scholarly journals Knockdown of HSPA9 Induces Apoptosis and Increases TP53 Levels in Human CD34+ Hematopoietic Progenitor Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 526-526
Author(s):  
Tuoen Liu ◽  
Kilannin Krysiak ◽  
Cara Lunn Shirai ◽  
Matthew Ndonwi ◽  
Matthew J. Walter
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Erythroid Differentiation ◽  
Fold Change ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract HSPA9, a gene located on chromosome 5q31.2, is commonly deleted in patients with myelodysplastic syndromes (MDS). MDS patients with a deletion of the long arm of chromosome 5 [del(5q)] typically present with cytopenias, including anemia, and have increased levels of apoptosis in their bone marrow contributing to ineffective hematopoiesis. Recent evidence suggests that upregulation of TP53 in MDS bone marrow cells may contribute to the cytopenias and accererated apoptosis observed in patients. While the mechanisms of TP53 activation in MDS are likely to be multifactorial, gene haploinsufficiency has been shown to contribute. Previous reports have shown that knockdown of RPS14, a chromosome 5q33.1 gene, in human CD34+ cells (or heterozygous knockout in mouse bone marrow cells) results in upregulation of TP53 and an increase in apoptosis. It is not known whether additional del(5q) candidate genes contribute to TP53 activation in del(5q)-associated MDS. In order to determine whether HSPA9 gene deletion also results in TP53 activation, we used lentiviral shRNA vectors to knockdown the expression of HSPA9 in primary human CD34+ hematopoietic progenitor cells. The HSPA9 protein level was reduced to ~20% (sh960) and ~50% (sh433) compared to the control lentiviral shRNA (shGFP). Knockdown of HSPA9 significantly inhibited the growth (fold change sh960 compared to shGFP = 0.16, p<0.01; sh433 compared to shGFP = 0.39, p=0.06, N=3) and erythroid differentiation (CD71+ expression fold change sh960 compared to shGFP =0.26, p<0.001; sh433 compared to shGFP = 0.52, p<0.01, N=3) of human CD34+ cells grown in media promoting erythroid differentiation after 7 days in culture. In addition, knockdown of HSPA9 by sh960 significantly increased apoptosis (AnnexinV+ cells) in CD34+ cells compared to shGFP (fold change = 2, p<0.01, N=3). The increased apoptosis observed following HSPA9 knockdown was associated with increased TP53 expression (fold change sh960 compared to shGFP = 2 by intracellular flow cytometry, p<0.05, N=3), TP53 activity (sh960 compared to shGFP, p<0.05; sh433 compared to shGFP, p=0.06 by TP53 luciferase reporter assay), and increased mRNA expression of the pro-apoptotic TP53 target gene BAX (fold change sh960 compared to shGFP = 1.8 by Q-RT-PCR, p<0.05). Gene expression profiling of CD34+ cells transduced with sh433 vs. control knockdown revealed that 26 well-annotated TP53-induced genes are also expressed higher in HSPA9 knockdown cells compared to controls by gene set enrichment (FWER p=0.01), further implicating TP53 activation in HSPA9 knockdown cells. We show that treatment of primary human CD34+ cells with MKT-077, a rhodacyanine dye with inhibitory effects on HSPA9 protein by binding to its nucleotide binding domain, also results in dose-dependent growth inhibition, enhanced apoptosis, and reduced erythroid differentiation, similar to that observed following HSPA9 knockdown. HSPA9 has been shown to physically interact with TP53 in the cytoplasm, and knockdown of HSPA9 changes localization of TP53 from the cytoplasm to the nucleus in non-hematopoietic cells. The loss of cytoplasmic sequestration of TP53 by HSPA9 following HSPA9 knockdown is thought to contribute to many of the cellular phenotypes induced by HSPA9 knockdown. We show by immunoprecipitation that HSPA9 also interacts with TP53 in primary human CD34+ cells, providing a possible mechanism for regulation of TP53 by HSPA9 in hematopoietic cells. These findings indicate that HSPA9 knockdown may contribute to TP53 activation and increased apoptosis observed in del(5q)-associated MDS. The data also suggest that genetic (i.e., mutation or deletion) or functional inactivation of TP53 may be necessary for disease progression to occur in MDS patients with del(5q). Disclosures No relevant conflicts of interest to declare.

