Impact of Erythropoietin Modulation Using Hyperbaric Oxygen on Umbilical Cord Blood CD34+ Cell Homing

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1870-1870 ◽  
Author(s):  
Omar S. Aljitawi ◽  
Tara L. Lin ◽  
George Vielhauer ◽  
Aaron Cheung ◽  
Maegan L. Capitano ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Western Blot ◽  
Umbilical Cord Blood ◽  
Murine Model ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Post Transplant ◽  
Cd34 Cells ◽  
Epor Expression

Abstract Background: Previously, we demonstrated that host pre-treatment with hyperbaric oxygen (HBO) improves engraftment of umbilical cord blood (UCB) CD34+ cells in a transplant murine model. Recent data suggest that erythropoietin (EPO) determines the fate of hematopoietic progenitor cells in favor of erythroid differentiation. Herein, we evaluated mechanisms underlying the favorable effect of HBO therapy on UCB CD34+ cell engraftment. We hypothesized that HBO modulates EPO which in turn enhances UCB CD34 homing to the marrow, thereby enhancing engraftment. Materials/methods: We examined EPO receptor (EPOR) expression on UCB CD34+ cells by flow cytometry and western blot. Transmigration assays were used to evaluate EPO effects on UCB CD34+ cell migration toward Stromal Cell-Derived Factor 1 (SDF-1; CXCL12) which has been shown to mediate CD34+ chemotaxis to the marrow. Using an NSG transplant murine model, we evaluated serum EPO levels by ELISA and early UCB bone marrow/spleen homing by flow cytometry. Bone marrow UCB cell differentiation was examined 1 and 2 weeks post-transplant using CFU-assay. Results: We found that on average 17.6 (4.9-38.7) % of CD34+ enriched UCB cells express EPOR. EPOR expression was confirmed by western blot. The percentage of EPOR positive cells was significantly higher in hematopoietic stem cells (HSC) defined as Lin- CD34+ CD38- CD45RA- CD90+ CD49f+ (45.7+/-1.4) compared to hematopoietic progenitor cells defined as Lin- CD34+ CD38+ (25.1+/-0.7) and multipotent progenitors defined as Lin- CD34+ CD38- CD45RA- CD90- CD49f- (27.2+/-0.3). Also, exposure of UCB CD34+ cells to EPO during cell culture inhibited their migration toward SDF-1 (Figure-1). HBO treated mice demonstrated significantly lower serum EPO blood levels compared to control mice (727.4+/- 42.02 versus1576 +/- 80.90 pg/mL, p <0.0001) measured 3-hours post-transplant. A higher percentage of human CD34+ was seen in the bone marrow of HBO treated mice 3-hours post-transplant (6.8+/-1.0 versus 3.4+/-0.6), p=0.01). HBO treated mice demonstrated significantly lower numbers of BFU-E ( 26.7+/-8.7 versus 75.3+/-19.3, p =0.043) and increasing numbers of CFU-G/M (166.0+/-27.9 versus 74.8+/-32.6, p =0.05). Conclusions: EPOR is enriched in UCB HSC. HBO lowers host blood EPO and as a result improves UCB CD34+ cell early bone marrow homing and enhances myeloid differentiation. Figure 1. EPO effects on UCB CD34+ cell transmigration toward SDF-1 gradient. Figure 1. EPO effects on UCB CD34+ cell transmigration toward SDF-1 gradient. Disclosures No relevant conflicts of interest to declare.

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.

Osteomyelosclerosis, Anemia and Extramedullary Hematopoiesis in Mice Deficient for the Transcription Factor NFAT (Nuclear Factor of Activated T Cells) c2

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3726-3726
Author(s):  
Satu Kyttälä ◽  
Wolfgang Bauer ◽  
Michael Haase ◽  
Ivonne Habermann ◽  
Gerhard Ehninger ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Extramedullary Hematopoiesis ◽  
Hematopoietic Progenitor Cells ◽  
Nuclear Factor ◽  
Bone Marrow Stroma ◽  
Hematopoietic Progenitor ◽  
Activated T Cells ◽  
Marrow Stroma

