Kit Deficiency Regulates Stable Human Hematopoietic Stem Cell Engraftment in Mice

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 653-653
Author(s):  
Claudia Waskow ◽  
Susann Rahmig ◽  
Nehir Cosgun
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Myeloid Cell ◽  
Cell Types ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Kit Receptor ◽  
Human Hscs

Abstract Humanized mice are required for in-depth analysis of human hematopoietic stem cell (HSC) function and immunobiology. The currently available options are problematic because stable engraftment of substantial numbers of human HSCs and the continuous generation of human myeloid cell types remain difficult to achieve. We generated three novel recipient mouse strains that combine immune deficiency with a functionally impaired endogenous HSC compartment mediated by a defective Kit receptor: BALB/c Rag2- Il2rg-KitWv/Wv (BRgWv), NOD/SCID Il2rg- (NSG) KitWv/Wv (NSGWv) and NSG KitW41/W41 (NSGW41). We find that the mutant Kit receptor opens up stem cell niches across species barriers and allows for robust and sustained engraftment of human HSCs after transfer into adult mice without the necessity for irradiation conditioning prior to transplantation. Following stable engraftment in the mouse bone marrow niches, human HSCs give rise to lymphoid cells and to robust numbers of erythroid and myeloid lineage cells over extended periods of time in primary and secondary recipient mice. Particularly in NSGW41 mice, we observe improved reconstitution of human myeloid cell types compared to control irradiated NSG mice. In the bone marrow, endogenous hematopoietic progenitor cells with a defective Kit receptor are largely replaced by human Kit-proficient hematopoietic progenitor cells because progenitor cell expansion requires normal signaling by Kit. Thus, human Kit-proficient donor cells have an advantage over endogenous murine Kit-mutant cells. Increased numbers of myeloid cells are found in the bone marrow and spleen of NSGW41 transplanted mice compared with grafts established in irradiated NSG mice. We conclude that Kit-signaling regulates HSC engraftment across the human-mouse species barrier and that Kit deficient mice show great potential for the study of human HSC functions including self-renewal, differentiation and mechanisms of innate immunity. Disclosures No relevant conflicts of interest to declare.

Modulation in the Expression of p70S6 Kinase Impairs the Engraftment and Self-Renewal of Hematopoietic Stem Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 252-252
Author(s):  
Joydeep Ghosh ◽  
Baskar Ramdas ◽  
Anindya Chatterjee ◽  
Peilin Ma ◽  
Michihiro Kobayashi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Mtorc1 Pathway ◽  
And Function

