Differential homing and engraftment properties of hematopoietic progenitor cells from murine bone marrow, mobilized peripheral blood, and fetal liver

Blood ◽  
2001 ◽  
Vol 98 (7) ◽  
pp. 2108-2115 ◽  
Author(s):  
Stephen J. Szilvassy ◽  
Todd E. Meyerrose ◽  
Penny L. Ragland ◽  
Barry Grimes
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Fetal Liver ◽  
Proliferative Potential ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Hematopoietic Reconstitution ◽  
Late Antigen ◽  
Mobilized Peripheral Blood

The rate of reconstitution following hematopoietic stem cell (HSC) transplantation differs widely depending on the tissue source of the cells infused. To test the hypothesis that variability in engraftment kinetics is related to differences in the efficiency with which intravenously transplanted HSCs “home” to the bone marrow (BM), the homing properties of murine fetal liver (FL), adult BM, and mobilized peripheral blood (MPB) cells were compared. Lethally irradiated mice transplanted with 2 × 106 FL, BM, or MPB cells exhibited sequentially slower recovery of circulating leukocytes and platelets that correlates with the progressively lower frequency of colony-forming cells (CFCs) in these tissues. However, differences in the rate and degree of early and long-term reconstitution were maintained even after infusing equal numbers of CFCs derived from FL, BM, and MPB. To compare the homing of progenitors from these tissues, cells were labeled with fluorescent PKH26 dye and injected into lethally irradiated hosts. Three hours later, PKH26+ cells were reisolated from the BM and spleen by fluorescence-activated cell sorting and assayed for in vitro CFCs. Despite the higher level of very late antigen (VLA)-2, VLA-4, and VLA-5 on Sca-1+c-kit+ cells from FL compared to BM, 10-fold fewer FL CFCs homed to hematopoietic organs than those from BM. MPB cells homed slightly better, but still less efficiently than BM cells. Therefore, clonogenic cells from different tissues exhibit striking variations in homing efficiency that does not necessarily correlate with engraftment kinetics. Homing is likely counterbalanced by intrinsic differences in proliferative potential that ultimately determine the rate of hematopoietic reconstitution.

scholarly journals Primitive Human Hematopoietic Cells Are Enriched in Cord Blood Compared With Adult Bone Marrow or Mobilized Peripheral Blood as Measured by the Quantitative In Vivo SCID-Repopulating Cell Assay

Blood ◽  
1997 ◽  
Vol 89 (11) ◽  
pp. 3919-3924 ◽  
Author(s):  
Jean C.Y. Wang ◽  
Monica Doedens ◽  
John E. Dick
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Hematopoietic Cells ◽  
Allogeneic Transplantation ◽  
Functional Assay ◽  
Hematopoietic Stem ◽  
Mobilized Peripheral Blood

Abstract We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 × 105 cells. This was significantly higher than the frequency of 1 SRC in 3.0 × 106 adult BM cells or 1 in 6.0 × 106 mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus, the findings reported here will have important clinical as well as biologic implications.

Differential Role for VLA-5 in Mobilization of Heamotopoieic Stem Cells Toward Peripheral Blood and Homing to Bone Marrow and Spleen.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2190-2190 ◽  
Author(s):  
Pieter K. Wierenga ◽  
Ellen Weersing ◽  
Bert Dontje ◽  
Gerald de Haan ◽  
Ronald P. van Os
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Late Antigen ◽  
Cell Mobilization

