scholarly journals Bradycardic mice undergo effective heart rate improvement after specific homing to the sino-atrial node and differentiation of adult muscle derived stem cells

2018 ◽  
Author(s):  
Pietro Mesirca ◽  
Daria Mamaeva ◽  
Isabelle Bidaud ◽  
Matthias Baudot ◽  
Romain Davaze ◽  
...  
Keyword(s):  
Stem Cells ◽  
Heart Rate ◽  
Heart Rhythm ◽  
Mutant Mice ◽  
Recipient Mouse ◽  
Wild Type ◽  
Cardiac Pacemakers ◽  
Lineage Differentiation

AbstractCurrent treatments for heart automaticity disorders still lack a safe and efficient source of stem cells to restore normal biological pacemaking. Since adult Muscle-Derived Stem Cells (MDSC) show multi-lineage differentiation in vitro including into spontaneously beating cardiomyocytes, we questioned whether they could effectively differentiate into cardiac pacemakers, a specific population of cardiomyocytes producing electrical impulses in the sino-atrial node (SAN) of adult heart. We show here that beating cardiomyocytes, differentiated from MDSC in vitro, exhibit typical characteristics of cardiac pacemakers: expression of markers of the SAN lineage Hcn4, Tbx3 and Islet1, as well as spontaneous calcium transients and hyperpolarization-activated “funny” current and L-type Cav1.3 channels. Pacemaker-like myocytes differentiated in vitro from Cav1.3-deficient mouse stem cells produced slower rate of spontaneous Ca2+ transients, consistent with the reduced activity of native pacemakers in mutant mice. In vivo, undifferentiated wild type MDSC migrated and homed with increased engraftment to the SAN of bradycardic mutant Cav1.3-/- within 2-3 days after systemic I.P. injection. The increased homing of MDSCs corresponded to increased levels of the chemokine SDF1 and its receptor CXCR4 in mutant SAN tissue and was ensued by differentiation of MDSCs into Cav1.3-expressing pacemaker-like myocytes within 10 days and a significant improvement of the heart rate maintained for up to 40 days. Optical mapping and immunofluorescence analyses performed after 40 days on SAN tissue from transplanted wild type and mutant mice showed MDSCs integrated as pacemaking cells both electrically and functionally within recipient mouse SAN. These findings identify MDSCs as directly transplantable stem cells that efficiently home, differentiate and improve heart rhythm in mouse models of congenital bradycardia.

Factor VIIa induces extracellular vesicles from the endothelium: A potential mechanism for its hemostatic effect

Blood ◽  
2021 ◽  
Author(s):  
Kaushik Das ◽  
Shiva Keshava ◽  
Shabbir A Ansari ◽  
Vijay Kumar Reddy Kondreddy ◽  
Charles Esmon ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Extracellular Vesicles ◽  
Factor Viia ◽  
Mutant Mice ◽  
In Vivo Studies ◽  
Cell Protein ◽  
Wild Type ◽  
Hemostatic Effect

Recombinant FVIIa (rFVIIa) is used as a hemostatic agent to treat bleeding disorders in hemophilia patients with inhibitors and other groups of patients. Our recent studies showed that FVIIa binds endothelial cell protein C receptor (EPCR) and induces protease-activated receptor 1 (PAR1)-mediated biased signaling. The importance of FVIIa-EPCR-PAR1-mediated signaling in hemostasis is unknown. In the present study, we show that FVIIa induces the release of extracellular vesicles (EVs) from endothelial cells both in vitro and in vivo. Silencing of EPCR or PAR1 in endothelial cells blocked the FVIIa-induced generation of EVs. Consistent with these data, FVIIa treatment enhanced the release of EVs from murine brain endothelial cells isolated from wild-type, EPCR overexpressors, and PAR1-R46Q mutant mice, but not EPCR-deficient or PAR1-R41Q mutant mice. In vivo studies revealed that administration of FVIIa to wild-type, EPCR overexpressors, and PAR1-R46Q mutant mice, but not EPCR-deficient or PAR1-R41Q mutant mice, increase the number of circulating EVs. EVs released in response to FVIIa treatment exhibit enhanced procoagulant activity. Infusion of FVIIa-generated EVs and not control EVs to platelet-depleted mice increased thrombin generation at the site of injury and reduced blood loss. Administration of FVIIa-generated EVs or generation of EVs endogenously by administering FVIIa augmented the hemostatic effect of FVIIa. Overall, our data reveal that FVIIa treatment, through FVIIa-EPCR-PAR1 signaling, releases EVs from the endothelium into the circulation, and these EVs contribute to the hemostatic effect of FVIIa.

