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 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.

CD133 Positive Mesenchymal Stem Cells in Mobilized Peripheral Blood and Cord Blood: Proliferation, Oct4 Expression and Plasticity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4256-4256
Author(s):  
Tatiana Tondreau ◽  
Nathalie Meuleman ◽  
Marielle Dejeneffe ◽  
Alain Delforge ◽  
Dominique Bron ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Cord Blood ◽  
Glial Cells ◽  
Peripheral Blood ◽  
Clinical Importance ◽  
Cell Fraction ◽  
Oct4 Expression ◽  
Mobilized Peripheral Blood

Abstract Background: Mesenchymal Stem Cells (MSC) can be isolated from bone marrow, adipose and fetal tissues, but their presence in Mobilized Peripheral Blood (MPB) and in Umbilical Cord Blood (UCB) remains controversial. Methods: In this study we evaluated whether MPB (n=6) and UCB (n=8) could be two other sources of MSC beside Bone Marrow (BM). CD133 positive cell fraction was isolated through immunomagnetic system and MNC were seeded in medium supplemented or not with 5% of conditioned medium durong the first 48 hours of adhesion. MSC derived from MPB or UCB were identified by their expression of mesenchymal (SH2, SH3) and hematopoietic markers (CD14, CD34, CD45 and CD62-E). MPB and CB-MSC were also tested for their capacity to generate CFU-F (Fibroblast colony-forming units) and to differentiate into adipocytes, osteocytes, chondrocytes and neuronal/glial cells after specific induction. Finally we tested through RT-PCR the gene expression of Oct4, a transcriptional binding factor present in undifferentiated cells with high proliferative capacity. Results: Through CFU-F, we observed that the selection of CD133 positive cells allows to obtain a great amount of MSC in comparison with the MNC fraction seeded (MPB-MSC, 139 versus 72 and UCB-MSC 165 versus 8 after the primoculture). After four passages, more than 1.108 cells were obtained by the cell expansion assays from 1.106 cells seeded in primoculture. During the first culture, whatever the medium used or cell fraction seeded, the cell population was composed of osteoblasts, osteoclasts, marcophages and hematopoietic cells with less than 5%SH3 positive cells. After two passages, cells derived from MPB or UCB expressed mesenchymal markers (SH3 and SH2) and Oct4. Under appropriate conditions these cells were able to differentiate into adipocytes, osteocytes, chondrocytes and neuronal/glial cells. Conclusion: These results indicate that MSC are present in MPB and UCB, with similar characteristics to bone marrow (self-renewal capacity and multi-diffentiation potential), and may have a major clinical importance due to their accessibility. They could be an attractive target for cellular and gene therapies Mesenchymal marker expression (%) T0 (%) second passage (%) MPB SH2 2,7±1,1 96,1±2,9 SH3 4,9±1,5 95,7±3,3 UCB SH2 1,4±0,6 98±0,7 SH3 5,2±1,8 96,5±2,5

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.

Inhibition of PDGFRβ by Imatinib Mesylate Suppresses Proliferation and Alters Differentiation of Human Mesenchymal Stem Cells In Vitro.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2563-2563
Author(s):  
Fernando Fierro ◽  
Thomas Illmer ◽  
Duhoui Jing ◽  
Philip Le Coutre ◽  
Gerhard Ehninger ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Tyrosine Kinase ◽  
Hematopoietic Stem Cells ◽  
Imatinib Mesylate ◽  
Dependent Manner ◽  
Differentiation Potential ◽  
Hematopoietic Stem

