Abstract 22: Identification of the Nature of Cardiac Resident c-kit
            +
            Cells

Identifying a bona fide population of cardiac stem cells (CSCs) is a critical step for developing cell-based therapies for heart failure patients. For more than a decade, c-kit, a receptor tyrosine kinase expressed in certain types of hematopoietic stem cells, has been recognized as a marker of resident CSCs in mammals. It was shown that c-kit + cells are multipotent, with differentiation potential to become cardiomyocytes, endothelial, and smooth muscle cells in vitro and after cardiac injury. Here, we provide new insights into the nature of cardiac resident c-kit + cells. By targeting the c-kit locus with several reporter genes in mice, we unexpectedly found that c-kit + cells rarely co-localizes with cardiac progenitor marker Nkx2.5 or myocardial marker cTnT. Instead, c-kit labels an endocardial population from embryonic stage to adulthood. After acute cardiac injury, the c-kit + cells still retain their endothelial identity and do not become cardiomyocytes. Our study supports the notion that cardiac c-kit + cells are in fact endothelial cells and not CSCs. This finding suggests an urgent need to re-evaluate the mechanisms by which c-kit + cells contribute to heart repair or regeneration given their endothelial identity.

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Abstract 15142: Endogenous Cardiac Stem Cells’ Activation in Response to Injury Potentiates Their Regenerative Ability

Circulation ◽

10.1161/circ.132.suppl_3.15142 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Andrew J Smith ◽

Iolanda Aquila ◽

Beverley J Henning ◽

Mariangela Scalise ◽

Bernardo Nadal-Ginard ◽

...

Keyword(s):

Stem Cells ◽

Cardiac Injury ◽

Fold Increase ◽

Cardiac Stem Cells ◽

Post Injury ◽

Activated State ◽

Potential Benefits ◽

Therapeutic Avenue

The identification of resident, endogenous cardiac stem cells (eCSCs) has re-shaped our understanding of cardiac cellular physiology, while offering a significant potential therapeutic avenue. The biology of these cells must be better understood to harness their potential benefits. We used an acute dose (s.c.; 5mgkg-1) of isoproterenol (ISO) to induce diffuse cardiac injury, with associated eCSC activation, in rats. As peak eCSC activation was at 24 hours post ISO-injury, c-kitpos eCSCs were isolated, characterised and their potential for growth and regenerative potential was assessed in vitro and in vivo, respectively. Activated eCSCs showed increased cell cycling activity (51+1% in S- or G2/M phases vs. 9+2% of quiescent), Ki67 expression (56+7% vs. 10+1%) and TERT expression (14-fold increase vs. quiescent). When directly harvested in culture, activated eCSCs showed augmented proliferation, clonogenicity and cardiosphere formation compared to quiescent eCSCs. Activated eCSCs showed increases in expression of numerous growth factors, particularly HGF, IGF-1, TGF-β, periostin, PDGF-AA and VEGF-A. Furthermore, significant alterations were found in the miRnome, notably increased miR-146b and -221, and decreased miR-192 and -351. ISO+5FU was administrated to mice to induce a model of chronic dilated cardiomyopathy, which is characterized by the ablation of eCSCs and the absence of cardiomyocyte replenishment. In these mice with chronic heart failure, freshly isolated quiescent eCSCs or activated eCSCs (2d post-ISO) were injected through the tail vein. 28 days after injection, activated but not quiescent eCSCs re-populated the resident CSC pool, promoted robust new cardiomyocyte formation and improved cardiac function when compared to saline-treated mice. Dual-labelling with BrdU and EdU at selected stages after ISO injury determined that activated eCSCs returned to a quiescent level by 10 weeks post-injury. In conclusion, CSCs rapidly switch from a quiescent to an activated state to match the myocardial needs for myocyte replacement after injury and then spontaneously go back to quiescence. Harnessing the molecules regulating this process may open up future novel approaches for effective myocardial regeneration.