scholarly journals A Role for Mac-1 (CDIIb/CD18) in Immune Complex–stimulated Neutrophil Function In Vivo: Mac-1 Deficiency Abrogates Sustained Fcγ Receptor–dependent Neutrophil Adhesion and Complement-dependent Proteinuria in Acute Glomerulonephritis

1997 ◽  
Vol 186 (11) ◽  
pp. 1853-1863 ◽  
Author(s):  
Tao Tang ◽  
Alexander Rosenkranz ◽  
Karel J.M. Assmann ◽  
Michael J. Goodman ◽  
Jose-Carlos Gutierrez-Ramos ◽  
...  
Keyword(s):  
Immune Complex ◽  
Mutant Mice ◽  
Polymorphonuclear Neutrophil ◽  
Complement C3 ◽  
Acute Glomerulonephritis ◽  
Wild Type ◽  
Filamentous Actin ◽  
Deficient Mice

Mac-1 (αmβ2), a leukocyte adhesion receptor, has been shown in vitro to functionally interact with Fcγ receptors to facilitate immune complex (IC)–stimulated polymorphonuclear neutrophil (PMN) functions. To investigate the relevance of Mac-1–FcγR interactions in IC-mediated injury in vivo, we induced a model of Fc-dependent anti–glomerular basement membrane (GBM) nephritis in wild-type and Mac-1–deficient mice by the intravenous injection of anti-GBM antibody. The initial glomerular PMN accumulation was equivalent in Mac-1 null and wild-type mice, but thereafter increased in wild-type and decreased in mutant mice. The absence of Mac-1 interactions with obvious ligands, intercellular adhesion molecule 1 (ICAM-1), and C3 complement, is not responsible for the decrease in neutrophil accumulation in Mac-1– deficient mice since glomerular PMN accumulation in mice deficient in these ligands was comparable to those in wild-type mice. In vitro studies showed that spreading of Mac-1–null PMNs to IC-coated dishes was equivalent to that of wild-type PMNs at 5–12 min but was markedly reduced thereafter, and was associated with an inability of mutant neutrophils to redistribute filamentous actin. This suggests that in vivo, Mac-1 is not required for the initiation of Fc-mediated PMN recruitment but that Mac-1–FcγR interactions are required for filamentous actin reorganization leading to sustained PMN adhesion, and this represents the first demonstration of the relevance of Mac-1–FcγR interactions in vivo. PMN-dependent proteinuria, maximal in wild-type mice at 8 h, was absent in Mac-1 mutant mice at all time points. Complement C3–deficient mice also had significantly decreased proteinuria compared to wild-type mice. Since Mac-1 on PMNs is the principal ligand for ic3b, an absence of Mac-1 interaction with C3 probably contributed to the abrogation of proteinuria in Mac-1–null mice.

scholarly journals Mesenchymal stem cells express serine protease inhibitor to evade the host immune response

Blood ◽  
2011 ◽  
Vol 117 (4) ◽  
pp. 1176-1183 ◽  
Author(s):  
Najib El Haddad ◽  
Dean Heathcote ◽  
Robert Moore ◽  
Sunmi Yang ◽  
Jamil Azzi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
T Cell ◽  
Protease Inhibitor ◽  
Serine Protease ◽  
Serine Protease Inhibitor ◽  
Cytotoxic T Cell ◽  
Wild Type

Abstract Clinical trials using mesenchymal stem cells (MSCs) have been initiated worldwide. An improved understanding of the mechanisms by which allogeneic MSCs evade host immune responses is paramount to regulating their survival after administration. This study has focused on the novel role of serine protease inhibitor (SPI) in the escape of MSCs from host immunosurveillance through the inhibition of granzyme B (GrB). Our data indicate bone marrow–derived murine MSCs express SPI6 constitutively. MSCs from mice deficient for SPI6 (SPI6−/−) exhibited a 4-fold higher death rate by primed allogeneic cytotoxic T cells than did wild-type MSCs. A GrB inhibitor rescued SPI6−/− MSCs from cytotoxic T-cell killing. Transduction of wild-type MSCs with MigR1-SPI6 also protected MSCs from cytotoxic T cell–mediated death in vitro. In addition, SPI6−/− MSCs displayed a shorter lifespan than wild-type MSCs when injected into an allogeneic host. We conclude that SPI6 protects MSCs from GrB-mediated killing and plays a pivotal role in their survival in vivo. Our data could serve as a basis for future SPI-based strategies to regulate the survival and function of MSCs after administration and to enhance the efficacy of MSC-based therapy for diseases.