Abstract Recent data show that the tyrosine kinase inhibitor Imatinib mesylate (IM) also affects normal hematopoietic stem cells (HSC), T lymphocyte activation and dendritic cell function not relying on the specific inhibition of bcr-abl activity. Mesenchymal stem cells (MSC) have been identified in the bone marrow (BM) as multipotent non-hematopoietic progenitor cells that differentiate into osteoblasts, adipocytes, chondrocytes, tenocytes, skeletal myocytes, and cells of visceral mesoderm. MSC interact with HSC, influencing their homing and differentiation through cell-cell contact and the production of factors including chemokines We evaluated possible effects of IM in vitro on human bone marrow-derived MSC. Screening the activity of fourty-two receptor tyrosine kinases by a phospho-receptor tyrosine kinase (RTK)-array revealed an exclusive inhibition of platelet-derived growth factor receptor (PDGFRβ) by IM which consequently affects downstream targets of PDGFRβ as Akt and Erk1/2 signalling pathways in a concentration and time dependent manner. Furthermore, perinuclear multivesicular bodies harbouring PDGFRβ were found within 18–20 hours culture of MSC in the presence of 5 μM IM. Cell proliferation and clonogenicity (evaluated as the capability to form colony forming units - fibroblasts (CFU-F)) of MSC were significantly inhibited by IM in a concentration dependent fashion. IM inhibits significantly the differentiation process of MSC into osteoblasts as evaluated by decreased alkaline phosphatase activity and reduced calcium phosphate precipitates. In contrary, differentiation of MSC into adipocytes was strongly favoured in presence of IM. All these functional deficits described, probably contribute to an observed 50% reduction in the support of clonogenic hematopoietic stem cells, as evaluated by a long term culture-initiating cells (LTC-IC)-based assay. In summary our experiments show that IM inhibits the capacity of human MSC to proliferate and to differentiate into the osteogenic lineage, favouring adipogenesis. This effect is mainly mediated by an inhibition of PDGFRβ autophosphorylation leading to a more pronounced inhibition of PI3K/Akt compared to Erk1/2 signalling. This work confirms the role of PDGFRβ recently described for the proliferation and differentiation potential of MSC and provides a first possible explanation for the altered bone metabolism found in certain patients treated with IM.

Cripto Selectively Expands a Distinct Population of Hematopoietic Stem Cells Expressing the Cell Surface Receptor GRP78 and Strongly Induces An Immature Phenotype In Vivo After Ex Vivo Culture

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 405-405
Author(s):  
Kenichi Miharada ◽  
Göran Karlsson ◽  
Jonas Larsson ◽  
Emma Larsson ◽  
Kavitha Siva ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Distinct Population ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Total Bone Marrow

