scholarly journals Pulsation Activates Mechanosensitive Piezo1 to Form Long-Term Hematopoietic Stem Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 445-445
Author(s):  
Giorgia Scapin ◽  
Dhvanit I Shah
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Blood Vessels ◽  
Circumferential Strain ◽  
Hematopoietic Stem ◽  
Dorsal Wall ◽  
Ventral Wall ◽  
Dnmt3b Expression

The temporal and spatial origin and development of long-term, self-renewing hematopoietic stem cells (LT-HSC) remain a mystery. The first set of definitive HSCs is born from the hemogenic endothelial cells residing in the ventral wall of the dorsal aorta (DA) of the aorta-gonad-mesonephros region during embryonic development. Blood flow- and shear-stress-mediated nitric oxide-induced vasodilation are responsible for the endothelial-to-HSC transition (EHT). However, it remains unknown why the ventral wall, and not the dorsal wall, of the DA is the restricted site of the EHT when blood flows through the entire DA and exerts shear stress on both the ventral and dorsal sides of the DA. Using single-particle tracking and fast Fourier Transform analyses of pulsating blood vessels, we demonstrate that the circumferential strain in the ventral wall, and not dorsal wall, is concurrent with and responsible for the magnitude, the site, and timing of the HSC formation. We extended our findings by developing a bioreactor to establish the functional link between pulsation in the blood vessels and HSC formation. Using serial transplant, limiting dilution, and serial replating assays, we found that pulsation mediated circumferential stretching of hemogenic endothelial cells or Piezo1 activation (Yoda1) yields 3-times higher amounts of Long Term (LT)-HSC formation; which reconstitute to normal multi-lineage adult blood. Using delayed-type hypersensitivity assay, adult globin expression, MPO enzyme activity, immunoglobulins, and T-cell receptor rearrangement analyses, we found that circumferential stretching or Piezo1 activation-derived HSCs reconstitute to functional T and B cells, adult erythrocytes, and myeloid cells. Our Piezo1fl/flxScl-Cre conditional knockout, gene-silencing, & confocal imaging further demonstrate that circumferential stretching of blood vessels activates Piezo1; which enhances epigenetic regulator Dnmt3b expression to stimulate the EHT. Our CUT&RUN CHIP-Sequencing & MASSArray methylation analyses demonstrate that Dnmt3b suppresses endothelial genes during EHT. To analyze the conserved role of PIEZO1-mediated mechanosensitive mechanisms in human hematopoiesis, we employed directed differentiation of constitutive RUNX1-mCherry human induced pluripotent stem cells (iPSCs) to hemogenic endothelial cells. We found that Yoda1-mediated PIEZO1 activation stimulated human endothelial-to-hematopoietic transition. In conclusion, pulsation-mediated circumferential strain activates Piezo1 to stimulate the endothelial-to-HSC transition via the induction of Dnmt3b expression. This leads to the formation of long-term self-renewing HSCs, which can engraft and reconstitute to multi-lineage, adult blood upon serial transplantations. Our identification of a novel biomechanical cue unravels the physiological mystery in HSC formation in the ventral wall of the DA. We also establish its cross-talk with mechanosensitive and epigenetic mechanisms to produce functional, long-term HSCs that reconstitute to form normal adult blood. This yields the therapeutic promise of developing transgene-free LT-HSC-based cellular therapies for the treatment of human blood disorders. Disclosures No relevant conflicts of interest to declare.

Hemangiogenic Role of ELF4 in Bone Marrow Post Myeloablation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1783-1783
Author(s):  
Mariela Sivina ◽  
Takeshi Yamada ◽  
Natalie Dang ◽  
H. Daniel Lacorazza
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Hematopoietic Stem Cells ◽  
Blood Vessels ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Huvec Cells

