scholarly journals CD34-positive cells and their subpopulations characterized by flow cytometry analyses on the bone marrow of healthy allogenic donors

2009 ◽  
Vol 127 (1) ◽  
pp. 12-18 ◽  
Author(s):  
Jerusa Martins Carvalho ◽  
Marlon Knabben de Souza ◽  
Valéria Buccheri ◽  
Cláudia Viviane Rubens ◽  
José Kerbauy ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Antigen Expression ◽  
Cross Sectional Study ◽  
Cross Sectional ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Before And After ◽  
Marrow Cells

CONTEXT AND OBJECTIVE: Counting and separating hematopoietic stem cells from different sources has importance for research and clinical assays. Our aims here were to characterize and quantify hematopoietic cell populations in marrow donors and to evaluate CD34 expression and relate this to engraftment. DESIGN AND SETTING: Cross-sectional study on hematopoietic stem cell assays, using flow cytometry on donor bone marrow samples, for allogenic transplantation patients at two hospitals in São Paulo. METHODS: Immunophenotyping of marrow cells was performed in accordance with positive findings of CD34FITC, CD117PE, CD38PE, CD7FITC, CD33PE, CD10FITC, CD19PE, CD14FITC, CD13PE, CD11cPE, CD15FITIC, CD22PE, CD61FITC and CD56PE monoclonal antibodies in CD45PerCP+ cells, searching for differentiation and maturation regions. CD34+ sorting cells were analyzed for CD38 and CD117. Rh-123 retention was done before and after sorting. Antigen expression and CD34+ cells were correlated with engraftment. RESULTS: In region R1, 0.1% to 2.8% of cells were CD34+/CD45+ and 1.1%, CD34+/CD45-. The main coexpressions of CD45+ cells were CD38, CD22, CD19 and CD56 in R2 and CD33, CD11c, CD14, CD15 and CD61 in R3 and R4. After sorting, 2.2x10(6) CD34+ cells were equivalent to 4.9% of total cells. Coexpression of CD34+/CD38+ and CD34+/CD117+ occurred in 94.9% and 82% of events, respectively. There was a positive relationship between CD34+ cells and engraftment. More than 80% of marrow cells expressed high Rh-123. CD34+ cell sorting showed that cells in regions of more differentiated lineages retained Rh-123 more intensively than in primitive lineage regions. CONCLUSION: We advocate that true stem cells are CD34+/CD45-/CD38-/low-Rh-123 accumulations.

Increased Engraftment after Double Cord Blood Transplantation in NOD/SCID Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1199-1199
Author(s):  
Alma J. Nauta ◽  
Alwine B. Kruisselbrink ◽  
Roelof Willemze ◽  
Willem E. Fibbe
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Cord Blood ◽  
Cord Blood Transplantation ◽  
Human Platelets ◽  
Hematopoietic Stem ◽  
Cell Dose ◽  
Blood Transplantation ◽  
Cd34 Cells

Abstract Umbilical cord blood (UCB) is considered as an attractive alternative source of hematopoietic stem cells for allogeneic stem cell transplantations in patients who lack HLA-matched donors. However, the low cell dose adversely affects the speed of hematopoietic recovery and therefore limits the application of UCB transplantation in adults. Although ex-vivo expansion of cord blood cells has been explored as a strategy to increase the cell dose, compromised engraftment potential of expanded cells has been demonstrated. Another approach to overcome cell dose limitations is transplantation of multiple, unrelated UCB units. To investigate the effect of multiple cord transplantation on engraftment, NOD/SCID mice were transplanted with human hematopoietic progenitor cells (CD34+) derived from two UCB units with HLA disparity. During the first six weeks after transplantation the number of human platelets in peripheral blood was quantified by flow cytometry. Six weeks after transplantation, the mice were sacrificed and the percentage and donor origin of human CD45+ cells in blood, and in bone marrow was determined by flow cytometry. Transplantation of CD34+ cells derived from two UCB donors resulted in significantly higher number of human platelets in peripheral blood than transplantation of CD34+ cells from either donor alone, ranging from 3.92x106/ml to 10.29x106/ml (mean 6.4x106 ± 2.55x106/ml) and 0.11x106/ml to 3.12.106/ml (mean 1.42x106 ± 1.17x106/ml), respectively. Furthermore, the overall human cell engraftment level in bone marrow after double cord blood transplantation ranged from 7.01% to 64.34% (mean 29.6 ± 21.5%) a nearly 7-fold increase compared to single cord blood transplantation ranging from 0.27% to 13.5% (mean 4.6 ± 3.8%) Although consistently higher engraftment levels were reached after double cord blood transplantation, two different patterns were observed: in 2 out of 4 experiments cells from one donor predominated the engraftment (ratio 3:1), while in two other experiments the two units contributed equally to BM engraftment. The mechanism underlying these effects are <S>is</S> not yet clear. It is not very likely that the single donor predominance results from an unequal amount of hematopoietic stem cells in the cord blood units because each cord blood showed comparable levels of engraftment as a single unit. Alternatively, the unequal engraftment may result from an immunological competition or a graft versus graft stimulatory effect between the cords during the engraftment process and further studies are required to determine if the contribution of both units is dependent on the degree of HLA matching between the two cords. Taken together, these results demonstrate that double cord blood transplantation may represent a means of achieving increased engraftment, making multiple cord blood transplantation a promising strategy to improve the outcome of UCB transplantation. Studies are underway to unravel the mechanisms underlying the enhanced engraftment.

