Engraftment potential of different sources of human hematopoietic progenitor cells in BNX Mice

Human hematopoietic progenitor cells (HPCs) from mobilized peripheral blood mononuclear cells (PBMCs), adult bone marrow (ABM), and fetal bone marrow (FBM) were evaluated for their ability to produce multilineage human hematopoietic engraftment in vivo. Sublethally irradiated BNX (beige/nude/xid) mice were injected with either unfractionated cells or CD34+ cells purified from these sources. The presence of human cells in the mouse PB, BM, and spleen was evaluated by flow cytometry at either 6 to 8 weeks or 6 months postinjection. Recipients with > or = 1% human cells in any of these tissues were considered chimeric. Of 26 mice injected with FBM, 4 showed up to 73% human cells in the BM or spleen at 6 months. The phenotypes of these cells included CD13/33+ myelomonocytic cells (38%), CD19+ B cells (67%), and CD34+ progenitor cells (28%). In contrast, ABM gave rise to a mean of 5% human cells in the PB in 2 of 42 (4%) recipients at 6 to 8 weeks. These circulating human cells were predominantly CD3+, whereas CD13/33+ and CD34+ cells were detected in the BM for up to 6 months. A total of 18% of mice injected with PBMCs showed a mean of 36% human cells in the PB. Both the BM and spleens of PBMC-injected mice contained CD3+ cells in a proportion similar to that observed in the PB. These CD3+ cells were phenotypically mature CD4+,CD8-or CD4-,CD8+ T cells and coexpressed a variety of Vbeta T-cell receptor (TCR) genes. The percentage of CD3+ cells in the circulation of chimeric recipients injected with either FBM, ABM, or PBMCs correlated well with the input CD3+ cell dose for each of these HPC sources (r = .99). The high levels of engraftment of CD3+ cells in recipients of PBMCs and the long-term multilineage engraftment of FBM recipients have important implications for developing strategies to study the regulation of these human cells in vivo.

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CIRCULATING HEMATOPOIETIC PROGENITOR CELLS IN PATIENTS AFFECTED BY CHORNOBYL ACCIDENT

Experimental Oncology ◽

10.31768/2312-8852.2016.38(4):242-244 ◽

2016 ◽

Vol 38 (4) ◽

pp. 242-244 ◽

Cited By ~ 1

Author(s):

N M Bilko ◽

I S Dyagil ◽

I S Russu ◽

D I Bilko

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Peripheral Blood ◽

Functional Activity ◽

Mononuclear Cells ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Scientific Center ◽

Cleanup Workers

High radiation sensitivity of stem cells and their ability to accumulate sublethal radiation damage provides the basis for investigation of hematopoietic progenitors using in vivo culture methodology. Unique samples of peripheral blood and bone marrow were derived from the patients affected by Chornobyl accident during liquidation campaign. Aim: To investigate functional activity of circulating hematopoietic progenitor cells from peripheral blood and bone marrow of cleanup workers in early and remote periods after the accident at Chornobyl nuclear power plant (CNPP). Materials and Methods: The assessment of the functional activity of circulating hematopoietic progenitor cells was performed in samples of peripheral blood and bone marrow of 46 cleanup workers, who were treated in the National Scientific Center for Radiation Medicine of the Academy of Medical Sciences of Ukraine alongside with 35 non radiated patients, who served as a control. Work was performed by culturing peripheral blood and bone marrow mononuclear cells in the original gel diffusion capsules, implanted into the peritoneal cavity of CBA mice. Results: It was shown that hematopoietic progenitor cells could be identified in the peripheral blood of liquidators of CNPP accident. At the same time the number of functionally active progenitor cells of the bone marrow was significantly decreased and during the next 10 years after the accident, counts of circulating progenitor cells in the peripheral blood as well as functionally active hematopoietic cells in bone marrow returned to normal levels. Conclusion: It was shown that hematopoietic progenitor cells are detected not only in the bone marrow but also in the peripheral blood of liquidators as a consequence of radiation exposure associated with CNPP accident. This article is a part of a Special Issue entitled “The Chornobyl Nuclear Accident: Thirty Years After”.