Abstract Adhesion molecules have been implicated in the interactions of hematopoietic stem and progenitor cells with the bone marrow extracellular matrix and stromal cells. In this study we examined the role of very late antigen-5 (VLA-5) in the process of stem cell mobilization and homing after stem cell transplantation. In normal bone marrow (BM) from CBA/H mice 79±3 % of the cells in the lineage negative fraction express VLA-5. After mobilization with cyclophosphamide/G-CSF, the number of VLA-5 expressing cells in mobilized peripheral blood cells (MPB) decreases to 36±4%. The lineage negative fraction of MPB cells migrating in vitro towards SDF-1α (M-MPB) demonstrated a further decrease to 3±1% of VLA-5 expressing cells. These data are suggestive for a downregulation of VLA-5 on hematopoietic cells during mobilization. Next, MPB cells were labelled with PKH67-GL and transplanted in lethally irradiated recipients. Three hours after transplantation an increase in VLA-5 expressing cells was observed which remained stable until 24 hours post-transplant. When MPB cells were used the percentage PKH-67GL+ Lin− VLA-5+ cells increased from 36% to 88±4%. In the case of M-MPB cells the number increased from 3% to 33±5%. Although the increase might implicate an upregulation of VLA-5, we could not exclude selective homing of VLA-5+ cells as a possible explanation. Moreover, we determined the percentage of VLA-5 expressing cells immediately after transplantation in the peripheral blood of the recipients and were not able to observe any increase in VLA-5+ cells in the first three hours post-tranpslant. Finally, we separated the MPB cells in VLA-5+ and VLA-5− cells and plated these cells out in clonogenic assays for progenitor (CFU-GM) and stem cells (CAFC-day35). It could be demonstared that 98.8±0.5% of the progenitor cells and 99.4±0.7% of the stem cells were present in the VLA-5+ fraction. Hence, VLA-5 is not downregulated during the process of mobilization and the observed increase in VLA-5 expressing cells after transplantation is indeed caused by selective homing of VLA-5+ cells. To shed more light on the role of VLA-5 in the process of homing, BM and MPB cells were treated with an antibody to VLA-5. After VLA-5 blocking of MPB cells an inhibition of 59±7% in the homing of progenitor cells in bone marrow could be found, whereas homing of these subsets in the spleen of the recipients was only inhibited by 11±4%. For BM cells an inhibition of 60±12% in the bone marrow was observed. Homing of BM cells in the spleen was not affected at all after VLA-5 blocking. Based on these data we conclude that mobilization of hematopoietic progenitor/stem cells does not coincide with a downregulation of VLA-5. The observed increase in VLA-5 expressing cells after transplantation is caused by preferential homing of VLA-5+ cells. Homing of progenitor/stem cells to the bone marrow after transplantation apparantly requires adhesion interactions that can be inhibited by blocking VLA-5 expression. Homing to the spleen seems to be independent of VLA-5 expression. These data are indicative for different adhesive pathways in the process of homing to bone marrow or spleen.

Homing and Engraftment of Mobilized Hematopoietic Stem Cells: Involvement of VLA-5.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4943-4943
Author(s):  
Pieter K. Wierenga ◽  
Gerald de Haan ◽  
Bert Dontje ◽  
Ellen Weersing ◽  
Ronald van Os
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Hematopoietic Reconstitution