Gfi-1 Is Overexpressed on Chronic Myeloid Leukemic Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2928-2928
Author(s):  
Hui Cheng ◽  
Wei Chang ◽  
Ming Huang ◽  
Ying Sun ◽  
Yicheng Zhang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Fluorescence Intensity ◽  
Zinc Finger Protein ◽  
Interleukin 2 ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Control Samples

Abstract Gfi-1 oncogene was first discovered as an integration site for Moloney murine leukemia virus(MoMulv) in virally infected cells that were selected for interleukin-2 (IL-2) independence; In synergy with pim-1 and L-Myc genes, Gfi-1 was involved in the pathogenesis of lymphoid malignancies. Recent studies also indicated that Gfi-1 regulates self-renewal and engraftment of hematopoietic stem cells and the development of neutrophils. Normally, Gfi-1 zinc finger protein is expressed almost exclusively on thymocytes or stem cells. Chronic myeloid leukemia (CML) is a typical hematopoietic stem cell disease, we ask whether Gfi-1 is expressed on CML cells and the possible role in the leukemogenesis. By RT-PCR and flow cytometry, we analysed the expression of Gfi-1 in the transcription and the protein levels respectively on bone marrow mononuclear cells(MNC) from 53 CML patients. Control samples were taken from the ribs of 5 patients undergoing thoracic surgery for lung cancer. Results Gfi-1 mRNA was detected in K562 cell line and all the MNCs from CML patients but not or very weak in control MNCs; Flow cytometry study show that both the positive percentage (87.2±7.5% Vs 12.4±6.6%, p<0.01) and the relative fluorescence intensity (represented by mean fluorescence intensity, 58.94 ±22.09 Vs 18.67±7.88, p<0.01) were significantly increased in CML MNCs. Considering the complexity of the composition of bone marrow MNCs, we also sorted CD34+ cells from CML or control samples and examined by Rt-PCR and flow cytometry; The Gfi-1 was found to be overexpressed in CD34+ cells of CML; On confocal microscopy, Gfi-1 was mainly sublocalized in nucleus. Conclusion Gfi-1 was overexpressed in MNCs or CD34+ cells from CML patients; Gfi-1 may be implicated in the leukemogenesis of CML and potentially be targeted in the future for treatment of CML.

scholarly journals Exploration of a 3D Scaffold Culture System for Ex Vivo Blood Production

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4371-4371
Author(s):  
Charlotte E Severn ◽  
Ashley M Toye
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Small Sample ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Adult Peripheral Blood