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Synergy of NUP98-HOXA10 Fusion Gene and NrasG12D Mutation Preserves the Stemness of Hematopoietic Stem Cells on Culture Condition

Cells ◽

10.3390/cells8090951 ◽

2019 ◽

Vol 8 (9) ◽

pp. 951 ◽

Cited By ~ 2

Author(s):

Yong Dong ◽

Chengxiang Xia ◽

Qitong Weng ◽

Tongjie Wang ◽

Fangxiao Hu ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Pluripotent Stem Cells ◽

Culture Condition ◽

Fusion Gene ◽

Nras Mutation ◽

Hematopoietic Stem ◽

Bona Fide

Natural hematopoietic stem cells (HSC) are susceptible and tend to lose stemness, differentiate, or die on culture condition in vitro, which adds technical challenge for maintaining bona fide HSC-like cells, if ever generated, in protocol screening from pluripotent stem cells. It remains largely unknown whether gene-editing of endogenous genes can genetically empower HSC to endure the culture stress and preserve stemness. In this study, we revealed that both NUP98-HOXA10HD fusion and endogenous Nras mutation modifications (NrasG12D) promoted the engraftment competitiveness of HSC. Furthermore, the synergy of these two genetic modifications endowed HSC with super competitiveness in vivo. Strikingly, single NAV-HSC successfully maintained its stemness and showed robust multi-lineage engraftments after undergoing the in vitro culture. Mechanistically, NUP98-HOXA10HD fusion and NrasG12D mutation distinctly altered multiple pathways involving the cell cycle, cell division, and DNA replication, and distinctly regulated stemness-related genes including Hoxa9, Prdm16, Hoxb4, Trim27, and Smarcc1 in the context of HSC. Thus, we develop a super-sensitive transgenic model reporting the existence of HSC at the single cell level on culture condition, which could be beneficial for protocol screening of bona fide HSC regeneration from pluripotent stem cells in vitro.

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

Blood ◽

10.1182/blood.v108.11.2563.2563 ◽

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.

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Hematopoietic stem cells express Tie-2 receptor in the murine fetal liver

Blood ◽

10.1182/blood.v96.12.3757.h8003757_3757_3762 ◽

2000 ◽

Vol 96 (12) ◽

pp. 3757-3762 ◽

Cited By ~ 1

Author(s):

Hsiang-Chun Hsu ◽

Hideo Ema ◽

Mitsujiro Osawa ◽

Yukio Nakamura ◽

Toshio Suda ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Receptor Tyrosine Kinase ◽

Fetal Liver ◽

Newborn Mice ◽

Hematopoietic Stem ◽

Fetal Liver Cells

Tie-2 receptor tyrosine kinase expressed in endothelial and hematopoietic cells is believed to play a role in both angiogenesis and hematopoiesis during development of the mouse embryo. This article addressed whether Tie-2 is expressed on fetal liver hematopoietic stem cells (HSCs) at day 14 of gestation. With the use of anti–Tie-2 monoclonal antibody, its expression was detected in approximately 7% of an HSC population of Kit-positive, Sca-1–positive, lineage-negative or -low, and AA4.1-positive (KSLA) cells. These Tie-2–positive KSLA (T+ KSLA) cells represent 0.01% to 0.02% of fetal liver cells. In vitro colony and in vivo competitive repopulation assays were performed for T+ KSLA cells and Tie-2–negative KSLA (T− KSLA) cells. In the presence of stem cell factor, interleukin-3, and erythropoietin, 80% of T+ KSLA cells formed colonies in vitro, compared with 40% of T− KSLA cells. Long-term multilineage repopulating cells were detected in T+ KSLA cells, but not in T− KSLA cells. An in vivo limiting dilution analysis revealed that at least 1 of 8 T+ KSLA cells were such repopulating cells. The successful secondary transplantation initiated with a limited number of T+ KSLA cells suggests that these cells have self-renewal potential. In addition, engraftment of T+ KSLA cells in conditioned newborn mice indicates that these HSCs can be adapted equally by the adult and newborn hematopoietic environments. The data suggest that T+ KSLA cells represent HSCs in the murine fetal liver.