Abstract NFAT (Nuclear Factor of Activated T cells) is a family of five calcium-induced, calcineurin-dependent transcription factors, characterized as master regulators of inducible gene expression in T lymphocytes. In various tissues (including bone, cartilage and adipose tissue), NFAT factors have also been found to regulate processes of cellular adaptation and differentiation. NFAT family members are also expressed in human CD34+ hematopoietic progenitor cells as well as in mesenchymal stem cells; however, a biological function of NFAT in these cells, such as a role in hematopoietic differentiation, has not yet been described. To determine a potential role for NFAT in hematopoiesis, we characterized the steady-state hematopoiesis in NFATc2 knockout (KO) mice by analyzing peripheral blood, bone marrow and extramedullary tissue (spleen and liver) of KO and age-matched wildtype (WT) mice by cytology, histopathology as well as flow cytometry. No significant abnormalities were observed in KO mice of young age (6–12 weeks). NFATc2 KO mice of older age (>12 months), however, were found to be anemic, with a significant decrease in red blood cell counts as well as in hemoglobin and hematocrit values, and also showed decreased numbers of peripheral blood platelets as compared to the WT. In contrast, no significant differences between WT and KO were found in total counts of white blood cells or in leukocyte differentials. Femora of NFATc2 KO mice appeared macroscopically pale and, upon elutriation, gave yield to less than 50% of bone marrow cells compared to the WT, indicating bone marrow hypoplasia. Further analysis of bone marrow differentials by morphology and flow cytometry showed a profound decrease in erythroid cells as well as in megakaryocytes. Histopathological examination of KO bone marrow sections revealed evidence of bone marrow sclerosis, as indicated by an increase in bone trabeculae and osteoblasts within the marrow space. In some mice, an increase in reticulin fiber content was also noted. Interestingly, both liver and spleens of NFATc2 KO mice showed clear evidence for extramedullary hematopoiesis, suggesting that displacement of hematopoietic cells by the bone marrow stroma may be a possible cause of the observed cytopenias. These results show that NFATc2 knockout mice over their lifespan develop a phenotype, which clinically resembles human osteomyelofibrosis/-sclerosis. The underlying mechanism for the observed hematopoietic abnormalities has to be defined, in particular the respective contribution of hematopoietic and bone marrow stroma cells. Given that NFAT factors are expressed at considerable levels in both hematopoietic progenitor cells and mesenchymal stem cells, our results should encourage further studies on the yet undefined role of these transcription factors within the bone marrow hematopoietic niche.

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

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.

scholarly journals Interleukin-10 increases Bcl-2 expression and survival in primary human CD34+ hematopoietic progenitor cells

Blood ◽  
1996 ◽  
Vol 88 (7) ◽  
pp. 2549-2558 ◽  
Author(s):  
RM Weber-Nordt ◽  
R Henschler ◽  
E Schott ◽  
J Wehinger ◽  
D Behringer ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Survival ◽  
Progenitor Cells ◽  
Colony Growth ◽  
Interleukin 10 ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Gm Csf ◽  
Human Cd34 ◽  
Cd34 Cells

Bcl-2 expression has been shown in hematopoietic progenitor cells. Through the use of Bcl-2 specific antisense oligonucleotides we herein report the biologic importance of Bcl-2 expression in primary human CD34+ hematopoietic progenitor cells committed to the myeloid lineage. In bone marrow or peripheral blood derived CD34+ cells Bcl-2 specific antisense decreased cell survival and inhibited the outgrowth of mixed myeloid colonies. A short-term overnight pretreatment of CD34+ cells with 25 mumol/L of Bcl-2 antisense in liquid culture completely ablated the growth of granulocyte-macrophage colony-forming cells (GM-CFC) in a subsequent 14 days methylcellulose colony assay. Control experiments using corresponding Bcl-2 sense or nonsense oligonucleotides did not significantly impair cell survival or growth of GM-colony-forming unit. Western blot analyses revealed the Bcl-2 antisense dependent inhibition of expression of the Bcl-2 protein in CD34+ progenitor cells. Furthermore, regulation of Bcl-2 expression by various cytokines including interleukin-10 (IL-10) was studied. IL-10′s effects on the formation of mixed myeloid colonies were examined in the absence or presence of Bcl-2 specific antisense. In the absence of Bcl-2 antisense IL-10 significantly extended the colony forming potential of mixed myeloid colonies to 14 days. In the presence of Bcl-2 antisense rhIL-10 completely restored GM-CSF driven colony growth. Fluorescent microscopy, Western blot analysis, and reverse transcriptase-polymerase chain reaction revealed the IL-10 dependent increase in cellular expression of Bcl-2 protein and Bcl-2 mRNA transcripts in CD34+ cells. Thus these results show that Bcl-2 expression is necessary for the formation of GM-CSF-dependent colony growth in vitro and that rhIL-10 increases Bcl-2 expression and survival in primary human CD34+ hematopoietic progenitor cells that are committed to the myeloid lineage.