Abstract Abstract 405 Cripto is a member of the EGF-CFC soluble protein family and has been identified as an important factor for the proliferation/self-renewal of ES and several types of tumor cells. The role for Cripto in the regulation of hematopoietic cells has been unknown. Here we show that Cripto is a potential new candidate factor to increase self-renewal and expand hematopoietic stem cells (HSCs) in vitro. The expression level of Cripto was analyzed by qRT-PCR in several purified murine hematopoietic cell populations. The findings demonstrated that purified CD34-KSL cells, known as highly concentrated HSC population, had higher expression levels than other hematopoietic progenitor populations including CD34+KSL cells. We asked how Cripto regulates HSCs by using recombinant mouse Cripto (rmCripto) for in vitro and in vivo experiments. First we tested the effects of rmCripto on purified hematopoietic stem cells (CD34-LSK) in vitro. After two weeks culture in serum free media supplemented with 100ng/ml of SCF, TPO and 500ng/ml of rmCripto, 30 of CD34-KSL cells formed over 1,300 of colonies, including over 60 of GEMM colonies, while control cultures without rmCripto generated few colonies and no GEMM colonies (p<0.001). Next, 20 of CD34-KSL cells were cultured with or without rmCripto for 2 weeks and transplanted to lethally irradiated mice in a competitive setting. Cripto treated donor cells showed a low level of reconstitution (4–12%) in the peripheral blood, while cells cultured without rmCripto failed to reconstitute. To define the target population and the mechanism of Cripto action, we analyzed two cell surface proteins, GRP78 and Glypican-1, as potential receptor candidates for Cripto regulation of HSC. Surprisingly, CD34-KSL cells were divided into two distinct populations where HSC expressing GRP78 exhibited robust expansion of CFU-GEMM progenitor mediated by rmCripto in CFU-assay whereas GRP78- HSC did not respond (1/3 of CD34-KSL cells were GRP78+). Furthermore, a neutralization antibody for GRP78 completely inhibited the effect of Cripto in both CFU-assay and transplantation assay. In contrast, all lineage negative cells were Glypican-1 positive. These results suggest that GRP78 must be the functional receptor for Cripto on HSC. We therefore sorted these two GRP78+CD34-KSL (GRP78+HSC) and GRP78-CD34-KSL (GRP78-HSC) populations and transplanted to lethally irradiated mice using freshly isolated cells and cells cultured with or without rmCripto for 2 weeks. Interestingly, fresh GRP78-HSCs showed higher reconstitution than GRP78+HSCs (58–82% and 8–40%, p=0.0038) and the reconstitution level in peripheral blood increased rapidly. In contrast, GRP78+HSC reconstituted the peripheral blood slowly, still at a lower level than GRP78-HSC 4 months after transplantation. However, rmCripto selectively expanded (or maintained) GRP78+HSCs but not GRP78-HSCs after culture and generated a similar level of reconstitution as freshly transplanted cells (12–35%). Finally, bone marrow cells of engrafted recipient mice were analyzed at 5 months after transplantation. Surprisingly, GRP78+HSC cultured with rmCripto showed higher reconstitution of the CD34-KSL population in the recipients' bone marrow (45–54%, p=0.0026), while the reconstitution in peripheral blood and in total bone marrow was almost the same. Additionally, most reconstituted CD34-KSL population was GRP78+. Interestingly freshly transplanted sorted GRP78+HSC and GRP78-HSC can produce the GRP78− and GRP78+ populations in the bone marrow and the ratio of GRP78+/− cells that were regenerated have the same proportion as the original donor mice. Compared to cultured cells, the level of reconstitution (peripheral blood, total bone marrow, HSC) in the recipient mice was almost similar. These results indicate that the GRP78 expression on HSC is reversible, but it seems to be “fixed” into an immature stage and differentiate with lower efficiency toward mature cells after long/strong exposure to Cripto signaling. Based on these findings, we propose that Cripto is a novel factor that maintains HSC in an immature state and may be a potent candidate for expansion of a distinct population of GRP78 expressing HSC. Disclosures: No relevant conflicts of interest to declare.

Novel In Vivo Evidence That Not Only Androgens But Also Pituitary Gonadotropins and Prolactin Directly Stimulate Murine Bone Marrow Stem Cells – Implications For Potential Treatment Strategies In Aplastic Anemias

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2476-2476
Author(s):  
Kasia Mierzejewska ◽  
Ewa Suszynska ◽  
Sylwia Borkowska ◽  
Malwina Suszynska ◽  
Maja Maj ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Sex Hormones ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Clonogenic Potential