Adhesion to E-selectin promotes growth inhibition and apoptosis of human and murine hematopoietic progenitor cells independent of PSGL-1

Blood ◽  
2004 ◽  
Vol 103 (5) ◽  
pp. 1685-1692 ◽  
Author(s):  
Ingrid G. Winkler ◽  
Karen R. Snapp ◽  
Paul J. Simmons ◽  
Jean-Pierre Lévesque
Keyword(s):  
Bone Marrow ◽  
Growth Inhibition ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Hematopoietic Progenitor Cells ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Mouse Bone Marrow ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells

AbstractAlthough both P- and E-selectin are constitutively expressed on bone marrow endothelial cells, their role in the regulation of hematopoiesis has only recently been investigated. We have previously shown that P-selectin glycoprotein ligand-l (PSGL-1/CD162) is expressed by primitive human bone marrow CD34+ cells, mediates their adhesion to P-selectin, and, more importantly, inhibits their proliferation. We now demonstrate that adhesion to E-selectin inhibits the proliferation of human CD34+ cells isolated either from human umbilical cord blood, adult mobilized blood, or steady-state bone marrow. Furthermore, a subpopulation, which does not contain the most primitive hematopoietic progenitor cells, undergoes apoptosis following E-selectin–mediated adhesion. The same phenomenon was observed in cells isolated from mouse bone marrow. Using lineage-negative Sca-1+ c-KIT+ bone marrow cells from PSGL-1–/– and wild-type mice, we establish that PSGL-1 is not the ligand involved in E-selectin–mediated growth inhibition and apoptosis. Moreover, stable transfection of the human myeloid cell line K562 (which does not express PSGL-1) with α(1,3) fucosyltransferase VII alone was sufficient to recapitulate the E-selectin–mediated growth inhibition and apoptosis observed in hematopoietic progenitor cells. These data demonstrate that an E-selectin ligand(s) other than PSGL-1 transduces growth inhibitory and proapoptotic signals and requires posttranslational fucosylation to be functional.

The Hemoglobin-Haptoglobin Receptor (CD163) Is Expressed by a Subpopulation of Erythroid and Granulo-Macrophagic Progenitors and Can Be Stimulated in Vitro and in Vivo by Physiological and Pharmacological Ligands to Stimulate Hematopoiesis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1221-1221
Author(s):  
Kathryn Matthews ◽  
Nicole Worsham ◽  
Neeta Rugg ◽  
Jose A. Cancelas ◽  
David Bell
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Fold Increase ◽  
Hematopoietic Progenitor ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Marrow Cells