Abstract The human body produces 2.5 million red blood cells (RBC) every second in the bone marrow, where differentiation of immature erythroblasts to reticulocytes occurs largely within erythroblastic islands. Erythropoiesis can be routinely replicated in 2D liquid culture using hematopoietic stem cells (HSC) isolated from adult peripheral blood. However current 2D culture methodologies eventually exhaust the input of HSC. The use of 3D scaffolds to better mimic erythropoiesis in the bone marrow would increase the RBC yield and longevity of ex vivo cultures, whilst reducing use of exogenous cytokines and minimizing handling requirements. As a starting point we are utilizing a porous collagen coated synthetic polyurethane (PU) scaffold (0.5cm by 0.5cm) for static 3D cultures provided by the Mantalaris group at Imperial College London (Mortera-Blanco et al., 2011). The PU scaffolds were seeded with lineage depleted or CD34+ population isolated from adult peripheral blood and maintained in serum-free erythroid expansion media with SCF, IL-3 and Dexamethasone, alone, or with erythropoietin (EPO) or thrombopoietin (TPO). Advantages of using the lineage depleted population is that it provides a larger diversity of stem cells for establishment of the niche, potentially facilitating the use of rare patient blood samples which may only be available in small sample volumes with low numbers of CD34+ cells. Scaffolds are productive using both cellular inputs, with significant cellular egress for up to 5 weeks regardless of whether exogenous EPO or TPO were included. As anticipated, the highest increase in cell production from the scaffolds was observed using CD34+ in the presence of EPO, which also provided a significant reduction in cell death. Histology and immunofluorescence were used to explore the cell populations within the scaffold. No mature macrophages were detected but GPA+ cells within the scaffold was observed until the end of culture, suggesting that cellular expansion is occurring without establishment of the classical macrophage niche. We also characterized the cells that continually egress from the CD34+ scaffold cultures using flow cytometry. Typically the cellular output exhibited approximately 20-60% CD34 positivity dropping to <15% post day 24 of culture and 10-30% GPA positivity, the remainder of the population was largely CD61 positive, with a contingent of CD14 positive monocytes. Mature cell surface markers for erythroid and megakaryocytic lineages were detected in up to half of the population, when the harvested 3D egress cells were further cultured in erythroid or megakaryocyte 2D culture systems. This suggests a high proportion of the cells that egress from the scaffold are megakaryocyte erythroid progenitors (MEPs) that are consistently expanding within the scaffold environment for the entire culture period. Since CD34+ expansion here has been achieved in the absence of macrophages, we wanted to explore their effects in 2D with the intention of introducing macrophages or macrophage derived functionality into our next generation scaffolds. Importantly, macrophage inclusion significantly increased proliferation of expanding erythroblasts compared to erythroblasts alone. Expansion of CD34+ cells in co-culture gave a statistically significant average fold increase of 528 compared to 301 for the control at day 7 (p = 0.0126 and 0.0162 for days 5 and 7 respectively (n=5)). Flow cytometry at the endpoint of the experiment showed a larger CD34+ population and a reduced GPA+ population when cells are in co-culture, suggesting the CD34+ cells are maintained in a more primitive state for longer. Therefore, co-culture has the additional benefit of improving early erythroblast expansion, alongside the recently reported enhanced expansion of erythroblasts during terminal differentiation (Ramos et al., 2013). In summary, we have demonstrated that a basic static PU scaffold can be utilized to increase hematopoietic stem cell culture longevity and facilitate generation of megakaryocyte or erythroid progenitors with expansion potential. This occurs in the absence of any detectable macrophage niche generation. We have also shown that co-culture with macrophages enhances erythroblast expansion in 2D. Further work is needed to determine whether inclusion of macrophages or macrophage derived proteins in our scaffolds will effectively boost progenitor production. Disclosures No relevant conflicts of interest to declare.

Fluorophore Conjugated Iron Oxide Nanoparticle Labeling and Analysis of Engrafting Human Hematopoietic Stem Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1213-1213
Author(s):  
Dustin Maxwell ◽  
David Hess ◽  
Jesper Bonde ◽  
Sarah A. Hohm ◽  
Ryan Lahey ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Iron Oxide ◽  
Cell Surface ◽  
Human Stem Cells ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Different Tissues