Abstract Regulation of hematopoietic stem cell (HSC) function(s) via the mammalian target of rapamycin complex1 (mTORC1) and its upstream regulators including PI3K and Akt has been described before. To this end, we and others have shown that hyperactivation and deficiency of the PI3K-mTORC1 pathway results in altered development, maintenance and function(s) of HSCs. However, the role of downstream effector of mTORC1, p70S6 kinase (S6K1), in HSC development and functions is unknown. Previous studies have implicated S6K1 as a regulator of ageing, by virtue of its ability to regulate cellular metabolic processes as well as protein translation. In certain cells, however S6K1 regulates cell survival and also acts as a negative regulator of PI3K-mTORC1 pathway, thus creating a negative feedback loop. Thus, how S6K1 impacts HSC ageing and stem cell functions remains an enigma. We have assessed the role of S6K1 in HSC development and function under steady-state as well as during recovery of hematopoietic system following myelosuppressive stress. We used a genetic model of S6K1 knockout mice (S6K1-/-). S6K1 deficiency in bone marrow hematopoietic cells resulted in decrease of absolute number of bone marrow hematopoietic progenitor cells as well as HSCs (Lin- Sca1+ c-Kit+; LSK) were significantly reduced relative to controls (n=14 in each group, p<0.04). Interestingly, in vitro, hematopoietic progenitor cells from S6K1-/- mice showed increased colony forming ability in response to cytokines which was associated with hyperactivation of Akt and ERK MAP kinase. To determine whether the reduced number of HSCs in S6K1-/- mice was due to deficiency of S6K1 in bone marrow microenvironment, we transplanted WT hematopoietic bone marrow cells into lethally irradiated WT or S6K1-/- mice. S6K1-/- mice transplanted with WT hematopoietic cells showed similar bone marrow cellularity and HSC numbers compared to controls suggesting that the bone marrow hypocellularity and reduced HSCs numbers in S6K1-/- mice were due to a cell intrinsic defect. To assess whether the reduced HSC number in S6K1-/- mice impacted the recovery of hematopoietic system following stress, WT and S6K1-/- mice were treated with a single dose of 5-fluorouracil (5-FU). In response to myelosuppressive stress, S6K1 deficiency resulted in increased frequency of HSCs in bone marrow despite a significant reduction in overall cellularity (n=12 in each group, p<0.02). Following administration of 5-FU, S6K1 deficiency resulted in increased cell cycle progression of HSCs in bone marrow and showed increased expression of CDK4 and CDK6 as compared to control suggesting that 5-FU administration results in upregulation of cell cycle regulatory genes in S6K1 deficient HSCs. Moreover, S6K1-/- mice showed more sensitivity to repeated injections of 5-FU (n=11 WT, 15 S6K1-/-, p<0.01). Given the differential role of S6K1 in HSCs and mature progenitors, we assessed the effect of S6K1 deficiency in HSC function. We performed competitive repopulation assay using S6K1 deficient HSCs. When transplanted into lethally irradiated primary and secondary recipients, S6K1 deficient HSCs show significantly reduced engraftment relative to controls (n=11-13 in each group; p<0.05). Interestingly, overexpression of S6K1 in wild type HSCs also resulted in reduced engraftment of HSCs in primary and secondary transplant recipients, suggesting that S6K1 overexpression in HSCs leads to decreased self-renewal. In summary, our study identifies S6K1 as a critical regulator of hematopoietic stem cell development and functions both under steady-state conditions as well as under conditions of genotoxic stress. Using both gain of function and loss of function approaches, we demonstrate that the level of expression and activation of S6K1in HSCs plays a critical role in the maintenance of HSC self-renewal and engraftment. Disclosures No relevant conflicts of interest to declare.

scholarly journals Arhgap21 Expression in Bone Marrow Niche Is Crucial for Hematopoietic Progenitor Homing and Short Term Reconstitution after Transplantation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1591-1591
Author(s):  
Juliana M. Xavier ◽  
Lauremilia Ricon ◽  
Karla Priscila Vieira ◽  
Longhini Ana Leda ◽  
Carolina Bigarella ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Progenitor Cells ◽  
Bone Marrow Niche ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Short Term ◽  
Hematopoietic Stem