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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 ◽

10.1182/blood.v90.2.658 ◽

1997 ◽

Vol 90 (2) ◽

pp. 658-668 ◽

Cited By ~ 63

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 <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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Flt3 Ligand Negatively Regulates In Vitro Migration, Intracellular Signaling Activated by SDF-1α/CXCR4 and In Vivo Homing of Hematopoietic Progenitor Cells.

Blood ◽

10.1182/blood.v104.11.2674.2674 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2674-2674

Author(s):

Seiji Fukuda ◽

Hal E. Broxmeyer ◽

Louis M. Pelus

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Hematopoietic Progenitor Cells ◽

Leukemia Cells ◽

Flt3 Ligand ◽

Hematopoietic Progenitor ◽

Short Term ◽

Hematopoietic Stem

Abstract The Flt3 receptor tyrosine kinase (Flt3) is expressed on primitive normal and transformed hematopoietic cells and Flt3 ligand (FL) facilitates hematopoietic stem cell mobilization in vivo. The CXC chemokine SDF-1α(CXCL12) attracts primitive hematopoietic cells to the bone marrow microenvironment while disruption of interaction between SDF-1α and its receptor CXCR4 within bone marrow may facilitate their mobilization to the peripheral circulation. We have previously shown that Flt3 ligand has chemokinetic activity and synergistically increases migration of CD34+ cells and Ba/F3-Flt3 cells to SDF-1α in short-term migration assays; this was associated with synergistic phosphorylation of MAPKp42/p44, CREB and Akt. Consistent with these findings, over-expression of constitutively active ITD (internal tandem duplication) Flt3 found in patients with AML dramatically increased migration to SDF-1α in Ba/F3 cells. Since FL can induce mobilization of hematopoietic stem cells, we examined if FL could antagonize SDF-1α/CXCR4 function and evaluated the effect of FL on in vivo homing of normal hematopoietic progenitor cells. FL synergistically increased migration of human RS4;11 acute leukemia cells, which co-express wild-type Flt3 and CXCR4, to SDF-1α in short term migration assay. Exogenous FL had no effect on SDF-1α induced migration of MV4-11 cells that express ITD-Flt3 and CXCR4 however migration to SDF-1α was partially blocked by treatment with the tyrosine kinase inhibitor AG1296, which inhibits Flt3 kinase activity. These results suggest that FL/Flt3 signaling positively regulates SDF-1α mediated chemotaxis of human acute leukemia cells in short-term assays in vitro, similar to that seen with normal CD34+ cells. In contrast to the enhancing effect of FL on SDF-1α, prolonged incubation of RS4;11 and THP-1 acute myeloid leukemia cells, which also express Flt3 and CXCR4, with FL for 48hr, significantly inhibited migration to SDF-1α, coincident with reduction of cell surface CXCR4. Similarly, prolonged exposure of CD34+ or Ba/F3-Flt3 cells to FL down-regulates CXCR4 expression, inhibits SDF-1α-mediated phosphorylation of MAPKp42/p44, CREB and Akt and impairs migration to SDF-1α. Despite reduction of surface CXCR4, CXCR4 mRNA and intracellular CXCR4 in Ba/F3-Flt3 cells were equivalent in cells incubated with or without FL, determined by RT-PCR and flow cytometry after cell permeabilization, suggesting that the reduction of cell surface CXCR4 expression is due to accelerated internalization of CXCR4. Furthermore, incubation of Ba/F3-Flt3 cells with FL for 48hr or over-expression of ITD-Flt3 in Ba/F3 cells significantly reduced adhesion to VCAM1. Consistent with the negative effect of FL on in vitro migration and adhesion to VCAM1, pretreatment of mouse bone marrow cells with 100ng/ml of FL decreased in vivo homing of CFU-GM to recipient marrow by 36±7% (P<0.01), indicating that FL can negatively regulate in vivo homing of hematopoietic progenitor cells. These findings indicate that short term effect of FL can provide stimulatory signals whereas prolonged exposure has negative effects on SDF-1α/CXCR4-mediated signaling and migration and suggest that the FL/Flt3 axis regulates hematopoietic cell trafficking in vivo. Manipulation of SDF-1α/CXCR4 and FL/Flt3 interaction could be clinically useful for hematopoietic cell transplantation and for treatment of hematopoietic malignancies in which both Flt3 and CXCR4 are expressed.

