhofer et al., J Virol, 2004). To develop an even more stringent approach to HIV gene therapy, we targeted hematopoietic stem cells. In 3 experimental groups of C57BL/6 mice (9 animals/group), we investigated the long-term toxicity of murine bone marrow cells transduced with M87o, a therapeutic vector designed to coexpress mbC46 and an HIV-derived RNA RRE-decoy to inhibit HIV replication. As controls we used the same vector containing an inactive C46 peptide and mock-transduced cells. Blood samples were collected monthly. Donor chimerism and transgene expression in multiple lineages were determined by FACS analysis and transgene integration was measured by real time PCR. Six months after transplantation, 4 mice per group were sacrificed and the remaining 5 mice per group were observed for another 6 months. In addition to the parameters mentioned above, we performed complete histopathology, blood counts and clinical biochemistry. Donor chimerism in all groups ranged from 82 – 94% (day 190 and day 349). In the M87o group, 60% of donor cells expressed mbC46. FACS data showed persisting transgene expression in T-cells (CD4, CD8, 65%), B-cells (B220, 46%), myeloid cells (CD11b, 68%), platelets (CD41, 19%), and RBC (60%) of the peripheral blood and bone marrow cells. Highly sustained gene marking (2–4 copies/genome) was noticed on day 190. To reveal latent malignant clones potentially originating from side effects of the genetic manipulation, 1x106 bone marrow cells from 4 primary recipients were transplanted into lethally irradiated secondary recipients (3 recipients/primary mouse) and these mice were observed for 8 months. All together, we could not observe any evidence for leukemogenic capacity. Analysis of peripheral blood and bone marrow showed a similar transgene expression pattern compared to the primary mice. To generate a complete chimerism of transgenic cells, we chose the human drug resistance gene methylguanine-methyltransferase (MGMT, P140K) to select for mbC46-transduced stem cells in vitro and in vivo. Different coexpression strategies were tested. Function of the MGMT protein was confirmed in a quantitative alkyltransferase assay and in a cytotoxicity assay using BCNU or temozolomide. In vitro selection of transduced 32D and PM1 cells with benzylguanine and BCNU showed >95% positive cells with evidence of polyclonal survival. Transduced PM1 cells underwent an HIV challenge assay. In vivo experiments in a murine bone marrow transplantation setting are ongoing to determine the potency and safety of combined retroviral expression of mbC46 and MGMT in relevant preclinical models. Successful conclusion of these studies will hopefully result in a phase I clinical trial testing the concept of generating an HIV-resistant autologous hematopoiesis.

Expression of Runx1 Isoforms in Sub-Populations of Human Blood and Bone Marrow Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4244-4244
Author(s):  
Maxim Yankelevich ◽  
Judith F. Margolin ◽  
Margaret A. Goodell
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Progenitor Cell ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Pcr Analysis ◽  
Cell Populations ◽  
Total Rna ◽  
Isoform Expression ◽  
A Minor

Abstract Runx1 is a pivotal regulator of hematopoiesis. Deficiency of Runx1 leads to complete block of definitive hematopoiesis in mouse embryos. Runx1 protein forms a heterodimer with CBFβ and binds to the specific DNA sequence TGT/cGGT to regulate the expression of a number of hematopoietic genes. Runx 1 has been recognized to have three different isoforms (a, b, and c) due to differential splicing and promoter utilization. The distal (Runx1c) promoter region was shown to have binding sites mediating repression of Runx1 transcription. Data on the pattern of Runx1 isoforms expression in different hematopoietic lineages are scant. The functional impact of different Runx1 isoforms on commitment and differentiation of blood cells is also essentially unknown. To further explore this, we studied Runx1 isoform expression in different subpopulations of human hematopoietic cells. Total RNA was extracted from peripheral blood and unstimulated bone marrow. Also, Lin-CD34+CD38+, and Lin-CD34+CD38− cell populations were sorted from G-CSF-stimulated bone marrow and total RNA extracted. Expression of Runx1 isoforms was assessed by semiquantitative RT-PCR analysis, using GAPDH as a control. All 3 isoforms were present in all studied cell populations. Runx1a was uniformly a minor form with lowest expression. Runx1c isoform was a dominant one in peripheral blood as opposed to Runx1b in bone marrow. Most interestingly, the ratio of Runx1b to other isoforms was significantly increased in Lin-CD34+CD38- subpopulation compared to Lin-CD34+CD38+ cells. The prevalence of Runx1c isoform in peripheral blood as compaired to bone marrow is most likely related to a different cellular composition of these tissues and suggests that Runx 1c isoform may be predominantly expressed in some of the mature cell lineages. Since Lin-CD34+CD38− cells represent most primitive progenitor cell population with multi-lineage repopulating ability, the observed overexpression of Runx1b may be related to transcriptional accessibility of these cells. Upregulation of Runx 1b with G-CSF in this subpopulation can not be ruled out as well. These preliminary data suggest that the expression of Runx1 alternative splicing isoforms is both lineage and differentiation stage-specific in human hematopoietic cells. We will also test whether Runx1 isoform expression has a consistent pattern in different types of leukemia and if it is resembles such of mature blood counterparts or primitive progenitors. These studies, as well as the studies of differential expression of Runx1 isoforms in different mature blood lineages and progenitor cell sub-populations are underway and results will be presented. These data may further shed a light on functional differences in Runx1 isoforms and mechanisms of hematopoiesis.