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Age-Associated Characteristics of Murine Hematopoietic Stem Cells

Journal of Experimental Medicine ◽

10.1084/jem.192.9.1273 ◽

2000 ◽

Vol 192 (9) ◽

pp. 1273-1280 ◽

Cited By ~ 477

Author(s):

Kazuhiro Sudo ◽

Hideo Ema ◽

Yohei Morita ◽

Hiromitsu Nakauchi

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Lymphoid Cells ◽

Differentiation Potential ◽

Self Renewal ◽

Hematopoietic Stem ◽

Functional Changes

Little is known of age-associated functional changes in hematopoietic stem cells (HSCs). We studied aging HSCs at the clonal level by isolating CD34−/lowc-Kit+Sca-1+ lineage marker–negative (CD34−KSL) cells from the bone marrow of C57BL/6 mice. A population of CD34−KSL cells gradually expanded as age increased. Regardless of age, these cells formed in vitro colonies with stem cell factor and interleukin (IL)-3 but not with IL-3 alone. They did not form day 12 colony-forming unit (CFU)-S, indicating that they are primitive cells with myeloid differentiation potential. An in vivo limiting dilution assay revealed that numbers of multilineage repopulating cells increased twofold from 2 to 18 mo of age within a population of CD34−KSL cells as well as among unseparated bone marrow cells. In addition, we detected another compartment of repopulating cells, which differed from HSCs, among CD34−KSL cells of 18-mo-old mice. These repopulating cells showed less differentiation potential toward lymphoid cells but retained self-renewal potential, as suggested by secondary transplantation. We propose that HSCs gradually accumulate with age, accompanied by cells with less lymphoid differentiation potential, as a result of repeated self-renewal of HSCs.

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Differentiation potential of human CD133 positive hematopoietic stem cells into motor neuron- like cells, in vitro

Journal of Chemical Neuroanatomy ◽

10.1016/j.jchemneu.2017.07.006 ◽

2017 ◽

Vol 86 ◽

pp. 35-40 ◽

Cited By ~ 3

Author(s):

Sepideh Alavi Moghaddam ◽

Behnam Yousefi ◽

Davood Sanooghi ◽

Faezeh Faghihi ◽

Nasim Hayati Roodbari ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Motor Neuron ◽

Differentiation Potential ◽

Hematopoietic Stem

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A novel microRNA-based strategy to expand the differentiation potency of stem cells

10.1101/826446 ◽

2019 ◽

Author(s):

María Salazar-Roa ◽

Marianna Trakala ◽

Mónica Álvarez-Fernández ◽

Fátima Valdés-Mora ◽

Cuiqing Zhong ◽

...

Keyword(s):

Stem Cells ◽

De Novo ◽

Cardiac Injury ◽

Dna Methyltransferases ◽

Short Exposure ◽

Differentiation Potential ◽

Differentiation Capacity ◽

Tetraploid Complementation

SUMMARYFull differentiation potential along with self-renewal capacity is a major property of pluripotent stem cells (PSCs). However, the differentiation capacity frequently decreases during expansion of PSCs in vitro. We show here that transient exposure to a single microRNA, expressed at early stages during normal development, improves the differentiation capacity of already-established murine and human PSCs. Short exposure to miR-203 in PSCs (miPSCs) results in expanded differentiation potency as well as improved efficiency in stringent assays such as tetraploid complementation and human-mouse interspecies chimerism. Mechanistically, these effects are mediated by direct repression of de novo DNA methyltransferases Dnmt3a and Dnmt3b, leading to transient and reversible erasing of DNA methylation. As a proof of concept, miR-203 improves differentiation and maturation of PSCs into cardiomyocytes in vitro as well as cardiac regeneration in vivo, after cardiac injury. These data support the use of transient exposure to miR-203 as a general and single method to reset the epigenetic memory in PSCs, and improve their use in regenerative medicine.