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Neutophil Gelatinase –Associated Lipocalin (LCN2) Affects Primary Melofibrosis Hematopoietic Progenitor Cells As Well As Microenvironmental Cells

Blood ◽

10.1182/blood.v122.21.2839.2839 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2839-2839

Author(s):

Min Lu ◽

Lijuan Xia ◽

Rona Singer Weinberg ◽

Ronald Hoffman

Keyword(s):

Progenitor Cells ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasm ◽

Cxcr4 Expression ◽

Hematopoietic Progenitor Cells ◽

Adherent Cell ◽

Hematopoietic Progenitor ◽

Cd34 Cells ◽

Cell Layers

Abstract Primary myelofibrosis (PMF) is a clonal myeloproliferative neoplasm (MPN) characterized by preferential proliferation of malignant hematopoietic progenitor cells which leads to excessive proliferation of marrow microenvironmental cells which are not involved by the malignant process. These events result in a clinical disorder characterized by anemia, a leukoerythroblastic blood picture, constitutive mobilization of CD34+ cells, extramedullary hematopoiesis, dacrocytosis, marrow megakaryocytic hyperplasia, progressive splenomegaly and reticulin and collagen marrow fibrosis. Several cytokines elaborated by PMF hematopoietic cells including TGF-b, vascular endothelial growth factor and tumor necrosis factor a have been implicated as playing a role in creation of the MF clinical phenotype. Neutrophil gelatinase associated Lipocalin-2 (LCN2) has been reported to have two distinct roles in the pathobiology of chronic myeloid leukemia (CML), suppressing residual normal HPC development and promoting CML proliferation (Devireddy LR et al, 2005, Cell). We, therefore hypothesized that LCN2 might also play a role in the development of the phenotypic features of PMF. Plasma LCN2 levels were measured by ELISA in 77 patients with PMF and were shown to be elevated as compared to 16 normal plasmas (P<0.001) Media conditioned by PMF MNC contained higher levels of LCN2 than media conditioned by normal MNC (p=0.03). The LCN2 receptor level was flow cytometrically analyzed and a significantly greater percentage of normal MNC and CD34+ cells than PMF MNC and CD34+ cells expressed the LCN2 receptor. The effect of increasing concentrations of recombinant LCN2 on CFU-GM and BFU-E derived colony by normal BM CD34+ cells as well as the PMF CD34+ cells was evaluated. The addition of LCN2 inhibited CFU-GM and BFU-E derived colony formation by normal CD34+ cells at a dose of 100 ng/ml (p=0.048 and p=0.043, respectively), while a similar dose of LCN2 did not influence the number of colonies cloned from PMF CD34+ cells. Previously our laboratory has reported that the expression of the chemokine receptor CXCR4 was decreased in PMF CD34+ cells which we hypothesized contributed to abnormal trafficking of CD34+ cells. LCN2 has been reported to affect CXCR4 expression by marrow CD34+ cells (Costa D, et al. 2010, Cytokine). CXCR4 expression by PMF CD34+ cells was lower than that of normal BM CD34+ cells. Incubation with LCN2, however, further reduced the expression of CXCR4 of PMF CD34+ cells by 10 to 50 % (p=0.012). By contrast, LCN2 increased CXCR4+ expression by normal CD34+ cells. We next evaluated the effects of LCN2 on the BM microenvironment. Normal BM mononuclear cells were plated in dishes exposed to the vary doses of LCN2 (10, 50, 100, 200 ng/ml) and the formation of adherent cell layers was monitored. Low doses of LCN2 (10 and 50 ng/ml) promoted the formation and proliferation of adherent cell layers composed of fibroblast-like cells after 1-3 weeks of culture. The fibroblast-like cells expressed vimentin and von Willebrand factor, indicating that they resembled mesenchymal stem cells, fibroblast and endothelial cells. We then examined the formation of adherent cell layers by normal BM MNC co-cultured with PMF, PV or normal MNCs separated by a 0.4 um trans-well for three weeks. The proliferation of confluent fibroblast-like cells was observed solely in BM MNC co-cultured with PMF MNC. These data indicate that LCN2 is generated in increased amounts by PMF mononuclear cells and likely plays a role in PMF biology by promoting malignant hematopoiesis but suppressing normal hemaopoiesis, suppressing CXCR4 expression by PMF CD34+ cells and promoting marrow fibroblast proliferation. Disclosures: No relevant conflicts of interest to declare.