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

Blood ◽  
1995 ◽  
Vol 85 (8) ◽  
pp. 2059-2068 ◽  
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)

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

Blood ◽  
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.

Identification of Highly Clonal Cells in CD34+ and Unselected Bone Marrow Samples From Patients with Myelodysplastic Syndrome Using a Sensitive Quantitative PCR Approach.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1760-1760
Author(s):  
Maximilian Mossner ◽  
Daniel Nowak ◽  
Ouidad Benlasfer ◽  
Jana Reins ◽  
Olaf Joachim Hopfer ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Progenitor Cells ◽  
Quantitative Pcr ◽  
X Chromosome ◽  
Hematologic Malignancies ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 1760 Poster Board I-786 Myelodysplastic syndromes (MDS) are clonal hematologic malignancies with molecular defects most probably arising in the hematopoietic stem or progenitor compartment. However, due to a frequent lack of trackable cytogenetic aberrations in a large proportion of MDS patients the capability to monitor the manifestation and origin of malignant MDS clones remains limited. To elucidate clonal dominance in a given cell population, the analysis of skewed X-chromosome inactivation patterns in females, based on the methylation analysis of X-chromosomal HUMARA alleles has been used widely. However, this method has several technical and biological drawbacks as methylation changes can be induced with increasing age leading to inaccuracy of the method in this context. Recently, the application of a quantitative PCR-based method to accurately detect single nucleotide polymorphism (SNP) allele-specific RNA transcription from the X-chromosome (Swierczek et al, Blood, 2008) has shown robust results for reliable calculation of X-chromosome allelic ratios. In our study we employed this method to assess clonality in CD34+ selected and unselected bone marrow cells derived from MDS patients and provide evidence for distinct clonal manifestations of MDS clones in hematopoietic progenitor cells of all analysed MDS samples as compared to healthy controls. Bone marrow (BM) cells were obtained from patients with MDS (IPSS-low/int-1-risk n=9, IPSS-int-2/high-risk n=9) after informed consent. Immunomagnetic selection of CD34+ cells was performed from the BM samples of MDS patients (IPSS-low/int-1-risk n=8, IPSS-int-2/high-risk n=10) and age related healthy donors (n=6) served as controls for normalization. Genomic DNA SNP genotyping using Taqman SNP Genotyping Assays (Applied Biosystems, Foster City, CA) was carried out in order to screen for informative clonality marker genes located on the X-chromosome, namely BTK, FHL1, IDS and MPP1. RNA transcripts from different alleles were quantified using SNP/allele-specific primers in a Taqman based quantitative PCR approach. Individual allelic ratios were calculated as previously described. Clonality values were assigned to 0 % according to an allelic ratio of 50/50 (polyclonal) up to 100 % for a ratio of 100/0 (clonal). All clonality values are presented as mean. Our analyses revealed a remarkably elevated proportion of clonal cells in all purified CD34+ cells from MDS low/int-1-risk (88 %) and MDS int-2/high-risk patients (98 %) compared to the cells from healthy donors (16 %, p<0.001). The degree of clonality in unselected BM samples was similarly increased in MDS low/int-1-risk (74 %) and MDS int-2/high-risk specimen (87 %, both p<0.001 as compared to controls). However, whereas all purified CD34+ samples from MDS patients appeared to be highly clonal, 2 of 9 (22 %) of the MDS low/int-1-risk samples exhibited distinctively lower clonality in unselected BM cells with values comparable to the healthy control group. Furthermore, we observed nearly identical high clonality values in 12 paired BM/CD34+ MDS samples, except for 1 of 6 MDS low/int-1-risk samples with significantly lower clonality in the unselected bone marrow as compared to purified CD34+ cells of the same patient. Our observation of specific clonality in both unselected bone marrow and purified CD34+ cells of MDS patients as compared to healthy controls underlines the proliferative manifestation of malignant MDS clones even in early hematopoietic progenitor cells. Furthermore, the high degree of clonality in all purified CD34+ cells suggests a clonal involvement of not only myeloid but also lymphoid lineages. Interestingly, we also identified 2 patients harboring polyclonal cells in the unselected bone marrow. In these cases differentiating cells in the bone marrow may be sustained by residual healthy hematopoietic progenitor cells. The determination whether patients with this constellation have a different clinical prognosis from patients with clonality of the complete bone marrow may be interesting to pursue. In summary, determination of clonality levels in distinct cell populations of hematologic malignancies using quantitative PCR appears to be highly suitable for monitoring the manifestation and origin of a malignant hematopoietic stem/precursor cell. 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.