Abstract The microenvironment of the bone marrow (BM) is essential for retention and migration of hematopoietic progenitor cells. ARHGAP21 is a negative regulator of RhoGTPAses, involved in cellular migration and adhesion, however the role of ARHGAP21 in hematopoiesis is unknown. In order to investigate whether downregulation of Arhgap21 in microenvironment modulates bone marrow homing and reconstitution, we generated Arhgap21+/-mice using Embryonic Stem cell containing a vector insertion in Arhgap21 gene obtained from GeneTrap consortium and we then performed homing and bone marrow reconstitution assays. Subletally irradiated (9.5Gy) Arhgap21+/- and wild type (WT) mice received 1 x 106 BM GFP+cells by IV injection. For homing assay, 19 hours after the transplant, Lin-GFP+ cells were analyzed by flow cytometry. In reconstitution and self-renew assays, the GFP+ cell percentage in peripheral blood were analyzed 4, 8, 12 and 16 weeks after transplantation. Hematopoietic stem cells [GFP+Lin-Sca+c-Kit+ (LSK)] were counted after 8 and 16 weeks in bone marrow after primary transplant and 16 weeks after secondary transplant. The percentage of Lin-GFP+ hematopoietic progenitor cells that homed to Arhgap21+/-recipient (mean± SD) (2.07 ± 0.85) bone marrow was lower than those that homed to the WT recipient (4.76 ± 2.60); p=0.03. In addition, we observed a reduction (WT: 4.22 ±1.39; Arhgap21+/-: 2.17 ± 0.69; p=0.001) of Lin- GFP+ cells in Arhgap21+/-receptor spleen together with an increase of Lin- GFP+ population in Arhgap21+/-receptor peripheral blood (WT: 8.07 ± 3.85; Arhgap21+/-: 14.07 ±5.20; p=0.01), suggesting that hematopoietic progenitor cells which inefficiently homed to Arhgap21+/-bone marrow and spleen were retained in the blood stream. In bone marrow reconstitution assay, Arhgap21+/-receptor presented reduced LSK GFP+ cells after 8 weeks (WT: 0.19 ±0.03; Arhgap21+/-0.12±0.05; p=0.02) though not after 16 weeks from primary and secondary transplantation. The reduced LSK percentage after short term reconstitution was reflected in the lower GFP+ cells in peripheral blood 12 weeks after transplantation (WT: 96.2 ±1.1; Arhgap21+/-94.3±1.6; p=0.008). No difference was observed in secondary transplantation, indicating that Arhgap21reduction in microenvironment does not affect normal hematopoietic stem cell self-renewal. The knowledge of the niche process in regulation of hematopoiesis and their components helps to better understand the disordered niche function and gives rise to the prospect of improving regeneration after injury or hematopoietic stem and progenitor cell transplantation. In previous studies, the majority of vascular niche cells were affected after sublethal irradiation, however osteoblasts and mesenchymal stem cells were maintained (Massimo Dominici et al.; Blood; 2009.). RhoGTPase RhoA, which is inactivated by ARHGAP21 (Lazarini et al.; Biochim Biophys acta; 2013), has been described to be crucial for osteoblasts and mesenchymal stem cell support of hematopoiesis (Raman et al.; Leukemia; 2013). Taken together, these results suggest that Arhgap21 expression in bone marrow niche is essential for homing and short term reconstitution support. Moreover, this is the first study to investigate the role of Arhgap21 in bone marrow niche. Figure 1 Reduced homing and short term reconstitution in Arhgap21 +/- recipients. Bone marrow cells from GFP+ mice were injected into wild-type and Arhgap21+/- sublethally irradiated mice. 19 hours after the transplant, a decreased homing was observed to both bone marrow (a) and spleen (b) together with an increase of retained peripheral blood (c) Lin-GFP+ cells. In serial bone marrow transplantation, Arhgap21+/- presented reduced bone marrow LSK GFP+ cells 8 weeks (d) and peripheral blood GFP+ cells 12 weeks (e) after primary transplantation, though not 16 weeks after primary (f) and 16 weeks after secondary (g) transplantations. The result is expressed by means ±SD of 2 independent experiments. Figure 1. Reduced homing and short term reconstitution in Arhgap21+/- recipients. Bone marrow cells from GFP+ mice were injected into wild-type and Arhgap21+/- sublethally irradiated mice. 19 hours after the transplant, a decreased homing was observed to both bone marrow (a) and spleen (b) together with an increase of retained peripheral blood (c) Lin-GFP+ cells. In serial bone marrow transplantation, Arhgap21+/- presented reduced bone marrow LSK GFP+ cells 8 weeks (d) and peripheral blood GFP+ cells 12 weeks (e) after primary transplantation, though not 16 weeks after primary (f) and 16 weeks after secondary (g) transplantations. The result is expressed by means ±SD of 2 independent experiments. Disclosures No relevant conflicts of interest to declare.

scholarly journals Hematopoietic Stem Cell Function in a Murine Model of Sickle Cell Disease

Anemia ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Elisabeth H. Javazon ◽  
Mohamed Radhi ◽  
Bagirath Gangadharan ◽  
Jennifer Perry ◽  
David R. Archer
Keyword(s):  
Cell Cycle ◽  
Lipid Peroxidation ◽  
Bone Marrow ◽  
Stem Cell ◽  
Sickle Cell Disease ◽  
Hematopoietic Stem Cell ◽  
Cell Function ◽  
Cell Disease ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem

Previous studies have shown that the sickle environment is highly enriched for reactive oxygen species (ROS). We examined the oxidative effects of sickle cell disease on hematopoietic stem cell function in a sickle mouse model.In vitrocolony-forming assays showed a significant decrease in progenitor colony formation derived from sickle compared to control bone marrow (BM). Sickle BM possessed a significant decrease in the KSL (c-kit+, Sca-1+, Lineage−) progenitor population, and cell cycle analysis showed that there were fewer KSL cells in the G0phase of the cell cycle compared to controls. We found a significant increase in both lipid peroxidation and ROS in sickle-derived KSL cells.In vivoanalysis demonstrated that normal bone marrow cells engraft with increased frequency into sickle mice compared to control mice. Hematopoietic progenitor cells derived from sickle mice, however, demonstrated significant impairment in engraftment potential. We observed partial restoration of engraftment by n-acetyl cysteine (NAC) treatment of KSL cells prior to transplantation. Increased intracellular ROS and lipid peroxidation combined with improvement in engraftment following NAC treatment suggests that an altered redox environment in sickle mice affects hematopoietic progenitor and stem cell function.