Sqstm1 Is Required to Retain Hematopoietic Stem Cell/ Progenitors As a Negative Regulator of Macrophage-Dependent Inflammatory Signaling in the Bone Marrow Osteoblastic Niche

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 350-350
Author(s):  
Kyung-Hee Chang ◽  
Amitava Sengupta ◽  
Ramesh C Nayak ◽  
Angeles Duran ◽  
Sang Jun Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Research Funding ◽  
Negative Regulator ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
P Retention

Abstract In the bone marrow (BM), hematopoietic stem cells and progenitors (HSC/P) reside in specific anatomical niches. Among these niches, a functional osteoblast (Ob)-macrophage (MΦ) niche has been described where Ob and MΦ (so called "osteomacs") are in direct relationship. A connection between innate immunity surveillance and traffic of hematopoietic stem cells/progenitors (HSC/P) has been demonstrated but the regulatory signals that instruct immune regulation from MΦ and Ob on HSC/P circulation are unknown. The adaptor protein sequestosome 1 (Sqstm1), contains a Phox bemp1 (PB1) domain which regulates signal specificities through PB1-PB1 scaffolding and processes of autophagy. Using microenvironment and osteoblast-specific mice deficient in Sqstm1, we discovered that the deficiency of Sqstm1 results in macrophage contact-dependent activation of Ob IKK/NF-κB, in vitro and in vivo repression of Ccl4 (a CCR5 binding chemokine that has been shown to modulate microenvironment Cxcl12-mediated responses of HSC/P), HSC/P egress and deficient BM homing of wild-type HSC/P. Interestingly, while Ccl4 expression is practically undetectable in wild-type or Sqstm1-/- Ob, primary Ob co-cultured with wild-type BM-derived MΦ strongly upregulate Ccl4 expression, which returns to normal levels upon genetic deletion of Ob Sqstm1. We discovered that MΦ can activate an inflammatory pathway in wild-type Ob which include upregulation of activated focal adhesion kinase (p-FAK), IκB kinase (IKK), nuclear factor (NF)-κB and Ccl4 expression through direct cell-to-cell interaction. Sqstm1-/- Ob cocultured with MΦ strongly upregulated p-IKBα and NF-κB activity, downregulated Ccl4 expression and secretion and repressed osteogenesis. Forced expression of Sqstm1, but not of an oligomerization-deficient mutant, in Sqstm1-/- Ob restored normal levels of p-IKBα, NF-κB activity, Ccl4 expression and osteogenic differentiation, indicating that Sqstm1 dependent Ccl4 expression depends on localization to the autophagosome formation site. Finally, Ob Sqstm1 deficiency results in upregulation of Nbr1, a protein containing a PB1 interacting domain. Combined deficiency of Sqstm1 and Nbr1 rescues all in vivo and in vitro phenotypes of Sqstm1 deficiency related to osteogenesis and HSC/P egression in vivo. Together, this data indicated that Sqstm1 oligomerization and functional repression of its PB1 binding partner Nbr1 are required for Ob dependent Ccl4 production and HSC/P retention, resulting in a functional signaling network affecting at least three cell types. A functional ‘MΦ-Ob niche’ is required for HSC/P retention where Ob Sqstm1 is a negative regulator of MΦ dependent Ob NF-κB activation, Ob differentiation and BM HSC/P traffic to circulation. Disclosures Starczynowski: Celgene: Research Funding. Cancelas:Cerus Co: Research Funding; P2D Inc: Employment; Terumo BCT: Research Funding; Haemonetics Inc: Research Funding; MacoPharma LLC: Research Funding; Therapure Inc.: Consultancy, Research Funding; Biomedical Excellence for Safer Transfusion: Research Funding; New Health Sciences Inc: Consultancy.

scholarly journals Adenovirus infection promotes the formation of glioma stem cells by glioblastoma cells through the TLR9/NEAT1/STAT3 pathway