Abstract The use of nanometer-sized iron oxide particles combined with molecular imaging techniques enable dynamic studies of homing and trafficking of human hematopoietic stem cells (HSC). Identifying clinically applicable strategies for loading nanoparticles into primitive HSC requires strictly defined culture conditions to maintain viability without inducing terminal differentiation. In the current study, fluorescent molecules were covalently linked to dextran-coated iron oxide nanoparticles (Feridex) to characterize human HSC labeling to monitor the engraftment process. Conjugating fluorophores to the dextran coat for FACS purification eliminated spurious signals from non-sequestered nanoparticle contaminants. A short-term defined incubation strategy was developed which allowed efficient labeling of both quiescent and cycling HSC, with no discernable toxicity in vitro or in vivo. Transplantation of purified primary human cord blood lineage-depleted and CD34+ cells into immunodeficient mice allowed detection of labeled human HSC in the recipient bones. Flow cytometry was used to precisely quantitate the cell populations that had sequestered the nanoparticles, and to follow their fate post-transplantation. Flow cytometry endpoint analysis confirmed the presence of nanoparticle-labeled human stem cells in the marrow. Using the current techniques, flow cytometry analysis gating on CD34+ or CD45+ expression and Fe[750] confirmed the presence of human cells residing in the BM. Using this method, as few as 1 × 105 Fe[750]+CD34+ cells residing in the bone marrow could be detected by fluorescence imaging. Beyond two weeks, the human cell expansion, egress from the marrow, and iron metabolism began to dilute the nanoparticle signal below the limit of detection of both techniques. The current studies provide a method by which investigators can track human stem cells to the marrow vs. different tissues of immune deficient mice. This has been extremely difficult in the past, because stem cells can alter their phenotype after engraftment. The fluorophore-tagged Feridex allows a clean recovery of labeled cells from different tissues by FACS for cell surface phenotype probing and other assays. Due to the fluorophore modification, quantitation of the number of cells that are engrafted in the bone marrow after transplantation is possible, and allows simultaneous probing of cell surface phenotype using flow cytometry, without the requirement for isolating cells based on a pre-determined cell surface marker. The use of fluorophore-labeled Feridex nanoparticles and the clinically relevant incubation procedure described in the current study offers an efficient and safe method to label both cycling and non-cycling human hematopoietic stem and progenitor cells without toxicity as well as to evaluate the homing, localization, phenotype, and short-term engraftment capabilities of defined human HSC subsets. The current data demonstrate that transient labeling of repopulating HSC subsets with fluorescent nanoparticles is a powerful and novel tool for dynamic tracking of human stem cells during the initial weeks after transplantation.

CD34+/CD33- cells reselected from macrophage inflammatory protein 1 alpha+interleukin-3--supplemented "stroma-noncontact" cultures are highly enriched for long-term bone marrow culture initiating cells

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1442-1449 ◽  
Author(s):  
CM Verfaillie ◽  
JS Miller
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Antigen Expression ◽  
Hematopoietic Stem ◽  
Interleukin 3 ◽  
Inflammatory Protein ◽  
Hla Dr ◽  
Macrophage Inflammatory Protein

Abstract Human hematopoietic stem cells are thought to express the CD34 stem cell antigen, low numbers of HLA-DR and Thy1 antigens, but no lineage commitment antigens, CD38, or CD45RA antigens. However, fluorescence- activated cell sorted CD34+ subpopulations contain not more than 1% to 5% primitive progenitors capable of initiating and sustaining growth in long-term bone marrow culture initiating cells (LTBMC-ICs). We have recently shown that culture of fresh human marrow CD34+/HLA-DR- cells separated from a stromal layer by a microporous membrane (“stroma- noncontact” culture) results in the maintenance of 40% of LTBMC-ICs. We hypothesized that reselection of CD34+ subpopulations still present after several weeks in stroma-noncontact cultures may result in the selection of cells more highly enriched for human LTBMC-ICs. Fresh marrow CD34+/HLA-DR- cells were cultured for 2 to 3 weeks in stroma- noncontact cultures. Cultured progeny was then sorted on the basis of CD34, HLA-DR, or CD33 antigen expression, and sorted cells evaluated for the presence of LTBMC-ICs by limiting dilution analysis. We show that (1) LTBMC-ICs are four times more frequent in cultured CD34+/HLA- DR- cells (4.6% +/- 1.7%) than in cultured CD34+/HLA-DR- cells (1.3% +/- 0.4%). This suggests that HLA-DR antigen expression may depend on the activation status of primitive cells rather than their lineage commitment. We then sorted cultured cells on the basis of the myeloid commitment antigen, CD33. (2) These studies show that cultured CD34+/CD33- cells contain 4% to 8% LTBMC-ICs, whereas cultured CD34+/CD33+bright cells contain only 0.1% +/- 0.03% LTBMC-ICs. Because LTBMC-ICs are maintained significantly better in stroma-noncontact cultures supplemented with macrophage inflammatory protein 1 alpha (MIP- 1 alpha) and interleukin-3 (IL-3) (Verfaillie et al, J Exp Med 179:643, 1994), we evaluated the frequency of LTBMC-ICs in CD34+/CD33- cells present in such cultures. (3) CD34+/CD33- cells present in MIP-1 alpha + IL-3-supplemented cultures contain up to 30% LTBMC-ICs. The increased frequency of LTBMC-ICs in cultured CD34+ subpopulations may be the result of terminal differentiation of less primitive progenitors, loss of cells that fail to respond to the culture conditions or recruitment of quiescent LTBMC-ICs. The capability to select progenitor populations containing up to 30% LTBMC-ICs should prove useful in studies examining the growth requirements, self-renewal, and multilineage differentiation capacity of human hematopoietic stem cells at the single-cell level.