Abstract VLA-5 has been implicated in the adhesive interactions of stem and progenitor cells with the bone marrow extracellular matrix and stromal cells and is therefore considered to play an important role in the hematopoietic reconstitution after stem cell transplantation. In normal bone marrow (BM) from CBA/H mice 79±3% of the cells in the lineage negative fraction express VLA-5. After mobilization with cyclophosphamide/G-GSF, the number of VLA-5 expressing cells in mobilized peripheral blood cells (MPB) decreases to 38±3%. Despite this low frequency of VLA-5+ cells, however, even when equal numbers of progenitor cells are transplanted MPB cells provide a much faster hematopoietic recovery compared to BM cells. To shed more light on the role of VLA-5 in the process of homing and engraftment, we investigated whether differences in homing potential of the stem cell subsets might be responsible for this enhanced reconstitution. At 3 hours post-transplant, however, no differences in homing efficiency of progenitor and stem cells from MPB and BM grafts in both bone marrow and spleen could be detected. It should be realized that MPB and BM grafts demonstrate different ratios of stem/progenitor cells which might be another explanation for the observed differences in repopulation potential. Furthermore, MPB cells migrating in vitro towards SDF-1α showed potent reconstitution while VLA-5 expression was reduced on these cells. In fact, in vitro treatment with SDF-1α showed further decrease in VLA-5 expressing cells (from 38% to 4%) in the lin- fraction. When equal numbers of MPB were transplanted with and without SDF-1α pretreatment, no difference in hematopoietic reconstitution was observed suggesting a minor role of VLA-5 in homing and engraftment. On the other hand, after VLA-5 blocking an inhibition of 59±7% in the homing of MPB progenitor cells in the bone marrow could be found, whereas homing in the spleen of the the recipients is only inhibited by 11±4%. To elucidate whether the observed enhanced reconstitution could be explained by a selective homing of VLA-5+ cells or a rapid upregulation of VLA-5 expression, cells were labelled with PKH67-GL and transplanted in lethally irradiated recipients. It could be demonstrated that at 3 hours post-transplant cells from MPB grafts showed a rapid increase from 38±3% up to 66±9% of VLA-5+ cells in the bone marrow of the recipient. In the spleen no significant increase in VLA-5+ cells was observed. When MPB cells were transplanted after pretreatment with SDF-1α an increase from 2±1% up to 33±5% of VLA-5+ cells in the bone marrow was detected. When calculating the number of cells recovered from bone marrow, a selective homing of VLA-5+ cells cannot be excluded. Therefore, we also assessed the number of VLA-5+ cells in the PKH+ fraction in peripheral blood from the recipient immediately (½-1 hour) after transplantation but found no increase during that time period. So far it can be concluded that MPB cells show low number of VLA-5+ cells but these cells possess an enhanced hematopoietic reconstitution potential. Homing of progenitor cells to the spleen seems to be less dependent on VLA-5 expression than homing to the bone marrow. A rapid upregulation of VLA-5 expression on engrafting MPB cells early after transplantation does not occur and hence our data are suggestive for the preferential homing of VLA-5+ cells in the bone marrow after transplantation.

G-CSF Mobilized Human Mesenchymal Stem Cells Are Found in the Peripheral Blood and Have Telomere Limited Growth Potential.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4246-4246
Author(s):  
Troy C. Lund ◽  
Lisa Baso ◽  
Paul J. Orchard
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Peripheral Blood ◽  
Peripheral Circulation ◽  
Growth Potential ◽  
Hematopoietic Stem ◽  
Marrow Cavity ◽  
Mobilized Peripheral Blood

Abstract Mesenchymal stem cells (MSC) are multipotent cells found lining the bone marrow cavity. Their primary function is to support the growth and differentiation of hematologic progenitors. MSC have been shown to differentiate into a variety of cell types including: bone, adipocytes, cartilage, neuron-like, and muscle-like cells. There is mounting evidence that these cells can, under the right circumstances, enter the peripheral circulation. However, MSC have not been routinely isolated from peripheral blood. Granulocyte colony stimulating factor (G-CSF) is commonly used to mobilize hematopoietic stem cells from the bone marrow into the peripheral circulation. We show that G-CSF mobilized peripheral blood also contains a small percentage of MSC although lower than that of bone marrow derived MSC (BMMSC): 0.012% vs 0.04%. Isolates were morphologically similar to BMMSC and were successfully expanded and shown to differentiate into osteogenic and adipogenic lineages in the appropriate differentiation conditions. FACS analysis showed that the cells reliably expressed cell surfaces markers commonly found on MSC including CD105, CD29, CD166, and CD13. They were negative for CD14, CD34, CD133, and CD45. Mobilized peripheral blood derived MSC (MPB-MSC) had limited expansion potential when compared with bone marrow isolated MSC. Most cells appeared to cease cell division 20–25 days after isolation. MPB-MSC did not have any detectable telomerase activity (as determined by TRAP assay) and consequently were found to have undergone significant telomere shortening (shown by Southern analysis.) The rarity of this cell in G-CSF-mobilized peripheral blood and the subsequent tremendous pressure to divide in cell culture are likely contributing factors leading to the telomere loss seen in MPB-MSC. This phenomenon probably also accounts for the observed senescence observed in vitro. While we have conclusively shown that MSC can be found in G-CSF mobilized peripheral blood, the use of such cells for transplant or gene therapy may be of limited potential due to the telomere-restricted capability of expansion.