2020 ◽  
Author(s):  
Jian Zang ◽  
Min-hua Zheng ◽  
Xiu-li Cao ◽  
Yi-zhe Zhang ◽  
Yu-fei Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Glioma Cells ◽  
Tumor Model ◽  
Glioma Stem Cells ◽  
Stem Cell Markers ◽  
Qrt Pcr ◽  
Lineage Differentiation ◽  
Stat3 Signaling

Abstract BackgroundGlioma stem cells (GSCs) are glioma cells with stemness and are responsible for a variety of malignant behaviors of glioma. Evidence has shown that signals from tumor microenvironment (TME) enhance stemness of glioma cells, but the identity of the signaling molecules and underlying mechanisms have been incompletely elucidated.MethodsHuman samples and glioma cell lines were cultured in vitro to determine the effects of viral infection by sphere formation, qRT-PCR, Western blot, FACS and immunofluorescence; for in vivo analysis, mice subcutaneous tumor model was carried; while bioinformatics analysis and qRT-PCR were applied for further mechanistic studies.ResultsIn this study, we show that infection of patient-derived glioma cells with adenovirus (ADV) increases the formation of tumor spheres. ADV infection upregulated stem cell markers, and the resultant tumor spheres held the capacities of self-renewal and multi-lineage differentiation, and had stronger potential to form xenograft tumors in immune-compromised mice. ADV promoted GSC formation likely via TLR9, because TLR9 was upregulated after ADV infection, and knockdown of TLR9 reduced ADV-induced GSCs. Consistently, MYD88, as well as total STAT3 and phosphorylated (p-)STAT3, were also upregulated in ADV-induced GSCs. Knockdown of MYD88 or pharmaceutical inhibition of STAT3 attenuated stemness of ADV-induced GSCs. Moreover, we found that ADV infection upregulated lncRNA NEAT1, which is downstream to TLRs and play important roles in cancer stem cells via multiple mechanisms including strengthening STAT3 signaling. Indeed, knockdown of NEAT1 impaired stemness of ADV-induced GSCs. Lastly, we show that HMGB1, a damage associated molecular pattern (DAMP) that also triggers TLR signaling, upregulated stemness markers in glioma cells.ConclusionsIn summary, our data indicate that ADV, which has been developed as vectors for gene therapy and oncolytic virus, promotes the formation of GSCs via TLR9/NEAT1/STAT3 signaling.

Hematopoietic stem cells with controllable tEpoR transgenes have a competitive advantage in bone marrow transplantation

Blood ◽  
2000 ◽  
Vol 95 (12) ◽  
pp. 3710-3715 ◽  
Author(s):  
Suzanne Kirby ◽  
William Walton ◽  
Oliver Smithies
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Erythropoietin Receptor ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor ◽  
Colony Forming Units

Abstract In a previous study, it was found that a truncated erythropoietin receptor transgene (tEpoR tg) enables multilineage hematopoietic progenitor amplification after treatment with erythropoietin (epo) in vitro and in vivo. This study used competitive bone marrow (BM) repopulation to show that tEpoR tg facilitates transplantation by hematopoietic stem cells (HSC). Individual multilineage colonies, committed myeloid progenitor colonies, and lymphoid colonies (pre-B colony-forming units) were grown from the marrow of animals 6 months after they received a 50/50 mixture of transgene and wild-type BM cells. In epo-treated recipients, the transgene-bearing cells significantly outcompeted the wild-type cells (84%-100% versus 16%-0%, respectively). In recipients treated with phosphate-buffered saline, the repopulation was minimally different from the donor mixture (49%-64% transgene versus 51%-36% wild-type). The epo-induced repopulation advantage is maintained in secondary transplants. In addition, neither accelerated HSC depletion nor uncontrollable proliferation occurred during epo-stimulated serial transplants of transgene-containing BM. Thus, the tEpoR tg functions in a benign fashion in HSC and allows for a significant and controllable repopulation advantage in vivo without excessive HSC depletion relative to wild-type BM.

scholarly journals Neurons Expressing the Highest Levels of γ-Synuclein Are Unaffected by Targeted Inactivation of the Gene