scholarly journals Host Conditioning With 5-Fluorouracil and kit-Ligand to Provide for Long-Term Bone Marrow Engraftment

Blood ◽  
1997 ◽  
Vol 89 (7) ◽  
pp. 2376-2383 ◽  
Author(s):  
Ronald van Os ◽  
Donald Dawes ◽  
John M.K. Mislow ◽  
Alice Witsell ◽  
Peter M. Mauch
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Mixed Chimerism ◽  
Kit Ligand ◽  
Donor Bone Marrow ◽  
Before And After ◽  
Total Body ◽  
Marrow Cells

Abstract Administration of kit-ligand (KL) before and after doses of 5-fluorouracil (5-FU) results in marrow failure in mice, presumably because of enhanced KL-induced cycling of stem cells, which makes them more susceptible to the effects of 5-FU. In attempt to capitalize on this effect on stem cells, we studied the ability of KL and 5-FU to allow stable donor engraftment of congenically marked marrow in a C57BL/6 (B6) mouse model. KL was administered subcutaneously at 50 μg/kg, 21 hours and 9 hours before and 3 hours after each of two doses of 5-FU (125 mg/kg) given 7 days apart to B6-recipients. Animals then received three injections of 107 congenic B6-Gpi-1a-donor bone marrow cells at 24, 48, and 72 hours after the second 5-FU dose. A separate group of animals received a single dose of either 1 × 107 or 3 × 107 donor marrow cells 24 hours after the last 5-FU dose. The level of engraftment was measured from Gpi-phenotyping at 1, 3, 6, and 8 months in red blood cells (RBCs) and at 8 months by phenotyping cells from the thymus, spleen, and marrow. Percent donor engraftment in RBCs appeared stable after 6 months. The percent donor engraftment in RBCs at 8 months was significantly higher in KL + 5-FU prepared recipients (33.0 ± 2.7), compared with 5-FU alone (18.5 ± 2.6, P &lt; .0005), or saline controls (17.8 ± 1.7, P &lt; .0001). In an additional experiment, granulocyte colony-stimulating factor (100 μg/dose) was added to a reduced dose of KL (12.5 μg/dose); engraftment was similar to KL alone. At 8 months after transplantation the levels of engraftment in other tissues such as bone marrow, spleen, and thymus correlated well with erythroid engraftment to suggest that multipotent long-term repopulating stem cells had engrafted in these animals. There are concerns for the toxicity of total body irradiation (TBI)- or busulfan-based regimens in young recipients of syngeneic or transduced autologous marrow who are transplanted for correction of genetic disease. In these recipients complete donor engraftment may not be needed. The results with KL and 5-FU are encouraging for the further refinement of non-TBI, nonbusulfan techniques to achieve stable mixed chimerism.

scholarly journals Long-term monoclonal reconstitution of erythropoiesis in genetically anemic W/Wv mice by injection of 5-fluorouracil-treated bone marrow cells of Pgk-1b/Pgk-1a mice

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1758-1763 ◽  
Author(s):  
T Nakano ◽  
N Waki ◽  
H Asai ◽  
Y Kitamura
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Electrophoretic Pattern ◽  
Phosphoglycerate Kinase ◽  
X Chromosomes ◽  
Hematopoietic Stem ◽  
Colony Forming Units ◽  
Marrow Cells