Abstract Abstract 1221 The receptor for the hemoglobin (Hb)-haptoglobin (Hp) complex, CD163, is expressed on the surface of a subpopulation of hematopoietic stem/progenitor cells (HPCs) (Matthews et al, 2006). The purpose of the studies presented here were two-fold – to demonstrate that the CD34+CD163+ double positive population could be isolated from normal adult bone marrow cells and these cells were functional as HPCs and, in addition, that these cells could be stimulated in vivo by ligands to CD163 to affect hematopoiesis. To investigate the clonogenic potential of CD34+/CD163+ HPCs, bone marrow CD34+ cells were examined for CD163 co-expression, sorted by fluorescence activated cell sorting (FACS) and plated into colony-forming assays (CFAs). 4.2% ± 1.4% (n=4) of CD34+ cells were found to co-express CD163 and this population consisted of two distinct sub-populations, CD34++ (hi)CD163+ and CD34+(lo)CD163+, each of which represented approximately half of the total CD34+CD163+ population. All three sorted populations (CD34+(all)CD163−, CD34++(hi) CD163+, CD34+(lo)CD163+) were plated into CFAs (n=4) and were assessed for erythroid and myeloid colony formation. The clonogenic efficiency of CD34++(hi)CD163+ had a 2.5-fold increase in the number CFU-E and CFU-GM when compared to both CD34+ (total) CD163− and CD34+(lo) CD163+ cells. In contrast, CD34+(hi an low)CD163+cells produced fewer BFU-E. To determine how the expression of CD163 expression on progenitor cells may play a role in hematopoiesis, we investigated the effects of the natural ligand to CD163 (Hb/Hp) as well as an agonistic antibody to CD163 (TBI 304) on HPCs in vivo. NOD-scid IL2R gammanull (NSG) mice (HuMurine Technologies) were engrafted with human CD34+cells and animals with < 30% human CD45+ cells in the peripheral blood were administered either 2 mg Hb/mouse, or 100 or 500 μg/mouse TBI 304 every 4 days. At study termination (day 14), bone marrow cells (BMC) were examined by flow cytometry and enriched for CD34+ cells for enumeration in CFAs. Hb administration resulted in an increase of human CD34+cells ranging from 4% to 7% of BMC and a corresponding 57% increase in colony-forming cells (CFC) when compared to control (PBS-administered) animals. In contrast, TBI 304 produced a dose dependent decrease in CD34+ and CFC, possibly reflecting a depletion of CD34+/CD163+ cells from overstimulation due to the longer circulating antibody. To investigate this, human CD34+ cell engrafted animals were given a single dose of 10 or 100 μg/mouse of TBI 304 and bone marrow cells were examined on day 7. TBI 304 provided a 3.5-fold increase in human CD34+ cells as well as a 1.8 to 6.7-fold increase in bone marrow erythroid lineage engraftment (huGlyA+, huCD36+ and huCD71+) and a 2-fold increase in erythroid and myeloid colony-forming cells. No overall toxicities were observed with the administration of TBI 304 or Hb. We have demonstrated that CD163 is expressed on a population of CD34+ hematopoietic progenitor cells, these cells have increased hematopoietic progenitor activity in vitro and that administration of physiological or pharmacological agonists of the CD163 receptor can measurably stimulate hematopoiesis in vivo. Disclosures: Matthews: Therapure Biopharma: Employment. Bell:Therapure Biopharma: Employment.

scholarly journals Functional differences between CD38- and DR- subfractions of CD34+ bone marrow cells

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1473-1481 ◽  
Author(s):  
LS Rusten ◽  
SE Jacobsen ◽  
O Kaalhus ◽  
OP Veiby ◽  
S Funderud ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Flow Cytometric Analysis ◽  
Hematopoietic Progenitor Cells ◽  
Proliferative Potential ◽  
Hematopoietic Progenitor ◽  
Erythroid Progenitor ◽  
Cd34 Cells ◽  
Marrow Cells

Abstract Several studies have previously demonstrated enrichment in primitive progenitor cells in subfractions of CD34+ bone marrow (BM) cells not expressing CD38 or HLA-DR (DR) antigens. However, no studies have directly compared these two cell populations with regard to their content of primitive and more committed progenitor cells. Flow cytometric analysis of immunomagnetic isolated CD34+ cells demonstrated little overlap between CD34+CD38- and CD34+DR- progenitor subpopulations in that only 12% to 14% of total CD34+DR- and CD34+CD38- cells were double negative (CD34+CD38-DR-). Although the number of committed myeloid progenitor cells (colony-forming units granulocyte- macrophage) was reduced in both subpopulations, only CD34+CD38- cells were significantly depleted in committed erythroid progenitor cells (burst-forming units-erythroid). In single-cell assay, CD34+CD38- cells showed consistently poorer response to single as opposed to multiple hematopoietic growth factors as compared with unfractionated CD34+ cells, indicating that the CD34+CD38- subset is relatively enriched in primitive hematopoietic progenitor cells. Furthermore, CD34+CD38- and CD34+DR- cells, respectively, formed 3.2-fold and 1.6-fold more high proliferative potential colony-forming cell (HPP-CFC) colonies than did unfractionated CD34+ cells. Finally, CD34+CD38-DR- cells were depleted in HPP-CFCs as compared with CD34+CD38+DR+ cells. The results of the present study suggest that both the CD38- and DR- subfractions of CD34+ bone marrow cells are enriched in primitive hematopoietic progenitor cells, with the CD34+CD38- subpopulation being more highly enriched than CD34+DR- cells.