The Cell Cycle in Hematopoietic Stem Cells, Lin−/C-kit+/Sca1+ Fraction, Is Regulated by Connexin 32, Specifically Expressed in the Stem Cell Compartment

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4775-4775
Author(s):  
Yoko Hirabayashi ◽  
Byung-Il Yoon ◽  
Isao Tsuboi ◽  
Yan Huo ◽  
Yukio Kodama ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Bone Marrow Cells ◽  
Wild Type ◽  
Cell Compartment ◽  
Hematopoietic Stem ◽  
In The Wild ◽  
Marrow Cells

Abstract Connexin (Cx) functions in the organization of cell-cell communication via gap junctions in multicellular organisms. Gap junctions have been implicated in the homeostatic regulation of various cellular functions, including growth control, cellular differentiation, apoptosis and the synchronization of electrotonic and metabolic functions. As Cxs are essential molecules for multicellular organisms, Cxs that organize cell-cell communication within the hematopoietic progenitor cell compartment are surmised to be present in bone marrow tissue. Recently, we first found that Cx32 is only Cx molecule expressed in the bone marrow in wild-type mice by means of comparison with Cx32-knockout (KO) mice, studied by a reverse biological approach. Cx32 is specifically expressed in primitive hematopoietic stem/progenitor cells, i.e., the lineage marker-negative (Lin−)/c-kit positive (c-kit+)/stem cell antigen-1-positive (Sca1+) (=LKS) fraction, and likely playing a role of restoration of stem/progenitor cell-quiescence, thereby preventing primitive stem cells from exhaustion. In this study, we present results on cell cycle analyses with respect to the function of Cx32; one for colony-forming progenitors by the method evaluating the cycling progenitor cells using incorporation of bromodeoxyuridine (BrdUrd) followed by ultraviolet-light cytocide and the other for primitive progenitor cells using a cell sorter with bioactive AT-rich DNA-binding dye Hoechst 33342. In the colonization assay on CFU-S-13 (primitive hematopoietic progenitor cells), the incorporation of BrdUrd starts from a higher percentage with rapid increase in Cx32-KO mice, suggesting suppression of cell cycle in these primitive hematopoietic progenitor cells with Cx32-mediated cell-cycle regulation in the wild-type steady state. This suppression may be attenuated in CFU-S-9, a differentiated progenitor cell compartment. The progenitor cells assayed by in vitro colonization on CFU-GM also showed accelerated cell cycle in the Cx32-KO mice. Following the incorporation of Hoechst 33342, the lineagedepleted bone marrow cells were analyzed by flow cytometry. The population sizes of the LKS fraction obtained were 0.052% in the Cx32-KO bone marrow cells and 0.035% in the wild-type bone marrow cells (p=0.0458&lt;0.05). The lineage-depleted bone marrow cells were analyzed their cell-cycle patterns by flow cytometry, and the G0/G1 was calculated for the LKS fractions in both, the Cx32-KO mice and wild-type mice. The percentage of G0/G1 calculated for the LKS fractions were significantly lower in the Cx32-KO mice than those in wild-type mice (60.6% vs. 87.9% for Cx32-KO vs. wild-type; p=0.001). The results suggest that Cx32 may have suppressive functions on the hematopoietic stem cell compartment, the LKS fraction, under the physiological function of Cx32. The Cx32 in the wild-type mice is, thus considered to be expressed in the primitive hematopoietic stem/progenitor cells to prevent from their exhaustion.

Sign in / Sign up

Export Citation Format

Share Document