Abstract The spleen colony-forming assay does not represent the number of hematopoietic stem cells with extensive self-maintaining capacity because five to 50 spleen colony-forming units (CFU-S) are necessary to rescue a genetically anemic (WB X C57BL/6)F1-W/Wv(WBB6F1-W/Wv) mouse. We investigated which is more important for the reconstitution of erythropoiesis, the transplantation of multiple CFU-S or that of a single stem cell with extensive self-maintaining potential. The electrophoretic pattern of hemoglobin was used as a marker of reconstitution and that of phosphoglycerate kinase (PGK), an X chromosome-linked enzyme, as a tool for estimating the number of stem cells. For this purpose, we developed the C57BL/6 congeneic strain with the Pgk-1a gene. Bone marrow cells were harvested after injection of 5- fluorouracil from C57BL/6-Pgk-1b/Pgk-1a female mice in which each stem cell had either A-type PGK or B-type PGK due to the random inactivation of one or two X chromosomes. When a relatively small number of bone marrow cells (ie, 10(3) or 3 X 10(3] were injected into 200-rad- irradiated WBB6F1-W/Wv mice, the hemoglobin pattern changed from the recipient type (Hbbd/Hbbs) to the donor type (Hbbs/Hbbs) in seven of 150 mice for at least 8 weeks. Erythrocytes of all these WBB6F1-W/Wv mice showed either A-type PGK alone or B-type PGK alone during the time of reconstitution, which suggests that a single stem cell with extensive self-maintaining potential may sustain the whole erythropoiesis of a mouse for at least 8 weeks.

scholarly journals Assessment of aldehyde dehydrogenase in viable cells

Blood ◽  
1995 ◽  
Vol 85 (10) ◽  
pp. 2742-2746 ◽  
Author(s):  
RJ Jones ◽  
JP Barber ◽  
MS Vala ◽  
MI Collector ◽  
SH Kaufmann ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Western Blotting ◽  
Aldehyde Dehydrogenase ◽  
Cell Populations ◽  
Intracellular Protein ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Viable Cells

Cytosolic aldehyde dehydrogenase (ALDH), an enzyme responsible for oxidizing intracellular aldehydes, has an important role in ethanol, vitamin A, and cyclophosphamide metabolism. High expression of this enzyme in primitive stem cells from multiple tissues, including bone marrow and intestine, appears to be an important mechanism by which these cells are resistant to cyclophosphamide. However, although hematopoietic stem cells (HSC) express high levels of cytosolic ALDH, isolating viable HSC by their ALDH expression has not been possible because ALDH is an intracellular protein. We found that a fluorescent aldehyde, dansyl aminoacetaldehyde (DAAA), could be used in flow cytometry experiments to isolate viable mouse and human cells based on their ALDH content. The level of dansyl fluorescence exhibited by cells after incubation with DAAA paralleled cytosolic ALDH levels determined by Western blotting and the sensitivity of the cells to cyclophosphamide. Moreover, DAAA appeared to be a more sensitive means of assessing cytosolic ALDH levels than Western blotting. Bone marrow progenitors treated with DAAA proliferated normally. Furthermore, marrow cells expressing high levels of dansyl fluorescence after incubation with DAAA were enriched for hematopoietic progenitors. The ability to isolate viable cells that express high levels of cytosolic ALDH could be an important component of methodology for identifying and purifying HSC and for studying cyclophosphamide-resistant tumor cell populations.

Human Embryonic Stem Cell (hESC)-Derived Hematopoietic Elements Are Capable of Engrafting Primary as well as Secondary Fetal Sheep Recipients.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2685-2685
Author(s):  
A. Daisy Narayan ◽  
Jessica L. Chase ◽  
Adel Ersek ◽  
James A. Thomson ◽  
Rachel L. Lewis ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Peripheral Blood ◽  
Fetal Sheep ◽  
Hematopoietic Stem ◽  
Blood Samples ◽  
Human Dna ◽  
Cd34 Cells ◽  
Hematopoietic Activity

Abstract We used transplantation into 10 and 20 pre-immune fetal sheep recipients (55–65 days-old, term: 145 days) to evaluate the in vivo potential of hematopoietic elements derived from hESC. The in utero human/sheep xenograft model has proven valuable in assessing the in vivo hematopoietic activity of stem cells from a variety of fetal and post-natal human sources. Five transplant groups were established. Non-differentiated hESC were injected in one group. In the second and third group, embroid bodies differentiated for 8 days were injected whole or CD34+ cells were selected for injection. In the fourth and fifth group, hESC were differentiated on S17 mouse stroma layer and injected whole or CD34+ cells were selected for injection. The animals were allowed to complete gestation and be born. Bone marrow and peripheral blood samples were taken periodically up to over 12 months after injection, and PCR and flowcytometry was used to determine the presence of human DNA/blood cells in these samples. A total of 30 animals were analyzed. One primary recipient that was positive for human hematopoietic activity was sacrificed and whole bone marrow cells were transplanted into a secondary recipient. We analyzed the secondary recipient at 9 months post-injection by PCR and found it to be positive for human DNA in its peripheral blood and bone marrow. This animal was further challenged with human GM-CSF and human hematopoietic activity was noted by flowcytometry analyses of bone marrow and peripheral blood samples. Further, CD34+ cells enriched from its bone marrow were cultured in methylcellulose and human colonies were identified by PCR. We therefore conclude that hESC are capable of generating hematopoietic cells that engraft in 1° sheep recipients. These cells also fulfill the criteria for long-term engrafting hematopoietic stem cells as demonstrated by engraftment and differentiation in the 20 recipient.