Ultrastructural Features of CD34+Hematopoietic Progenitor Cells from Bone Marrow, Peripheral Blood and Umbilical Cord Blood

2001 ◽  
Vol 42 (4) ◽  
pp. 699-708 ◽  
Author(s):  
Giorgio Lambertenghi Deliliers ◽  
Lorenza Caneva ◽  
Rossella Fumiatti ◽  
Federica Servida ◽  
Paolo Rebulla ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Peripheral Blood ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor

Expression of Complex Junction Proteins in Hematopoietic Progenitor Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1282-1282 ◽  
Author(s):  
Beate K. Straub ◽  
Volker Eckstein ◽  
Christine Grund ◽  
Katrin Miesala ◽  
Kerstin Horsch ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Adherens Junctions ◽  
Actin Binding ◽  
Hematopoietic Progenitor Cells ◽  
Bovine Bone ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Junction Protein

Abstract The interaction of hematopoietic stem cells with the microenvironment plays a pivotal role in regulating self-renewal and differentiation. N-cadherin-based adherens junctions have been reported to be involved in this process (Puch et al., 2001; Zhang et al., 2003). However, the molecular composition of such junctions in hematopoietic progenitor cells (HPC) remains to be defined as well as their function. CD34+ cells and CD34+/CD38− cells were isolated from human umbilical chord blood (CB). In immunoblotting, CD34+ cells were positive for the transmembrane proteins N- and E-cadherin, cadherin 11 (low amounts) and the cytoplasmic plaque proteins α- and β-catenin, protein p120ctn, vinculin, spectrin/fodrin and actin as markers for adherens junctions as well as for ezrin, moesin and drebrin, actin-binding proteins known to be enriched in cellular protrusions. Other classical cadherins such as VE-, P-, R-cadherin or cadherin-6 were negative. Moreover HPCs were positive for the intermediate filament cytoskeletal protein vimentin, the integral heparan sulphate proteoglycans syndecan-1 (CD138), syndecan-2 (fibroglycan), syndecan-3 (N-syndecan) and syndecan-4 (ryudocan, amphiglycan) and the gap junction protein connexin 43. CD34+/CD38− cells showed positive reactions with antibodies against vimentin, ezrin, vinculin, α- and β-catenin and with a pan cadherin antibody, with no obvious differences to CD34+/CD38+ and Kg1a cells. In parallel, we established single- and double-immunohistochemistry with antigen-retrieval-treated, formalin-fixed, paraffin-embedded bone marrow using peroxidase, alkaline phosphatase and glucose oxidase coupled secondary antibodies. The architecture of human, rat and bovine bone marrow tissue was analyzed with a panel of antibodies against components of adhering, gap and tight junctions. Immunohistochemical sections of bone marrow incubated with the various antibodies remarkably showed the presence of all these antigens in their natural microenvironment. Intercellular junctions of rat and bovine bone marrow were also observed with transmission electron microscopy. Our results show that HPC are equipped with components of cadherin-catenin-based adherens junctions. Immunoprecipitation and immunolocalisation experiments are underway to further elucidate the homo- and heterotypic intercellular junctional complexes between HPC and stromal cells in vitro and in vivo.

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