2003 ◽  
Vol 23 (22) ◽  
pp. 8233-8245 ◽  
Author(s):  
Natalia Ninkina ◽  
Katerina Papachroni ◽  
Darren C. Robertson ◽  
Oliver Schmidt ◽  
Liz Delaney ◽  
...  
Keyword(s):  
Es Cells ◽  
Culture Conditions ◽  
Sensory Function ◽  
Mutant Mice ◽  
Null Mutant ◽  
Wild Type ◽  
Complete Inactivation ◽  
Null Mutant Mice

ABSTRACT Homologous recombination in ES cells was employed to generate mice with targeted deletion of the first three exons of the γ-synuclein gene. Complete inactivation of gene expression in null mutant mice was confirmed on the mRNA and protein levels. Null mutant mice are viable, are fertile, and do not display evident phenotypical abnormalities. The effects of γ-synuclein deficiency on motor and peripheral sensory neurons were studied by various methods in vivo and in vitro. These two types of neurons were selected because they both express high levels of γ-synuclein from the early stages of mouse embryonic development but later in the development they display different patterns of intracellular compartmentalization of the protein. We found no difference in the number of neurons between wild-type and null mutant animals in several brain stem motor nuclei, in lumbar dorsal root ganglia, and in the trigeminal ganglion. The survival of γ-synuclein-deficient trigeminal neurons in various culture conditions was not different from that of wild-type neurons. There was no difference in the numbers of myelinated and nonmyelinated fibers in the saphenous nerves of these animals, and sensory reflex thresholds were also intact in γ-synuclein null mutant mice. Nerve injury led to similar changes in sensory function in wild-type and mutant mice. Taken together, our data suggest that like α-synuclein, γ-synuclein is dispensable for the development and function of the nervous system.

scholarly journals Generation and Analysis of Siah2 Mutant Mice

2003 ◽  
Vol 23 (24) ◽  
pp. 9150-9161 ◽  
Author(s):  
Ian J. Frew ◽  
Vicki E. Hammond ◽  
Ross A. Dickins ◽  
Julian M. W. Quinn ◽  
Carl R. Walkley ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Ubiquitin Ligase ◽  
Mutant Mice ◽  
Wild Type ◽  
Protein Substrates ◽  
Myeloid Progenitor ◽  
Neonatal Lethality ◽  
Myeloid Progenitor Cells

ABSTRACT Siah proteins function as E3 ubiquitin ligase enzymes to target the degradation of diverse protein substrates. To characterize the physiological roles of Siah2, we have generated and analyzed Siah2 mutant mice. In contrast to Siah1a knockout mice, which are growth retarded and exhibit defects in spermatogenesis, Siah2 mutant mice are fertile and largely phenotypically normal. While previous studies implicate Siah2 in the regulation of TRAF2, Vav1, OBF-1, and DCC, we find that a variety of responses mediated by these proteins are unaffected by loss of Siah2. However, we have identified an expansion of myeloid progenitor cells in the bone marrow of Siah2 mutant mice. Consistent with this, we show that Siah2 mutant bone marrow produces more osteoclasts in vitro than wild-type bone marrow. The observation that combined Siah2 and Siah1a mutation causes embryonic and neonatal lethality demonstrates that the highly homologous Siah proteins have partially overlapping functions in vivo.

Critical Role for DOCK2 in CD8+/TCR− Graft Facilitating Cells Enhancing Engraftment of Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 142-142
Author(s):  
Yujie Wen ◽  
Mary J Elliott ◽  
Yiming Huang ◽  
Deborah R Corbin ◽  
Yoshinori Fukui ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Signaling Pathways ◽  
Microarray Analysis ◽  
Control Analysis ◽  
Wild Type ◽  
Hematopoietic Stem