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Microrna 199b Regulates Mouse Hematopoietic Stem Cells Maintenance

Blood ◽

10.1182/blood-2019-126283 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3704-3704

Author(s):

Aldona A Karaczyn ◽

Edward Jachimowicz ◽

Jaspreet S Kohli ◽

Pradeep Sathyanarayana

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Quiescent State ◽

Differentiation Potential ◽

Self Renewal ◽

Hematopoietic Stem

The preservation of hematopoietic stem cell pool in bone marrow (BM) is crucial for sustained hematopoiesis in adults. Studies assessing adult hematopoietic stem cells functionality had been shown that for example loss of quiescence impairs hematopoietic stem cells maintenance. Although, miR-199b is frequently down-regulated in acute myeloid leukemia, its role in hematopoietic stem cells quiescence, self-renewal and differentiation is poorly understood. Our laboratory investigated the role of miR-199b in hematopoietic stem and progenitor cells (HSPCs) fate using miR-199b-5p global deletion mouse model. Characterization of miR-199b expression pattern among normal HSPC populations revealed that miR-199b is enriched in LT-HSCs and reduced upon myeloablative stress, suggesting its role in HSCs maintenance. Indeed, our results reveal that loss of miR-199b-5p results in imbalance between long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSCs) and multipotent progenitors (MMPs) pool. We found that during homeostasis, miR-199b-null HSCs have reduced capacity to maintain quiescent state and exhibit cell-cycle deregulation. Cell cycle analyses showed that attenuation of miR-199b controls HSCs pool, causing defects in G1-S transition of cell cycle, without significant changes in apoptosis. This might be due to increased differentiation of LT-HSCs into MPPs. Indeed, cell differentiation assay in vitro showed that FACS-sorted LT-HSCs (LineagenegSca1posc-Kitpos CD48neg CD150pos) lacking miR-199b have increased differentiation potential into MPP in the presence of early cytokines. In addition, differentiation assays in vitro in FACS-sorted LSK population of 52 weeks old miR-199b KO mice revealed that loss of miR-199b promotes accumulation of GMP-like progenitors but decreases lymphoid differentiation, suggesting that miR199b may regulate age-related pathway. We used non-competitive repopulation studies to show that overall BM donor cellularity was markedly elevated in the absence of miR-199b among HSPCs, committed progenitors and mature myeloid but not lymphoid cell compartments. This may suggest that miR-199b-null LT-HSC render enhanced self-renewal capacity upon regeneration demand yet promoting myeloid reconstitution. Moreover, when we challenged the self-renewal potential of miR-199b-null LT-HSC by a secondary BM transplantation of unfractionated BM cells from primary recipients into secondary hosts, changes in PB reconstitution were dramatic. Gating for HSPCs populations in the BM of secondary recipients in 24 weeks after BMT revealed that levels of LT-HSC were similar between recipients reconstituted with wild-type and miR-199b-KO chimeras, whereas miR-199b-null HSCs contributed relatively more into MPPs. Our data identify that attenuation of miR-199b leads to loss of quiescence and premature differentiation of HSCs. These findings indicate that loss of miR-199b promotes signals that govern differentiation of LT-HSC to MPP leading to accumulation of highly proliferative progenitors during long-term reconstitution. Hematopoietic regeneration via repopulation studies also revealed that miR-199b-deficient HSPCs have a lineage skewing potential toward myeloid lineage or clonal myeloid bias, a hallmark of aging HSCs, implicating a regulatory role for miR-199b in hematopoietic aging. Disclosures No relevant conflicts of interest to declare.