Abstract Background Hematopoietic stem/progenitor cells (HSPCs) are exposed in vivo to several growth factors, cytokines, chemokines, and bioactive lipids in bone marrow (BM) in addition to various sex hormones circulating in peripheral blood (PB). It is known that androgen hormones (e.g., danazol) is employed in the clinic to treat aplastic anemia patients. However, the exact mechanism of action of sex hormones secreted by the pituitary gland or gonads is not well understood. Therefore, we performed a complex series of experiments to address the influence of pregnant mare serum gonadotropin (PMSG), luteinizing hormone (LH), follicle-stimulating hormone (FSH), androgen (danazol) and prolactin (PRL) on murine hematopoiesis. In particular, from a mechanistic view we were interested in whether this effect depends on stimulation of BM-residing stem cells or is mediated through the BM microenvironment. Materials and Methods To address this issue, normal 2-month-old C57Bl6 mice were exposed or not to daily injections of PMSG (10 IU/mice/10 days), LH (5 IU/mice/10 days), FSH (5 IU/mice/10 days), danazol (4 mg/kg/10 days) and PRL (1 mg/day/5days). Subsequently, we evaluated changes in the BM number of Sca-1+Lin–CD45– that are precursors of long term repopulating hematopoietic stem cells (LT-HSCs) (Leukemia 2011;25:1278–1285) and bone forming mesenchymal stem cells (Stem Cell & Dev. 2013;22:622-30) and Sca-1+Lin–CD45+ hematopoietic stem/progenitor cells (HSPC) cells by FACS, the number of clonogenic progenitors from all hematopoietic lineages, and changes in peripheral blood (PB) counts. In some of the experiments, mice were exposed to bromodeoxyuridine (BrdU) to evaluate whether sex hormones affect stem cell cycling. By employing RT-PCR, we also evaluated the expression of cell-surface and intracellular receptors for hormones in purified populations of murine BM stem cells. In parallel, we studied whether stimulation by sex hormones activates major signaling pathways (MAPKp42/44 and AKT) in HSPCs and evaluated the effect of sex hormones on the clonogenic potential of murine CFU-Mix, BFU-E, CFU-GM, and CFU-Meg in vitro. We also sublethally irradiated mice and studied whether administration of sex hormones accelerates recovery of peripheral blood parameters. Finally, we determined the influence of sex hormones on the motility of stem cells in direct chemotaxis assays as well as in direct in vivo stem cell mobilization studies. Results We found that 10-day administration of each of the sex hormones evaluated in this study directly stimulated expansion of HSPCs in BM, as measured by an increase in the number of these cells in BM (∼2–3x), and enhanced BrdU incorporation (the percentage of quiescent BrdU+Sca-1+Lin–CD45– cells increased from ∼2% to ∼15–35% and the percentage of BrdU+Sca-1+Lin–CD45+ cells increased from 24% to 43–58%, Figure 1). These increases paralleled an increase in the number of clonogenic progenitors in BM (∼2–3x). We also observed that murine Sca-1+Lin–CD45– and Sca-1+Lin–CD45+ cells express sex hormone receptors and respond by phosphorylation of MAPKp42/44 and AKT in response to exposure to PSMG, LH, FSH, danazol and PRL. We also observed that administration of sex hormones accelerated the recovery of PB cell counts in sublethally irradiated mice and slightly mobilized HSPCs into PB. Finally, in direct in vitro clonogenic experiments on purified murine SKL cells, we observed a stimulatory effect of sex hormones on clonogenic potential in the order: CFU-Mix > BFU-E > CFU-Meg > CFU-GM. Conclusions Our data indicate for the first time that not only danazol but also several pituitary-secreted sex hormones directly stimulate the expansion of stem cells in BM. This effect seems to be direct, as precursors of LT-HSCs and HSPCs express all the receptors for these hormones and respond to stimulation by phosphorylation of intracellular pathways involved in cell proliferation. These hormones also directly stimulated in vitro proliferation of purified HSPCs. In conclusion, our studies support the possibility that not only danazol but also several other upstream pituitary sex hormones could be employed to treat aplastic disorders and irradiation syndromes. Further dose- and time-optimizing mouse studies and studies with human cells are in progress in our laboratories. Disclosures: No relevant conflicts of interest to declare.

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 Bone Marrow-Derived Mesenchymal Stem Cells Modified with Akt1 Ameliorates Acute Liver GVHD

2019 ◽  
Vol 21 (1) ◽  
Author(s):  
Lukun Zhou ◽  
Shuang Liu ◽  
Zhao Wang ◽  
Jianfeng Yao ◽  
Wenbin Cao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Liver Injury ◽  
Therapeutic Effects ◽  
Hematopoietic Stem ◽  
Ifn Γ ◽  
Immunomodulatory Function