scholarly journals Arsenic Primes Human Bone Marrow CD34+ Cells for Erythroid Differentiation

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Yuanyuan Zhang ◽  
Shasha Wang ◽  
Chunyan Chen ◽  
Xiao Wu ◽  
Qunye Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Arsenic Trioxide ◽  
Erythroid Differentiation ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Hematopoietic Progenitor Cells ◽  
Therapeutic Effects ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells

Arsenic trioxide exhibits therapeutic effects on certain blood malignancies, at least partly by modulating cell differentiation. Previousin vitrostudies in human hematopoietic progenitor cells have suggested that arsenic may inhibit erythroid differentiation. However, these effects were all observed in the presence of arsenic compounds, while the concomitant cytostatic and cytotoxic actions of arsenic might mask a prodifferentiating activity. To eliminate the potential impacts of the cytostatic and cytotoxic actions of arsenic, we adopted a novel protocol by pretreating human bone marrow CD34+ cells with a low, noncytotoxic concentration of arsenic trioxide, followed by assaying the colony forming activities in the absence of the arsenic compound. Bone marrow specimens were obtained from chronic myeloid leukemia patients who achieved complete cytogenetic remission. CD34+ cells were isolated by magnetic-activated cell sorting. We discovered that arsenic trioxide enhanced the erythroid colony forming activity, which was accompanied by a decrease in the granulomonocytic differentiation function. Moreover, in erythroleukemic K562 cells, we showed that arsenic trioxide inhibited erythrocyte maturation, suggesting that arsenic might have biphasic effects on erythropoiesis. In conclusion, our data provided the first evidence showing that arsenic trioxide could prime human hematopoietic progenitor cells for enhanced erythroid differentiation.

scholarly journals Effect of flt3 ligand on the ex vivo expansion of human CD34+ hematopoietic progenitor cells

Blood ◽  
1995 ◽  
Vol 86 (9) ◽  
pp. 3413-3420 ◽  
Author(s):  
HJ McKenna ◽  
P de Vries ◽  
K Brasel ◽  
SD Lyman ◽  
DE Williams
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Progenitor Cells ◽  
Flt3 Ligand ◽  
Hematopoietic Progenitor ◽  
Human Cd34

ligand for the tyrosine kinase receptor flt3/flk-2, referred to here as flt3 ligand (flt3L), was recently cloned. The effect of flt3L on purified human CD34+ progenitor cells was examined. flt3 receptor (flt3R) was detected on the surface of human bone marrow cells that were enriched for CD34 expression. The effects of flt3L and the c-kit ligand Steel factor (SLF) on hematopoietic progenitors were compared in clonal colony assays. Both factors synergized with Pixy321 (interleukin- 3 [IL-3]-granulocyte-macrophage colony-stimulating factor fusion protein) to induce granulocytic-monocytic (GM) and high proliferative potential (HPP) colonies and synergized with Pixy321 + erythropoietin (EPO) to induce multipotent granulocytic-erythroid-monocytic- megakaryocytic colonies. Although SLF had a potent effect on colony formation of erythroid restricted progenitor cells (burst-forming unit- erythroid), no effect by flt3L was observed. The addition of flt3L to irradiated long-term marrow cultures seeded with CD34+ cells augmented both total and progenitor cell production. Ex vivo expansion studies with isolated CD34+ bone marrow cells from normal donors showed that flt3L alone supported maintenance of both GM and HPP progenitors for 3 to 4 weeks in vitro. The addition of flt3L to a growth factor combination of IL-1 alpha + IL-3 + IL-6 + EPO resulted in a synergistic effect on progenitor cell expansion comparable to that observed with the addition of SLF to IL-1 alpha + IL-3 + IL-6 + EPO. These data show a function for flt3L in the regulation of both primitive multipotent and lineage-committed hematopoietic progenitor cells.