Abstract Bone marrow suppression is an important cause of death in patients exposed to radiation or in cancer patients treated with conventional chemotherapeutic agents. Myeloablative treatments (i.e. 5-fluorouracil administration) lead to apoptosis of blood forming cells and to regression of blood vessels in bone marrow. It is well known that hematological recovery post-bone marrow insult depends on the capacity of hematopoietic stem cells to regenerate the entire hematopoietic system, however, the transcriptional machinery involved in the regeneration of sinusoidal blood vessels in bone marrow from endothelial progenitor cells is largely unknown. Endothelial cells express the Tie2 receptor tyrosine kinase (a.k.a. Tek), which is involved in the angiogenic remodeling and vessel stabilization. Gene targeting of Tie2 showed that it is not required for differentiation and proliferation of definitive hematopoietic lineages in the embryo although Tie2 is needed during postnatal bone marrow hematopoiesis. ELF is a subgroup of the ETS family of transcription factors composed by ELF1, ELF2 (a.k.a. NERF), ELF3, ELF4 (a.k.a. MEF) and ELF5. ELF1 and ELF2 have been shown to regulate Tie2 expression in vitro. Recently we showed that ELF4 modulates the exit of hematopoietic stem cells (HSC) from quiescence (Lacorazza et al., Cancer Cell2006, 9:175–187). Given the high homology between ELF1 and ELF4 and the same origin of HSC and endothelial progenitor cells, we hypothesize that ELF4 regulates proliferation and Tie2 expression of endothelial cells. We used a luciferase gene reporter system in COS-7 and HEK cells to examine the capacity of ELF proteins to activate Tie2. ELF4 is the strongest activator of Tie2 expression following the hierarchy ELF4>ELF1>ELF2 variant 1>ELF2 variant 2. Site directed mutagenesis of each of the five ETS-binding sites (EBS) present in the Tie2 promoter shows that ELF4 binds preferentially to EBS 1, 3 and 5. Binding of ELF4 to the Tie2 promoter was confirmed by chromatin immunoprecipitation and EMSA. Although Elf1 gene expression is essentially normal in Elf4−/− bone marrow cells collected after 5-FU treatment, we detected diminished Tie2 expression compared to Elf4+/+ bone marrow cells. The association of this effect to human endothelial cells derived from umbilical cord (HUVEC cells) was investigated. All-trans retinoic acid (ATRA) and vascular-endothelial growth factor (VEGF) induced ELF4 expression in HUVEC cells in a dose and time dependent manner which was followed by increased Tie2 expression, suggesting that expression of ELF4 is modulated by angiogenic signals. Moreover, endothelial cells treated with ATRA showed rapid wound colonization in a wound assay. Expression of the pan-endothelial marker MECA-32 was determined by immunohistochemistry to correlate Tie2 with the regeneration of blood vessels: myeloablated Elf4−/− femurs exhibited a reduction of MECA-32 positive arterioles. Finally, temporal and spatial expression of Tie2 during hematological recovery post ablation was measured in bone marrow using transgenic Tie2-LacZ mice crossed to Elf4−/− mice. Collectively, our data suggests that ELF4 regulates Tie2 expression in endothelial cells but most importantly their proliferative capacity in response to angiogenic signals.

AGM-Derived Endothelial Cells and Notch Ligands Provide Embryonic Hematopoietic Stem Cell-Supportive Niches In Vitro

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1167-1167
Author(s):  
Brandon K Hadland ◽  
Barbara Varnum-Finney ◽  
Randall T Moon ◽  
Jason M Butler ◽  
Shahin Rafii ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Small Molecule Inhibition ◽  
Short And Long Term ◽  
High Level ◽  
Notch Ligands

Abstract Greater knowledge of embryonic niche signals regulating the establishment, maintenance, and expansion of hematopoietic stem cells (HSC) during development will be essential in deriving therapeutically useful HSC from pluripotent stem cells (PSC). To this end, we have used the murine embryo model to dissect components of embryonic hematopoietic microenvironments which are sufficient to support nascent HSC and their precursors in vitro. We demonstrate that Akt pathway-activated endothelial cells (ECs) derived from the AGM (aorta-gonad-mesonephros) region, a critical site of HSC emergence during development, can substantially increase short and long-term multilineage engraftment potential from isolated embryonic day 11 (E11) VE-Cadherin+/CD45+ AGM-derived hematopoietic cells by co-culture in vitro. Furthermore, preliminary experiments show that co-culture with AGM-ECs also promotes high level, multilineage engraftment capacity from VE-cadherin+/c-kit+ precursors isolated from younger embryos (E9-E10). These results suggest that endothelial cells from an embryonic HSC-producing niche provide signals sufficient to promote maturation of HSC from embryonic precursors and subsequently support early HSC expansion in vitro. Further dissection of required signals for embryonic HSC expansion identified a unique combination of Notch activation by immobilized Notch ligands, cytokines, and small molecule inhibition of the TGF-β pathway, which is sufficient to inhibit differentiation and enhance self-renewal of embryonic, definitive-stage hematopoietic precursors in vitro. Notably, these conditions significantly increased short and long-term, multilineage repopulating HSC from E11 VE-Cadherin+/CD45+, but not E9-10 VE-Cadherin+/c-kit+ AGM-derived hematopoietic cells, indicating AGM-ECs provide additional, yet to be identified, signals for HSC maturation from developmental precursors. These findings have important implications for dissecting critical niche signals for HSC formation and expansion that will be essential for addressing the elusive goal of deriving HSC from pluripotent precursors. Disclosures: Rafii: Angiocrine Bioscience: Founder Other.