scholarly journals Identification of BCR/ABL-negative primitive hematopoietic progenitor cells within chronic myeloid leukemia marrow

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 801-807 ◽  
Author(s):  
T Leemhuis ◽  
D Leibowitz ◽  
G Cox ◽  
R Silver ◽  
EF Srour ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Polymerase Chain Reaction Analysis ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem

Chronic myeloid leukemia (CML) is a malignant disorder of the hematopoietic stem cell. It has been shown that normal stem cells coexist with malignant stem cells in the bone marrow of patients with chronic-phase CML. To characterize the primitive hematopoietic progenitor cells within CML marrow, CD34+DR- and CD34+DR+ cells were isolated using centrifugal elutriation, monoclonal antibody labeling, and flow cytometric cell sorting. Polymerase chain reaction analysis of RNA samples from these CD34+ subpopulations was used to detect the presence of the BCR/ABL translocation characteristic of CML. The CD34+DR+ subpopulation contained BCR/ABL(+) cells in 11 of 12 marrow samples studied, whereas the CD34+DR- subpopulation contained BCR/ABL(+) cells in 6 of 9 CML marrow specimens. These cell populations were assayed for hematopoietic progenitor cells, and individual hematopoietic colonies were analyzed by PCR for their BCR/ABL status. Results from six patients showed that nearly half of the myeloid colonies cloned from CD34+DR- cells were BCR/ABL(+), although the CD34+DR- subpopulation contained significantly fewer BCR/ABL(+) progenitor cells than either low-density bone marrow (LDBM) or the CD34+DR+ fraction. These CD34+ cells were also used to establish stromal cell-free long-term bone marrow cultures to assess the BCR/ABL status of hematopoietic stem cells within these CML marrow populations. After 28 days in culture, three of five cultures initiated with CD34+DR- cells produced BCR/ABL(-) cells. By contrast, only one of eight cultures initiated with CD34+DR+ cells were BCR/ABL(-) after 28 days. These results indicate that the CD34+DR- subpopulation of CML marrow still contains leukemic progenitor cells, although to a lesser extent than either LDBM or CD34+DR+ cells.

scholarly journals Increased Hematopoietic Stem Cells/Hematopoietic Progenitor Cells Measured as Endogenous Spleen Colonies in Radiation-Induced Adaptive Response in Mice (Yonezawa Effect)

Dose-Response ◽  
2018 ◽  
Vol 16 (3) ◽  
pp. 155932581879015 ◽  
Author(s):  
Bing Wang ◽  
Kaoru Tanaka ◽  
Yasuharu Ninomiya ◽  
Kouichi Maruyama ◽  
Guillaume Varès ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Adaptive Response ◽  
Underlying Mechanism ◽  
Hematopoietic Progenitor Cells ◽  
X Rays ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Different Types ◽  
Radiation Induced

The existence of radiation-induced adaptive response (AR) was reported in varied biosystems. In mice, the first in vivo AR model was established using X-rays as both the priming and the challenge doses and rescue of bone marrow death as the end point. The underlying mechanism was due to the priming radiation-induced resistance in the blood-forming tissues. In a series of investigations, we further demonstrated the existence of AR using different types of ionizing radiation (IR) including low linear energy transfer (LET) X-rays and high LET heavy ion. In this article, we validated hematopoietic stem cells/hematopoietic progenitor cells (HSCs/HPCs) measured as endogenous colony-forming units-spleen (CFU-S) under AR inducible and uninducible conditions using combination of different types of IR. We confirmed the consistency of increased CFU-S number change with the AR inducible condition. These findings suggest that AR in mice induced by different types of IR would share at least in part a common underlying mechanism, the priming IR-induced resistance in the blood-forming tissues, which would lead to a protective effect on the HSCs/HPCs and play an important role in rescuing the animals from bone marrow death. These findings provide a new insight into the mechanistic study on AR in vivo.

scholarly journals Identification of BCR/ABL-negative primitive hematopoietic progenitor cells within chronic myeloid leukemia marrow

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 801-807 ◽  
Author(s):  
T Leemhuis ◽  
D Leibowitz ◽  
G Cox ◽  
R Silver ◽  
EF Srour ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Polymerase Chain Reaction Analysis ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem

Abstract Chronic myeloid leukemia (CML) is a malignant disorder of the hematopoietic stem cell. It has been shown that normal stem cells coexist with malignant stem cells in the bone marrow of patients with chronic-phase CML. To characterize the primitive hematopoietic progenitor cells within CML marrow, CD34+DR- and CD34+DR+ cells were isolated using centrifugal elutriation, monoclonal antibody labeling, and flow cytometric cell sorting. Polymerase chain reaction analysis of RNA samples from these CD34+ subpopulations was used to detect the presence of the BCR/ABL translocation characteristic of CML. The CD34+DR+ subpopulation contained BCR/ABL(+) cells in 11 of 12 marrow samples studied, whereas the CD34+DR- subpopulation contained BCR/ABL(+) cells in 6 of 9 CML marrow specimens. These cell populations were assayed for hematopoietic progenitor cells, and individual hematopoietic colonies were analyzed by PCR for their BCR/ABL status. Results from six patients showed that nearly half of the myeloid colonies cloned from CD34+DR- cells were BCR/ABL(+), although the CD34+DR- subpopulation contained significantly fewer BCR/ABL(+) progenitor cells than either low-density bone marrow (LDBM) or the CD34+DR+ fraction. These CD34+ cells were also used to establish stromal cell-free long-term bone marrow cultures to assess the BCR/ABL status of hematopoietic stem cells within these CML marrow populations. After 28 days in culture, three of five cultures initiated with CD34+DR- cells produced BCR/ABL(-) cells. By contrast, only one of eight cultures initiated with CD34+DR+ cells were BCR/ABL(-) after 28 days. These results indicate that the CD34+DR- subpopulation of CML marrow still contains leukemic progenitor cells, although to a lesser extent than either LDBM or CD34+DR+ cells.

Chemotactic and chemokinetic activities of stem cell factor on murine hematopoietic progenitor cells

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4100-4108 ◽  
Author(s):  
N Okumura ◽  
K Tsuji ◽  
Y Ebihara ◽  
I Tanaka ◽  
N Sawai ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Stem Cell Factor ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Murine Bone Marrow ◽  
Checkerboard Assay

We investigated the effects of stem cell factor (SCF) on the migration of murine bone marrow hematopoietic progenitor cells (HPC) in vitro using a modification of the checkerboard assay. Chemotactic and chemokinetic activities of SCF on HPC were evaluated by the numbers of HPC migrated on positive and negative gradients of SCF, respectively. On both positive and negative gradients of SCF, HPC began to migrate after 4 hours incubation, and their numbers then increased time- dependently. These results indicated that SCF functions as a chemotactic and chemokinetic agent for HPC. Analysis of types of colonies derived from the migrated HPC showed that SCF had chemotactic and chemokinetic effects on all types of HPC. When migrating activities of other cytokines were examined, interleukin (IL)-3 and IL-11 also affected the migration of HPC, but the degrees of each effect were lower than that of SCF. The results of the present study demonstrated that SCF is one of the most potent chemotactic and chemokinetic factors for HPC and suggest that SCF may play an important role in the flow of HPC into bone marrow where stromal cells constitutively produce SCF.

scholarly journals Interferon gamma selectively inhibits very primitive CD342+CD38- and not more mature CD34+CD38+ human hematopoietic progenitor cells.

1994 ◽  
Vol 180 (3) ◽  
pp. 1177-1182 ◽  
Author(s):  
H W Snoeck ◽  
D R Van Bockstaele ◽  
G Nys ◽  
M Lenjou ◽  
F Lardon ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Interferon Gamma ◽  
Bone Marrow Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Ifn Gamma ◽  
Hematopoietic Stem ◽  
First Time ◽  
Dose Dependent