Abstract Background Liver injury associated with acute graft-versus-host disease (aGVHD) is a frequent and severe complication of hematopoietic stem cell transplantation and remains a major cause of transplant-related mortality. Bone marrow-derived mesenchymal stem cells (BM-MSCs) has been proposed as a potential therapeutic approach for aGVHD. However, the therapeutic effects are not always achieved. In this study, we genetically engineered C57BL/6 mouse BM-MSCs with AKT1 gene and tested whether AKT1-MSCs was superior to control MSCs (Null-MSCs) for cell therapy of liver aGVHD. Results In vitro apoptosis analyses showed that, under both routine culture condition and high concentration interferon-γ (IFN-γ) (100ng/mL) stimulation condition, AKT1-MSCs had a survival (anti-apoptotic) advantage compared to Null-MSCs. In vivo imaging showed that AKT1-MSCs had better homing capacity and longer persistence in injured liver compared to Null-MSCs. Most importantly, AKT1-MSCs demonstrated an enhanced immunomodulatory function by releasing more immunosuppressive cytokines, such as IL-10. Adoptive transfer of AKT1-MSCs mitigated the histopathological abnormalities of concanavalin A(ConA)-induced liver injury along with significantly lowered serum levels of ALT and AST. The attenuation of liver injury correlated with the decrease of TNF-α and IFN-γ both in liver tissue and in the serum. Conclusions In summary, BM-MSCs genetically modified with AKT1 has a survival advantage and an enhanced immunomodulatory function both in vitro and in vivo and thus demonstrates the therapeutic potential for prevention and amelioration of liver GVHD and other immunity-associated liver injuries.

Ex Vivo Expanded Megakaryocyte Progenitors Generated From Human Mobilized Peripheral Blood CD34+ Stem Cells Can Be Cryopreserved and Transplanted Into NSG Mice to Generate Functional Platelets

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 482-482
Author(s):  
Holger Karsunky ◽  
Robert J. Tressler ◽  
Joy Chananukul
Keyword(s):  
Stem Cells ◽  
Peripheral Blood ◽  
Platelet Transfusion ◽  
Ex Vivo ◽  
High Dose ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Mobilized Peripheral Blood ◽  
Megakaryocyte Progenitors

Abstract Abstract 482 Thrombocytopenia is a common side effect of high-dose chemotherapy that can compromise cancer treatment by requiring treatment delay and/or dose reduction for the patient. Platelet transfusion is typically given to prevent severe hemorrhage. However, several factors including acquisition, banking, and associated risks of bacterial infections and alloimmunization are hampering reliance on platelet transfusion. Growth factors are also used to stimulate proliferation and differentiation of megakaryocytes to increase platelet production, but in severely myelosuppressed patients these have only had modest benefit. The limitations of these two modalities for the treatment of chemotherapy-induced thrombocytopenia indicates that additional treatment approaches are needed. We have developed a novel approach to reconstitute megakaryocytes and platelets in thrombocytopenic patients which is presented here. We have identified a scalable culture system using serum-free medium and a defined cytokine cocktail free of animal products to expand CD34+ hematopoietic stem cells from G-CSF mobilized peripheral blood donors in vitro and direct their development to the megakaryocyte lineage to yield committed human megakaryocyte progenitors (MKPs). These MKPs can be readily cryopreserved while retaining their capacity to generate CFU-MK and platelets in vitro. When infused into NSG mice, ex vivo expanded MKP generate clinically relevant platelet levels of platelets in blood within a few days with sustained platelet levels for several weeks. The platelets generated from MKP in vivo are also functional as assessed by CD62P expression in responses to ADP stimulation in vitro. Our results present a compelling approach for the development of off-the-shelf storable MKPs for the treatment of thrombocytopenia. Disclosures: Karsunky: Cellerant Therapeutics Inc.: Employment, Patents & Royalties. Tressler:Cellerant Therapeutics, Inc.: Employment, Equity Ownership. Chananukul:Cellerant Therapeutics Inc.: Employment, Patents & Royalties.

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