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Hematopoietic stem cells express Tie-2 receptor in the murine fetal liver

Blood ◽

10.1182/blood.v96.12.3757 ◽

2000 ◽

Vol 96 (12) ◽

pp. 3757-3762 ◽

Cited By ~ 47

Author(s):

Hsiang-Chun Hsu ◽

Hideo Ema ◽

Mitsujiro Osawa ◽

Yukio Nakamura ◽

Toshio Suda ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Receptor Tyrosine Kinase ◽

Fetal Liver ◽

Newborn Mice ◽

Hematopoietic Stem ◽

Fetal Liver Cells

Abstract Tie-2 receptor tyrosine kinase expressed in endothelial and hematopoietic cells is believed to play a role in both angiogenesis and hematopoiesis during development of the mouse embryo. This article addressed whether Tie-2 is expressed on fetal liver hematopoietic stem cells (HSCs) at day 14 of gestation. With the use of anti–Tie-2 monoclonal antibody, its expression was detected in approximately 7% of an HSC population of Kit-positive, Sca-1–positive, lineage-negative or -low, and AA4.1-positive (KSLA) cells. These Tie-2–positive KSLA (T+ KSLA) cells represent 0.01% to 0.02% of fetal liver cells. In vitro colony and in vivo competitive repopulation assays were performed for T+ KSLA cells and Tie-2–negative KSLA (T− KSLA) cells. In the presence of stem cell factor, interleukin-3, and erythropoietin, 80% of T+ KSLA cells formed colonies in vitro, compared with 40% of T− KSLA cells. Long-term multilineage repopulating cells were detected in T+ KSLA cells, but not in T− KSLA cells. An in vivo limiting dilution analysis revealed that at least 1 of 8 T+ KSLA cells were such repopulating cells. The successful secondary transplantation initiated with a limited number of T+ KSLA cells suggests that these cells have self-renewal potential. In addition, engraftment of T+ KSLA cells in conditioned newborn mice indicates that these HSCs can be adapted equally by the adult and newborn hematopoietic environments. The data suggest that T+ KSLA cells represent HSCs in the murine fetal liver.

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In Vitro Myelo-Lymphoid Colony Formation by Mouse Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v112.11.3861.3861 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3861-3861

Author(s):

Jun Ooehara ◽

Hina Takano ◽

Shin-ichiro Takayanagi ◽

Hiromitsu Nakauchi ◽

Hideo Ema

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Culture Conditions ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Lymphoid Progenitors ◽

Marrow Cells

Abstract Hematopoietic stem cells (HSCs) clonally differentiate into all myeloid, B-lymphoid, and T-lymphoid lineages. Mouse HSCs are known to form in vitro colonies comprised of morphologically identifiable myeloid cells such as neutrophils, macrophages, erythroblasts, and megakaryocytes. Whether HSCs are able to differentiate along B-and T-lymphoid lineages in such colonies remains obscure. The co-culture systems with stromal cells such as S17, OP9, OP9/Delta cells have been shown to support B- and T-cell development. These systems have been used to identify subclasses of progenitors with lymphoid potentials. However, neither B cells nor T cells have been successfully generated from HSCs in vitro. This is most likely due to the lack of culture conditions which support HSCs to differentiate into a certain stage of lymphoid progenitors. In this study, we attempted to use serum-free single-cell culture to identify cytokines which fill the developmental gap between HSCs and lymphoid progenitors. Here we show that myelo-lymphoid colonies are formed by HSCs in the presence of thrombopoietin (TPO), interleukin (IL)-11, or IL-12 together with stem cell factor (SCF). CD34-negative/low, c-Kit-positive, Sca-1-positive, lineage marker-negative (CD34-KSL) bone marrow cells were individually cultured with a combination of cytokines for 7 days. All cells in each colony were transplanted into each from a group of lethally irradiated mice, along with compromised bone marrow cells. The recipient mice were periodically analyzed after transplantation to detect transient myeloid and lymphoid reconstitution. All myeloid, B-, and T-lymphoid progenitor activities were detected in single colonies formed in the presence of SCF+TPO, SCF+IL-11, SCF+IL-12. Only myeloid progenitor activity was predominantly detected in single colonies formed in the presence of SCF+IL-3, consistent with previous observations in blast colony assays. All these combinations of cytokines support self-renewal in HSCs to varying degrees. We conclude that TPO, IL-11, and IL-12 directly act on HSCs and support them to differentiate into progenitors with lymphoid differentiation potential. Early differentiation pathways in HSCs are likely to be used in common by myeloid and lymphoid lineages and be supported in common by multiple cytokines.

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