Role of Osteoclasts in Regulation of Human Hematopoietic Stem Cell Niche and Fate.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4253-4253
Author(s):  
Shmuel Yaccoby ◽  
Kenichiro Yata ◽  
Yun Ge ◽  
Bart Barlogie ◽  
Joshua Epstein ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cord Blood ◽  
Scid Mice ◽  
Common Mechanism ◽  
Hematopoietic Stem ◽  
Variable Expression ◽  
Cd34 Cells ◽  
Rabbit Bone

Abstract Recent studies indicate that osteoblasts play an important role in maintaining hematopoietic stem cells (HSCs) niche in the bone marrow microenvironment. The aim of study was to test the effect of osteoclasts on the fate of HSCs in a long term co-culture assay. To generate osteoclasts, peripheral blood mononuclear cells from mobilized donors were cultured for 6–10 days in αMEM media supplemented with 10% FCS, M-CSF and RANKL. After removal of non-adherent cells, the cultures contained 95% multinucleated osteoclasts and their precursors. These osteoclasts expressed TRAP and formed resorption pits on bone slices (Yaccoby et al., Cancer Res., 2004). CD34+ cells were purified from donor PBSCs and cord blood using immunomagnetic beads separation (&gt;95% purity). Adult and cord blood HSCs were co-cultured with osteoclasts for up to 3 and 10 months, respectively, in media lacking any cytokines. Because osteoclasts do not survive long without M-CSF and RANKL, the HSCs were transferred to fresh osteoclast cultures every 6–10 days. Unlike their tight adherence to stromal cells, HSCs did not adhere to the osteoclasts and were easily recovered from co-cultures by gentle pipetting. Following 1 to 3 weeks of co-culture, committed HSCs rapidly differentiated into various hematopoietic cell lineage, followed by phagocytosis of terminal differentiated hematopoietic cells by the osteoclasts. The remaining HSCs were highly viable (&gt;90% by trypan blue exclusion) and gradually lost their CD34 expression, so that the cultures contained subpopulations of HSCs expressing CD34−/lowCD38+ and CD34−/lowCD38−. Quantitive real time RT-PCR (qRT-PCR) revealed loss of expression of CD34 and reduced expression of CD45 by HSCs co-cultured with osteoclasts longer than 6 weeks. Variable expression of CD34 on HSCs was previously reported in murine but not human HSCs (Tajima et al., Blood, 2001). The co-cultured HSCs showed reduced capacity of generating in vitro hematopoietic colonies, and did not differentiate into osteoclasts upon stimulation with M-CSF and RANKL. We next tested the long term engraftment of these co-cultured HSCs in 2 animal models. In the first model, cord blood and adult HSCs from 2 donors recovered after &gt;6 weeks in co-culture were injected I.V. into irradiated NOD/SCID mice. In the second novel model, co-cultured cord blood and adult HSCs from 2 donors were injected directly into rabbit bones implanted subcutaneously in SCID mice (SCID-rab model), 6–8 weeks after rabbit bone implantation. After 2–4 months, 10%±3% human CD45-expressing cells were identified in the NOD/SCID mice femora and 8%±4% in the SCID-rab mice rabbit bone. Our study suggests that osteoclasts promote rapid differentiation of committed HSCs and induce conversion of CD34+ cells to CD34− stem cells with self renewal potential. Intriguingly, long term co-culture of primary CD138-selected myeloma plasma cells (n=16) with osteoclasts resulted in dedifferentiation of tumor cells from a mature CD45− phenotype to an immature, CD45-expressing cells, suggesting a common mechanism of osteoclast-induced HSC and myeloma cell plasticity. This indicates that osteoclasts are important bone marrow component regulating human HSC niche, plasticity and fate.

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