scholarly journals Transcription factor Gfi-1 induced by G-CSF is a negative regulator of CXCR4 in myeloid cells

Blood ◽  
2007 ◽  
Vol 110 (7) ◽  
pp. 2276-2285 ◽  
Author(s):  
Maria De La Luz Sierra ◽  
Paola Gasperini ◽  
Peter J. McCormick ◽  
Jinfang Zhu ◽  
Giovanna Tosato
Keyword(s):  
Bone Marrow ◽  
Dna Sequences ◽  
Peripheral Blood ◽  
Cell Function ◽  
Myeloid Cell ◽  
Cxcr4 Expression ◽  
Negative Regulator ◽  
Hematopoietic Stem

The mechanisms underlying granulocyte-colony stimulating factor (G-CSF)–induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood remain elusive. We provide evidence that the transcriptional repressor growth factor independence-1 (Gfi-1) is involved in G-CSF–induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood. We show that in vitro and in vivo G-CSF promotes expression of Gfi-1 and down-regulates expression of CXCR4, a chemokine receptor essential for the retention of hematopoietic stem cells and granulocytic cells in the bone marrow. Gfi-1 binds to DNA sequences upstream of the CXCR4 gene and represses CXCR4 expression in myeloid lineage cells. As a consequence, myeloid cell responses to the CXCR4 unique ligand SDF-1 are reduced. Thus, Gfi-1 not only regulates hematopoietic stem cell function and myeloid cell development but also probably promotes the release of granulocytic lineage cells from the bone marrow to the peripheral blood by reducing CXCR4 expression and function.

Organ-Selective Homing Defines Engraftment Kinetics of Murine Hematopoietic Stem Cells and Is Compromised by Ex Vivo Expansion

Blood ◽  
1999 ◽  
Vol 93 (5) ◽  
pp. 1557-1566 ◽  
Author(s):  
Stephen J. Szilvassy ◽  
Michael J. Bass ◽  
Gary Van Zant ◽  
Barry Grimes
Keyword(s):  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Hematopoietic Reconstitution ◽  
Dramatic Decline ◽  
Stem And Progenitor Cells ◽  
Clonogenic Cells

Abstract Hematopoietic reconstitution of ablated recipients requires that intravenously (IV) transplanted stem and progenitor cells “home” to organs that support their proliferation and differentiation. To examine the possible relationship between homing properties and subsequent engraftment potential, murine bone marrow (BM) cells were labeled with fluorescent PKH26 dye and injected into lethally irradiated hosts. PKH26+ cells homing to marrow or spleen were then isolated by fluorescence-activated cell sorting and assayed for in vitro colony-forming cells (CFCs). Progenitors accumulated rapidly in the spleen, but declined to only 6% of input numbers after 24 hours. Although egress from this organ was accompanied by a simultaneous accumulation of CFCs in the BM (plateauing at 6% to 8% of input after 3 hours), spleen cells remained enriched in donor CFCs compared with marrow during this time. To determine whether this differential homing of clonogenic cells to the marrow and spleen influenced their contribution to short-term or long-term hematopoiesis in vivo, PKH26+ cells were sorted from each organ 3 hours after transplantation and injected into lethally irradiated Ly-5 congenic mice. Cells that had homed initially to the spleen regenerated circulating leukocytes (20% of normal counts) approximately 2 weeks faster than cells that had homed to the marrow, or PKH26-labeled cells that had not been selected by a prior homing step. Both primary (17 weeks) and secondary (10 weeks) recipients of “spleen-homed” cells also contained approximately 50% higher numbers of CFCs per femur than recipients of “BM-homed” cells. To examine whether progenitor homing was altered upon ex vivo expansion, highly enriched Sca-1+c-kit+Lin−cells were cultured for 9 days in serum-free medium containing interleukin (IL)-6, IL-11, granulocyte colony-stimulating factor, stem cell factor, flk-2/flt3 ligand, and thrombopoietin. Expanded cells were then stained with PKH26 and assayed as above. Strikingly, CFCs generated in vitro exhibited a 10-fold reduction in homing capacity compared with fresh progenitors. These studies demonstrate that clonogenic cells with differential homing properties contribute variably to early and late hematopoiesis in vivo. The dramatic decline in the homing capacity of progenitors generated in vitro underscores critical qualitative changes that may compromise their biologic function and potential clinical utility, despite their efficient numerical expansion.