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Murine Hematopoietic Progenitor Cells With Colony-Forming or Radioprotective Capacity Lack Expression of the β2-Integrin LFA-1

Blood ◽

10.1182/blood.v93.1.107.401k14_107_112 ◽

1999 ◽

Vol 93 (1) ◽

pp. 107-112 ◽

Cited By ~ 2

Author(s):

Johannes F.M. Pruijt ◽

Yvette van Kooyk ◽

Carl G. Figdor ◽

Roel Willemze ◽

Willem E. Fibbe

Keyword(s):

Bone Marrow ◽

Steady State ◽

Progenitor Cells ◽

Mononuclear Cells ◽

Hematopoietic Progenitor Cells ◽

Colony Stimulating Factor ◽

Hematopoietic Progenitor ◽

Β2 Integrin ◽

Stimulating Factor

Recently, we have demonstrated that antibodies that block the function of the β2-integrin leukocyte function-associated antigen-1 (LFA-1) completely abrogate the rapid mobilization of hematopoietic progenitor cells (HPC) with colony-forming and radioprotective capacity induced by interleukin-8 (IL-8) in mice. These findings suggested a direct inhibitory effect of these antibodies on LFA-1–mediated transmigration of stem cells through the bone marrow endothelium. Therefore, we studied the expression and functional role of LFA-1 on murine HPC in vitro and in vivo. In steady state bone marrow ± 50% of the mononuclear cells (MNC) were LFA-1neg. Cultures of sorted cells, supplemented with granulocyte colony-stimulating factor (G-CSF)/granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-1/IL-3/IL-6/stem cell factor (SCF) and erythropoietin (EPO) indicated that the LFA-1neg fraction contained the majority of the colony-forming cells (CFCs) (LFA-1neg 183 ± 62/7,500 cells v LFA-1pos 29 ± 17/7,500 cells,P < .001). We found that the radioprotective capacity resided almost exclusively in the LFA-1neg cell fraction, the radioprotection rate after transplantation of 103, 3 × 103, 104, and 3 × 104 cells being 63%, 90%, 100%, and 100% respectively. Hardly any radioprotection was obtained from LFA-1pos cells. Similarly, in cytokine (IL-8 and G-CSF)–mobilized blood, the LFA-1neg fraction, which comprised 5% to 10% of the MNC, contained the majority of the colony-forming cells, as well as almost all cells with radioprotective capacity. Subsequently, primitive bone marrow-derived HPC, represented by Wheat-germ-agglutinin (WGA)+/Lineage (Lin)−/Rhodamine (Rho)− sorted cells, were examined. More than 95% of the Rho− cells were LFA-1neg. Cultures of sorted cells showed that the LFA-1neg fraction contained all CFU. Transplantation of 150 Rho− LFA-1neg or up to 600 Rho−LFA-1pos cells protected 100% and 0% of lethally irradiated recipient mice, respectively. These results show that primitive murine HPC in steady-state bone marrow and of cytokine-mobilized blood do not express LFA-1.

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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 ◽

10.1182/blood.v90.2.658.658_658_668 ◽

1997 ◽

Vol 90 (2) ◽

pp. 658-668 ◽

Cited By ~ 8

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

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 <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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Evaluation of ex vivo expansion potential of cord blood and bone marrow hematopoietic progenitor cells using cell tracking and limiting dilution analysis

Blood ◽

10.1182/blood.v85.8.2059.bloodjournal8582059 ◽

1995 ◽

Vol 85 (8) ◽

pp. 2059-2068 ◽

Cited By ~ 115

Author(s):