scholarly journals Functional differences between CD38- and DR- subfractions of CD34+ bone marrow cells

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1473-1481 ◽  
Author(s):  
LS Rusten ◽  
SE Jacobsen ◽  
O Kaalhus ◽  
OP Veiby ◽  
S Funderud ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Flow Cytometric Analysis ◽  
Hematopoietic Progenitor Cells ◽  
Proliferative Potential ◽  
Hematopoietic Progenitor ◽  
Erythroid Progenitor ◽  
Cd34 Cells ◽  
Marrow Cells

Several studies have previously demonstrated enrichment in primitive progenitor cells in subfractions of CD34+ bone marrow (BM) cells not expressing CD38 or HLA-DR (DR) antigens. However, no studies have directly compared these two cell populations with regard to their content of primitive and more committed progenitor cells. Flow cytometric analysis of immunomagnetic isolated CD34+ cells demonstrated little overlap between CD34+CD38- and CD34+DR- progenitor subpopulations in that only 12% to 14% of total CD34+DR- and CD34+CD38- cells were double negative (CD34+CD38-DR-). Although the number of committed myeloid progenitor cells (colony-forming units granulocyte- macrophage) was reduced in both subpopulations, only CD34+CD38- cells were significantly depleted in committed erythroid progenitor cells (burst-forming units-erythroid). In single-cell assay, CD34+CD38- cells showed consistently poorer response to single as opposed to multiple hematopoietic growth factors as compared with unfractionated CD34+ cells, indicating that the CD34+CD38- subset is relatively enriched in primitive hematopoietic progenitor cells. Furthermore, CD34+CD38- and CD34+DR- cells, respectively, formed 3.2-fold and 1.6-fold more high proliferative potential colony-forming cell (HPP-CFC) colonies than did unfractionated CD34+ cells. Finally, CD34+CD38-DR- cells were depleted in HPP-CFCs as compared with CD34+CD38+DR+ cells. The results of the present study suggest that both the CD38- and DR- subfractions of CD34+ bone marrow cells are enriched in primitive hematopoietic progenitor cells, with the CD34+CD38- subpopulation being more highly enriched than CD34+DR- cells.

scholarly journals Functional activity of animal bone marrow cells after their treatment with nanocomplexes

2021 ◽  
Vol 29 (2) ◽  
pp. 9-21
Author(s):  
A. M. Goltsev ◽  
T. G. Dubrava ◽  
Yu. O. Gaevska ◽  
N. M. Babenko ◽  
M. O. Bondarovych ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Toxic Effect ◽  
Progenitor Cells ◽  
Functional Activity ◽  
Therapeutic Dose ◽  
Bone Marrow Cells ◽  
Earth Metal ◽  
Hematopoietic Progenitor Cells ◽  
National Academy Of Sciences ◽  
Hematopoietic Progenitor