scholarly journals Molecular Modulation of Fetal Liver Hematopoietic Stem Cell Mobilization into Fetal Bone Marrow in Mice

2020 ◽  
Vol 2020 ◽  
pp. 1-12
Author(s):  
Huihong Zeng ◽  
Jiaoqi Cheng ◽  
Ying Fan ◽  
Yingying Luan ◽  
Juan Yang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Vascular Endothelial Cells ◽  
Fetal Liver ◽  
Bone Marrow Niche ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Fetal Bone

Development of hematopoietic stem cells is a complex process, which has been extensively investigated. Hematopoietic stem cells (HSCs) in mouse fetal liver are highly expanded to prepare for mobilization of HSCs into the fetal bone marrow. It is not completely known how the fetal liver niche regulates HSC expansion without loss of self-renewal ability. We reviewed current progress about the effects of fetal liver niche, chemokine, cytokine, and signaling pathways on HSC self-renewal, proliferation, and expansion. We discussed the molecular regulations of fetal HSC expansion in mouse and zebrafish. It is also unknown how HSCs from the fetal liver mobilize, circulate, and reside into the fetal bone marrow niche. We reviewed how extrinsic and intrinsic factors regulate mobilization of fetal liver HSCs into the fetal bone marrow, which provides tools to improve HSC engraftment efficiency during HSC transplantation. Understanding the regulation of fetal liver HSC mobilization into the fetal bone marrow will help us to design proper clinical therapeutic protocol for disease treatment like leukemia during pregnancy. We prospect that fetal cells, including hepatocytes and endothelial and hematopoietic cells, might regulate fetal liver HSC expansion. Components from vascular endothelial cells and bones might also modulate the lodging of fetal liver HSCs into the bone marrow. The current review holds great potential to deeply understand the molecular regulations of HSCs in the fetal liver and bone marrow in mammals, which will be helpful to efficiently expand HSCs in vitro.

High-resolution tracking of cell division suggests similar cell cycle kinetics of hematopoietic stem cells stimulated in vitro and in vivo

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 855-862 ◽  
Author(s):  
Robert A. J. Oostendorp ◽  
Julie Audet ◽  
Connie J. Eaves
Keyword(s):  
Stem Cells ◽  
Fetal Liver ◽  
Flow Cytometric Analysis ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Flow Cytometric ◽  
Differentiation Status ◽  
Kinetics Of

The kinetics of proliferation of primitive murine bone marrow (BM) cells stimulated either in vitro with growth factors (fetal liver tyrosine kinase ligand 3 [FL], Steel factor [SF], and interleukin-11 [IL-11], or hyper–IL-6) or in vivo by factors active in myeloablated recipients were examined. Cells were first labeled with 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and then incubated overnight prior to isolating CFSE+ cells. After 2 more days in culture, more than 90% of the in vivo lymphomyeloid repopulating activity was associated with the most fluorescent CFSE+ cells (ie, cells that had not yet divided), although this accounted for only 25% of the repopulating stem cells measured in the CFSE+ “start” population. After a total of 4 days in culture (1 day later), 15-fold more stem cells were detected (ie, 4-fold more than the day 1 input number), and these had become (and thereafter remained) exclusively associated with cells that had divided at least once in vitro. Flow cytometric analysis of CFSE+ cells recovered from the BM of transplanted mice indicated that these cells proliferated slightly faster (up to 5 divisions completed within 2 days and up to 8 divisions completed within 3 days in vivo versus 5 and 7 divisions, respectively, in vitro). FL, SF, and ligands which activate gp130 are thus efficient stimulators of transplantable stem cell self-renewal divisions in vitro. The accompanying failure of these cells to accumulate rapidly indicates important changes in their engraftment potential independent of accompanying changes in their differentiation status.

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