CM Traycoff ◽

ST Kosak ◽

S Grigsby ◽

EF Srour

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Progenitor Cells ◽

Cell Tracking ◽

Ex Vivo ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Cells In Culture ◽

Cd34 Cells ◽

Limiting Dilution

In the absence of conclusive assays capable of determining the functionality of ex vivo expanded human hematopoietic progenitor cells, we combined cell tracking with the membrane dye PKH2, immunostaining for CD34, and limiting dilution analysis to estimate the frequency of long-term hematopoietic culture-initiating cells (LTHC-ICs) among de novo-generated CD34+ cells. Umbilical cord blood (CB) and bone marrow (BM) CD34+ cells were stained with PKH2 on day 0 and cultured with stem cell factor (SCF) and interleukin-3 (IL-3) in short-term stromal cell-free suspension cultures. Proliferation of CD34+ cells in culture was tracked through their PKH2 fluorescence relative to day 0 and the continued expression of CD34. As such, it was possible to identify cells that had divided while maintaining the expression of CD34 (CD34+PKH2dim) and others that expressed CD34 but had not divided (CD34+PKH2bright). In all such cultures, a fraction of both BM and CB CD34+ cells failed to divide in response to cytokines and persisted in culture for up to 10 days as CD34+PKH2bright cells. Between days 5 and 7 of culture, CD34+PKH2bright and CD34+PKH2dim cells were sorted in a limiting dilution scheme into 96-well plates prepared with medium, SCF, IL-3, IL-6, granulocyte-macrophage colony-stimulating factor, and erythropoietin. Cells proliferating in individual wells were assayed 2 weeks later for their content of clonogenic progenitors and the percentage of negative wells was used to calculate the frequency of LTHC-ICs in each population. Among fresh isolated BM and CB CD34+ cells, the frequencies of LTHC-ICs were 2.01% +/- 0.98% (mean +/- SEM) and 7.56% +/- 2.48%, respectively. After 5 to 7 days in culture, 3.00% +/- 0.56% of ex vivo-expanded BM CD34+PKH2bright cells and 4.46% +/- 1.10% of CD34+PKH2dim cells were LTHC-ICs. In contrast, the frequency of LTHC-IC in ex vivo expanded CB CD34+ cells declined drastically, such that only 3.87% +/- 2.06% of PKH2bright and 2.29% +/- 1.75% of PKH2dim cells were determined to be initiating cells after 5 to 7 days in culture. However, when combined with a calculation of the net change in the number of CD34+ cells in culture, the sum total of LTHC-ICs in both BM and CB cells declined in comparison to fresh isolated cells, albeit to a different degree between the two tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

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A phase I trial of monoclonal antibody M195 in acute myelogenous leukemia: specific bone marrow targeting and internalization of radionuclide.

Journal of Clinical Oncology ◽

10.1200/jco.1991.9.3.478 ◽

1991 ◽

Vol 9 (3) ◽

pp. 478-490 ◽

Cited By ~ 157

Author(s):

D A Scheinberg ◽

D Lovett ◽

C R Divgi ◽

M C Graham ◽

E Berman ◽

...

Keyword(s):

Monoclonal Antibody ◽

Bone Marrow ◽

Phase I ◽

Progenitor Cells ◽

Phase I Trial ◽

Whole Body ◽

Hematopoietic Progenitor Cells ◽

Target Cells ◽

Hematopoietic Progenitor

Ten patients with myeloid leukemias were treated in a phase I trial with escalating doses of mouse monoclonal antibody (mAb) M195, reactive with CD33, a glycoprotein found on myeloid leukemia blasts and early hematopoietic progenitor cells but not on normal stem cells. M195 was trace-labeled with iodine-131 (131I) to allow detailed pharmacokinetic and dosimetric studies by serial sampling of blood and bone marrow and whole-body gamma-camera imaging. Total doses up to 76 mg were administered safely without immediate adverse effects. Absorption of M195 onto targets in vivo was demonstrated by biopsy, pharmacology, flow cytometry, and imaging; saturation of available sites occurred at doses greater than or equal to 5 mg/m2. The entire bone marrow was specifically and clearly imaged beginning within hours after injection; optimal imaging occurred at the lowest dose. Bone marrow biopsies demonstrated significant dose-related uptake of M195 as early as 1 hour after infusion in all patients, with the majority of the dose found in the marrow. Tumor regressions were not observed. An estimated 0.33 to 1.0 rad/mCi 131I was delivered to the whole body, 1.1 to 6.1 rad/mCi was delivered to the plasma, and up to 34 rad/mCi was delivered to the red marrow compartment. 131I-M195 was rapidly modulated, with a majority of the bound immunoglobulin G (IgG) being internalized into target cells in vivo. These data indicate that whole bone marrow ablative doses of 131I-M195 can be expected. The rapid, specific, and quantitative delivery to the bone marrow and the efficient internalization of M195 into target cells in vivo also suggest that the delivery of other isotopes such as auger or alpha emitters, toxins, or other biologically important molecules into either leukemia cells or normal hematopoietic progenitor cells may be feasible.