To assess the effects of interferon gamma (IFN-gamma) on very primitive hematopoietic progenitor cells, CD34(2+)CD38- human bone marrow cells were isolated and cultured in a two-stage culture system, consisting of a primary liquid culture phase followed by a secondary semisolid colony assay. CD34(2+)CD38- cells needed at least the presence of interleukin 3 (IL-3) and kit ligand (KL) together with either IL-1, IL-6, or granulocyte-colony-stimulating factor (G-CSF) in the primary liquid phase in order to proliferate and differentiate into secondary colony-forming cells (CFC). Addition of IFN-gamma to the primary liquid cultures inhibited cell proliferation and generation of secondary CFC in a dose-dependent way. This was a direct effect since it was also seen in primary single cell cultures of CD34(2+)CD38- cells. The proliferation of more mature CD34+CD38+ cells, however, was not inhibited by IFN-gamma, demonstrating for the first time that IFN-gamma is a specific and direct hematopoietic stem cell inhibitor. IFN-gamma, moreover, preserves the viability of CD34(2+)CD38- cells in the absence of other cytokines. IFN-gamma could, therefore, play a role in the protection of the stem cell compartment from exhaustion in situations of hematopoietic stress and may be useful as stem cell protecting agent against chemotherapy for cancer.

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

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2674-2674
Author(s):  
Seiji Fukuda ◽  
Hal E. Broxmeyer ◽  
Louis M. Pelus
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Hematopoietic Progenitor Cells ◽  
Leukemia Cells ◽  
Flt3 Ligand ◽  
Hematopoietic Progenitor ◽  
Short Term ◽  
Hematopoietic Stem

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

Human Embryonic Stem Cell-Derived Mesenchymal Stroma Cells (hES-MSC) Share Basic Properties with Bone Marrow MSC, They Have Potent Stroma Support Capacities but Lack Immune-Modulatory Function. Implications for off-the Shelf ES-MSC Therapies.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3858-3858 ◽  
Author(s):  
Ou Li ◽  
Ariane Tormin ◽  
Jan Claas Brune ◽  
Berit Sundberg ◽  
Johan Hyllner ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Human Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Cell Types ◽  
Es Cell ◽  
Nsg Mice ◽  
Mesenchymal Stroma ◽  
Mesenchymal Stroma Cells

Abstract Abstract 3858 Mesenchymal stroma cells (MSC) have a high potential for novel cell therapy approaches in clinical transplantation due to their intriguing properties, e.g. high proliferation and differentiation capacity, stromal support and immune-modulation. Commonly, bone marrow-derived MSC (BM-MSC) are used for clinical MSC cell therapies. However, BM-derived MSC have a restricted proliferative capacity and cultured BM-MSC are heterogeneous and thus difficult to standardize. Human embryonic stem cell-derived mesenchymal stroma cells (hES-MSC) have recently been developed and might represent an alternative and unlimited source of hMSCs. We therefore aimed to characterize human ES-cell-derived MSC, i.e. the hES-MSC line hES-MP002.5 (Cellartis) and compare its properties with normal human bone marrow (BM) derived MSC. We found that hES-MP cells have lower yet reasonable CFU-F capacity when compared with BM-MSC (6+3 vs 25+1 CFU-F per 100 cells). hES-MP cells showed similar immunophenotypic properties compared with BM-MSC (flow cytometry): Both cell types were positive for CD105, CD73, CD166, HLA Class I, CD44, CD146 and CD90, and cells were negative for surface markers such as CD45, CD34, CD14, CD31, CD19, and HLA-DR. hES-MP, like BM-MSC, could be differentiated into adipocytes, osteoblasts and chondrocytes upon induction in vitro. In order to test whether MSC were capable of homing to the bone marrow after intravenous injection, hES-MP and BM-MSC were markerd with GFP, and sorted GFP-positive cells were injected intravenously into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. GFP-positive cells were not detected in the bone marrow 24 hours after injection, neither when hES-MP cells were injected, nor - and as expected - when cultured BM-MSC were used. Intra-femoral transplantation into NSG mice using GFP expressing hES-MP and BM-MSC on the other hand demonstrated successful long-term engraftment (8 weeks) for both cell types. Morphology and intra-femoral localization of hES-MP were similar compared to BM-MSC. LTC-IC and co-transplantation experiments with cord blood CD34+ hematopoietic cells demonstrated furthermore that hES-MP, like BM-MSC, possess potent stroma support function both in vitro and in vivo. However, hES-MP showed no or only little activity in mixed lymphocyte cultures and PHA lymphocyte stimulation assays. In summary, our data demonstrate that MSC derived from hES cells have biological properties and potent stroma functions similar to conventional BM-MSC. Thus, ES-cell derived MSC might be an attractive and reliable alternative and unlimited source for obtaining MSC for clinical cell therapy. However, hES-MP probably have no or only little immuno-modulative capacity, which may limit their potential clinical use. Disclosures: Hyllner: Cellartis AB: Employment.

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