Background. Previously, the antitumor activity of nanocomplexes (NCs) containing nanoparticles of rare earth metal orthovanadates GdYEuVO4 and cholesterol has been approved when applied in 9:1 ratio (the cells-to-NCs), which can be considered as a conditionally therapeutic dose. Therefore, studying the potential risks of NCs exposure in terms of functional activity of hematopoietic progenitor cells is relevant. Рurpose – determining a toxic effect of NCs on functional activity of hematopoietic cells of bone marrow (BM). Materials and Methods. The study was performed in BM cells of CBA/H mice. Nanocomplexes were synthesized at Institute for Scintillation Materials of the National Academy of Sciences of Ukraine. BM cells with NCs were incubated in the ratios as follows: 9BM:1NCs; 1BM:1NCs; 1BM:9NCs, followed by assessing the number of apoptotic/necrotic cells in BM using FITC Annexin V Apoptosis Detection Kit I (BD, USA) by means of “FACS Calibur” flow cytometer (“BD”, USA). Hematopoietic progenitor cells of BM were functionally evaluated in vivo by determining the content of colony-forming units of the spleen (CFUs) and the number of myelokaryocytes in lethally irradiated recipients on day 8 after administering BM cells, pre-incubated with NCs. Survival of irradiated recipient mice after BM administration was recorded 12 days long. Results and discussion. The dose-dependent effect of functional potential in- hibition for BM hematopoietic progenitor cells under NCs influence has been established. Although, in vitro processing the BM cells with a conditionally therapeutic dose of NCs (9BM:1NCs) before administration to irradiated animal caused remodeling of cell membranes and contributed to apoptotic manifes- tations, but it did not lead to strong changes in their colony-forming potential and did not reduce the number of BM cells in animals if compared with the introduced BM cells without NCs treatment. Increasing the NCs concentration five- and tenfold significantly reduced the colony-forming potential of BM cells, caused BM hypoplasia and a crucial reduction in the survival of recipient animals, indicating possible toxic effects of this compound when administered at high concentrations. Conclusions. The toxic effect of NCs is detected only when certain concen- trations, significantly exceeding the conditionally therapeutic dose previously determined when treating the experimental oncology diseases, are used.

scholarly journals A novel 37-Kd adhesive membrane protein from cloned murine bone marrow stromal cells and cloned murine hematopoietic progenitor cells

Blood ◽  
1993 ◽  
Vol 82 (5) ◽  
pp. 1436-1444 ◽  
Author(s):  
Y Shiota ◽  
JG Wilson ◽  
K Harjes ◽  
ED Zanjani ◽  
M Tavassoli
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Stromal Cell ◽  
Hematopoietic Cells ◽  
Marrow Stromal Cells ◽  
Hematopoietic Progenitor Cells ◽  
Single Chain ◽  
Hematopoietic Progenitor

Abstract The adhesion of hematopoietic progenitor cells to bone marrow stromal cells is critical to hematopoiesis and involves multiple effector molecules. Stromal cell molecules that participate in this interaction were sought by analyzing the detergent-soluble membrane proteins of GBI/6 stromal cells that could be adsorbed by intact FDCP-1 progenitor cells. A single-chain protein from GBI/6 cells having an apparent molecular weight of 37 Kd was selectively adsorbed by FDCP-1 cells. This protein, designated p37, could be surface-radiolabeled and thus appeared to be exposed on the cell membrane. An apparently identical 37- Kd protein was expressed by three stromal cell lines, by Swiss 3T3 fibroblastic cells, and by FDCP-1 and FDCP-2 progenitor cells. p37 was selectively adsorbed from membrane lysates by a variety of murine hematopoietic cells, including erythrocytes, but not by human erythrocytes. Binding of p37 to cells was calcium-dependent, and was not affected by inhibitors of the hematopoietic homing receptor or the cell-binding or heparin-binding functions of fibronectin. It is proposed that p37 may be a novel adhesive molecule expressed on the surface of a variety of hematopoietic cells that could participate in both homotypic and heterotypic interactions of stromal and progenitor cells.

scholarly journals Gene transfer into hematopoietic progenitor cells from normal and cyclic hematopoietic dogs using retroviral vectors

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 717-722 ◽  
Author(s):  
MA Eglitis ◽  
PW Kantoff ◽  
JD Jolly ◽  
JB Jones ◽  
WF Anderson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Retroviral Vectors ◽  
Hematopoietic Progenitor Cells ◽  
Neomycin Phosphotransferase ◽  
Drug Resistant ◽  
Hematopoietic Progenitor ◽  
Exogenous Genes ◽  
Murine Leukemia