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Targeted transduction of CD34+ hematopoietic progenitor cells in nonpurified human mobilized peripheral blood mononuclear cells

The Journal of Gene Medicine ◽

10.1002/jgm.1290 ◽

2009 ◽

Vol 11 (3) ◽

pp. 185-196 ◽

Cited By ~ 16

Author(s):

Min Liang ◽

Nonia Pariente ◽

Kouki Morizono ◽

Irvin S. Y. Chen

Keyword(s):

Progenitor Cells ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Peripheral Blood Mononuclear ◽

Blood Mononuclear Cells ◽

Mobilized Peripheral Blood

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Adhesion of Hematopoietic Progenitor Cells to Human Mesenchymal Stromal Cells as a Model for Interaction between Stem Cells and Their Niche.

Blood ◽

10.1182/blood.v108.11.1399.1399 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1399-1399

Author(s):

Wolfgang Wagner ◽

Frederik Wein ◽

Christoph Roderburg ◽

Vladimir Benes ◽

Anke Diehlmann ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Progenitor Cells ◽

Hematopoietic Progenitor Cells ◽

Cell Contact ◽

Hematopoietic Progenitor ◽

Adhesion Proteins ◽

Cd34 Cells ◽

Cell Cell

Abstract Objective: The significant role of direct contact between hematopoietic progenitor cells (HPC) and the cellular microenvironment for maintaining “stemness” has been demonstrated. Human mesenchymal stromal cell (MSC) feeder layers represent a surrogate model for this interaction. The molecular composition of this heterotypic cell-cell contact is yet unknown. Methods: To define this cell-cell contact between HPC and MSC, we have studied adhesion of various fractions of HPC with different preparations of MSC by using a novel assay based on gravitational force upon inversion. Adherent and non-adherent cells were then separated. Gene expression analysis by microarray (GeneChip Human Genome U133_Plus_2.0, Affymetrix) of the two populations was performed and the relationship to long-term hematopoietic culture initiating cell (LTC-IC) frequency examined. Results: HPC subsets with higher self-renewing capacity demonstrated significantly higher adherence to MSC from human bone marrow (CD34+vs. CD34−, CD34+/CD38−vs. CD34+/CD38+, slow dividing fraction vs. fast dividing fraction). LTC-IC frequency was significantly higher in the adherent fraction than in the non-adherent CD34+ cells, thus providing evidence for specific adhesive interaction of primitive HPC with MSC. Genes coding for adhesion proteins and extracellular matrix were highly expressed in the adherent fraction compared to non-adherent CD34+ cells. These genes included fibronectin1 (FN1), cadherin11, VCAM1, connexin43 and ITGBL1. Furthermore, affinity of CD34+ cells was analyzed on human MSC isolated from bone marrow (BM), adipose tissue (AT) and cord blood (CB). Affinity to BM-MSC was significantly higher compared to AT-MSC and CB-MSC. Gene expression in different MSC preparations (BM-MSC, AT-MSC and CB-MSC) correlated in various adhesion proteins with the differences observed in affinity of HPC (including cadherin11, VCAM1, N-cadherin, ITGB1, ITGA1, ITGA5, SDF-1 and osteopontin). Western blot analysis also confirmed higher protein expression of FN1, cadherin11, N-cadherin and ITGB1 in BM-MSC compared to AT-MSC and CB-MSC. Conclusion: MSC represent a model for the human hematopoietic niche. Primitive subsets of HPC have significantly higher affinity to BM-MSC. The essential role of specific junction proteins (cadherin11, VCAM1, N-cadherin) for stabilization of cell-cell contact is indicated by their significant higher expression on both sides of the heterotypic interaction.

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