Abstract Abstract 142 CD8+/TCR− graft facilitating cells (FC) have a potent capability to facilitate relatively small numbers of highly purified hematopoietic stem cells (HSC) in both allogeneic and syngeneic recipients. The mechanisms by which FC promote HSC engraftment have not been fully elucidated. We previously found that FC from non-obese diabetic mice (NOD) were compromised in enhancing engraftment of syngeneic and allogeneic HSC compared with FC from diabetes free congenic NOR mice. We therefore compared gene expression profiling between NOD FC and control NOR FC by microarray analysis. Among 18 most significant differentially expressed genes revealed from 45101 genes by false discovery rate control analysis with a cut-off value at level 0.05, dedicator of cytokinesis 2 (DOCK2) was identified as the gene with the most significant difference (P = 1.07 × 10−7). DOCK2 is a hematopoietic cell-specific member of the Caenorhabditis elegans Ced-5, mammalian DOCK180 and Drosophila melanogaster myoblast city (CDM) family of guanine nucleotide exchange factors. DOCK2 activates the small GTPase Rac and is indispensable for migration of plasmacytoid dendritic cells (pDC). FC is a heterogeneous cell population, with a predominant subpopulation resembling plasmacytoid precursor DC (p-preDC FC). In vitro studies demonstrated that FC increased clonogenicity of HSC and improved the ability of the impaired stroma to support late cobblestone area formation by HSC, which suggests that FC homing to hematopoietic niche as a potential component might be a perquisite for FC to enhance HSC engraftment. Therefore, we hypothesized that signaling pathways controlling cell migration via DOCK2 are critical for FC to enhance HSC engraftment. To test our hypothesis, DOCK2 expression data from microarray analysis was further confirmed using relative quantitative real-time PCR and high content image analysis. The expression level of DOCK2 in FC was significantly lower in NOD mice compared with NOR mice (p < 0.01 vs. NOR FC). Functional phenotypes of DOCK2-deficient FC were determined by Transwell migration assay and colony-forming cell assay in vitro, and by co-transplantation of HSC with FC in vivo as well as enumeration of CellTrack Green labeled FC by flow cytometry in spleen, thymus, and bone marrow of femurs and tibias 18 hours post-transplantation. Deficiency of DOCK2 in FC did not affect the ability of FC in promoting HSC colony formation when the two were cultured together. However, DOCK2-deficient FC were compromised in migration to the α-cheemokine, stromal derived factor-1 (SDF-1) at dose 200 ng/ml (Fig. A, P < 0.01 vs. wild-type FC). Homing of FC to spleen and bone marrow of femurs and tibias was also significantly impaired in DOCK2-deficient FC. Moreover, deficiency of DOCK2 in FC abrogated enhancement of HSC engraftment by FC in the syngeneic and allogeneic in vivo assays (Fig. B, syngeneic model: 500 B6 HSC plus 30K B6 or DOCK2−/−FC into lethally irradiated B6 recipients; Fig. C, allogeneic model: 10K B6 HSC plus 30K B6 or DOCK2−/−FC into lethally irradiated B10.BR recipients, P < 0.05 vs. B6 HSC plus wild-type FC). Taken together, our results indicate that deficiency of DOCK2 in FC leads to the dysfunction in migration, and suggest that the signaling pathways involved in FC migration are crucial for FC to enhance HSC engraftment. Disclosures: Ildstad: Regenerex LLC: Equity Ownership.

Steel factor responsiveness regulates the high self-renewal phenotype of fetal hematopoietic stem cells

Blood ◽  
2007 ◽  
Vol 109 (11) ◽  
pp. 5043-5048 ◽  
Author(s):  
Michelle B. Bowie ◽  
David G. Kent ◽  
Michael R. Copley ◽  
Connie J. Eaves
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Wild Type ◽  
Developmentally Regulated ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Steel Factor ◽  
Reduced Rate

Abstract Fetal hematopoietic stem cells (HSCs) regenerate daughter HSCs in irradiated recipients more rapidly than do adult HSCs. However, both types of HSCs divide in vitro with the same cell-cycle transit times, suggesting different intrinsically determined self-renewal activities. To investigate the mechanism(s) underlying these differences, we compared fetal and adult HSC responses to Steel factor (SF) stimulation in vitro and in vivo. These experiments were undertaken with both wild-type cells and W41/W41 cells, which have a functionally deficient c-kit kinase. In vitro, fetal HSC self-renewal divisions, like those of adult HSCs, were found to be strongly dependent on c-kit activation, but the fetal HSCs responded to much lower SF concentrations in spite of indistinguishable levels of c-kit expression. Fetal W41/W41 HSCs also mimicked adult wild-type HSCs in showing the same reduced rate of amplification in irradiated adult hosts (relative to fetal wild-type HSCs). Assessment of various proliferation and signaling gene transcripts in fetal and adult HSCs self-renewing in vitro revealed a singular difference in Ink4c expression. We conclude that the ability of fetal HSCs to execute symmetric self-renewal divisions more efficiently than adult HSCs in vivo may be dependent on specific developmentally regulated signals that act downstream of the c-kit kinase.

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