Abstract The Moloney murine leukemia retrovirus-derived vector N2 was used to transfer the bacterial NeoR gene (conferring resistance to the neomycin analogue G418) into hematopoietic progenitor cells. Approximately 5% of day seven CFU-GM were resistant to 2,000 micrograms/ml G418, using a supernatant infection protocol in the absence of vector-producing cells. A greater proportion of CFU-GM colonies were recovered relative to uninfected controls as the stringency of selection was diminished. Enzyme activity was detected in drug-resistant colonies, confirming that the resistant colonies obtained after infection with N2 represented cells producing neomycin phosphotransferase. Activity in the CFU-GM colonies approached 50% of that of drug-resistant vector- producing cells on a per cell basis. To test the hypothesis that more rapidly cycling bone marrow cells would be more susceptible to vector infection, we treated progenitor cells obtained from cyclic hematopoietic (CH) dogs with the N2 vector. Despite the increased numbers of hematopoietic progenitor cells obtained from CH dogs, the proportion of G418-resistant CFU-GM did not increase over that obtained with N2-infected normal marrow. These results demonstrate that retroviral vectors can be used to transfer and express exogenous genes in canine hematopoietic progenitor cells.

scholarly journals Orderly Process of Sequential Cytokine Stimulation Is Required for Activation and Maximal Proliferation of Primitive Human Bone Marrow CD34+ Hematopoietic Progenitor Cells Residing in G0

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 658-668 ◽  
Author(s):  
Amy C. Ladd ◽  
Robert Pyatt ◽  
Andre Gothot ◽  
Susan Rice ◽  
Jon McMahel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Phase I ◽  
Progenitor Cells ◽  
Phase Ii ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells ◽  
Cytokine Stimulation ◽  
Phase Iv

Abstract Bone marrow (BM) CD34+ cells residing in the G0 phase of cell cycle may be the most suited candidates for the examination of cell cycle activation and proliferation of primitive hematopoietic progenitor cells (HPCs). We designed a double simultaneous labeling technique using both DNA and RNA staining with Hoechst 33342 and Pyronin Y, respectively, to isolate CD34+ cells residing in G0(G0CD34+ ). Using long-term BM cultures and limiting dilution analysis, G0CD34+ cells were found to be enriched for primitive HPCs. In vitro proliferation of G0CD34+ cells in response to sequential cytokine stimulation was examined in a two-step assay. In the first step, cells received a primary stimulation consisting of either stem cell factor (SCF), Flt3-ligand (FL), interleukin-3 (IL-3), or IL-6 for 7 days. In the second step, cells from each group were washed and split into four or more groups, each of which was cultured again for another week with one of the four primary cytokines individually, or in combination. Tracking of progeny cells was accomplished by staining cells with PKH2 on day 0 and with PKH26 on day 7. Overall examination of proliferation patterns over 2 weeks showed that cells could progress into four phases of proliferation. Phase I contained cytokine nonresponsive cells that failed to proliferate. Phase II contained cells dividing up to three times within the first 7 days. Phases III and IV consisted of cells dividing up to five divisions and greater than six divisions, respectively, by the end of the 14-day period. Regardless of the cytokine used for primary stimulation, G0CD34+ cells moved only to phase II by day 7, whereas a substantial percentage of cells incubated with SCF or FL remained in phase I. Cells cultured in SCF or FL for the entire 14-day period did not progress beyond phase III but proliferated into phase IV (with &lt;20% of cells remaining in phases I and II) if IL-3, but not IL-6, was substituted for either cytokine on day 7. G0CD34+ cells incubated with IL-3 for 14 days proliferated the most and progressed into phase IV; however, when SCF was substituted on day 7, cells failed to proliferate into phase IV. Most intriguing was a group of cells, many of which were CD34+, detected in cultures initially stimulated with IL-3, which remained as a distinct population, mostly in G0 /G1 , unable to progress out of phase II regardless of the nature of the second stimulus received on day 7. A small percentage of these cells expressed cyclin E, suggesting that their proliferation arrest may have been mediated by a cyclin-related disruption in cell cycle. These results suggest that a programmed response to sequential cytokine stimulation may be part of a control mechanism required for maintenance of proliferation of primitive HPCs and that unscheduled stimulation of CD34+ cells residing in G0 may result in disruption of cell-cycle regulation.

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