Transplanted adult hematopoietic stems cells differentiate into functional endothelial cells

Blood ◽  
2004 ◽  
Vol 103 (1) ◽  
pp. 13-19 ◽  
Author(s):  
Alexis S. Bailey ◽  
Shuguang Jiang ◽  
Michael Afentoulis ◽  
Christina I. Baumann ◽  
David A. Schroeder ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Hematopoietic Stem Cells ◽  
Blood Vessels ◽  
Cell Potential ◽  
Hematopoietic Stem ◽  
Adult Bone Marrow ◽  
Adult Bone

Abstract During early embryogenesis, blood vessels and hematopoietic cells arise from a common precursor cell, the hemangioblast. Recent studies have identified endothelial progenitor cells in the peripheral blood, and there is accumulating evidence that a subset of these cells is derived from precursors in the bone marrow. Here we show that adult bone marrow–derived, phenotypically defined hematopoietic stem cells (c-kit+, Sca-1+, lineage–) give rise to functional endothelial cells. With the exception of the brain, donor-derived cells are rapidly integrated into blood vessels. Durably engrafted endothelial cells express CD31, produce von Willebrand factor, and take up low-density lipoprotein. Analysis of DNA content indicates that donor-derived endothelial cells are not the products of cell fusion. Self-renewal of stem cells with hematopoietic and endothelial cell potential was revealed by serial transplantation studies. The clonal origin of both hematopoietic and endothelial cell outcomes was established by the transfer of a single cell. These results suggest that adult bone marrow–derived hematopoietic stem cells may serve as a reservoir for endothelial cell progenitors.

scholarly journals Piezo1-Sensitive Biomechanical Pulsation Stimulates Long-Term Hematopoietic Stem Cell Formation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3826-3826
Author(s):  
Giorgia Scapin ◽  
Jennifer Cillis ◽  
Taylor Patch ◽  
Priyanka R Dharampuriya ◽  
Elliott J. Hagedorn ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Flow ◽  
Stem Cell ◽  
Cell Formation ◽  
Time Lapse ◽  
Functional Link ◽  
Hematopoietic Stem ◽  
Biomechanical Forces ◽  
Dnmt3b Expression

Abstract The birth and development of hemat\opoietic stem cells (HSCs) remain a mystery. During fetal development, a subset of endothelial cells transitions to become HSCs in the aorta-gonad-mesonephros (AGM) region. Blood flow-mediated shear stress and activation of nitric oxide synthase (NOS) were demonstrated to stimulate the endothelial-to-HSC transition in the AGM. However, we showed that malbec (mlbbw306), a zebrafish mutant for cadherin 5, produces HSCs despite circulation arrest and the inhibition of NOS, suggesting that other biomechanical forces, mechanosensation pathways, or epigenetic mechanisms might regulate HSC formation and could have utility in developing HSCs. Using zebrafish, murine, and human models, we show that Piezo1-sensitive biomechanical stretching of hemogenic endothelial cells enhances Dnmt3b expression for long-term (LT)-HSC formation. Our microangiography and time-lapse confocal imaging established that cdh5-MO embryos have a heartbeat and pulsation in blood vessels despite the absence of blood flow. We also employed light sheet and time-lapse confocal microscopy followed by Fourier transform analyses to establish that although pulsation is independent of blood flow in the AGM, it is concurrent with the endothelial-to-hematopoietic transition. To establish the functional link between pulsation and HSC formation, we developed a bioreactor simulating the pulsating blood vessel conditions. We found that the biomechanical stretching of hemogenic endothelial cells or the pharmacological activation of Piezo1 yields three times higher amounts of LT-HSC formation; which reconstitute to normal multi-lineage adult blood even upon serial transplantation. Our gene-silencing, time-lapse imaging, explant culture, and computational analyses further demonstrated that biomechanical stretching activates Piezo1; which enhances epigenetic regulator Dnmt3b expression to stimulate the endothelial-to-HSC transition. Our results demonstrate how pulsation-mediated biomechanical forces stimulate cell-fate transitions and stem cell formation by activating mechanosensitive channels as well as epigenetic machinery. We present a model that addresses major challenges in HSC transplantation and cellular therapies for treating blood and bone marrow diseases. In addition, we report a scalable bioreactor with potential widespread use and a pharmacological target to develop and expand LT-HSCs. Disclosures No relevant conflicts of interest to declare.

scholarly journals Role of CXCL12-Expressing Bone Marrow Populations in Leukemic Stem Cell Regulation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 26-26 ◽  
Author(s):  
Puneet Agarwal ◽  
Hui Li ◽  
Andrew J Paterson ◽  
Jianbo He ◽  
Takashi Nagasawa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Hematopoietic Stem ◽  
Exon 2 ◽  
Knockout Animals

Abstract CXCL12 is the major chemoattractant for hematopoietic stem cells (HSC) in the bone marrow microenvironment (BMM), and plays a major role in HSC localization to their regulatory niches. Studies using genetic drivers/reporters have shown that CXCL12 deletion from perivascular mesenchymal stem cells (MSC) and CAR cells (using Prx1-Cre line) leads to loss of HSC quiescence, frequency and self-renewal potential, while deletion from endothelial cells (using Tek-Cre line) results in modest loss of HSC long-term repopulating activity. In contrast, deletion of CXCL12 from osteoprogenitors (using Osx-Cre line) resulted in HSC mobilization without any effect on HSC function, while deletion from mature osteoblasts (using OCN-Cre line) had no effect on HSC function (Greenbaum et al. Nature. 2013;495(7440):227-30; Ding et al. Nature. 2013;495(7440):231-5.). These studies have been useful in identifying MSC/CAR cells and endothelial cells as important HSC niche components but the BM niches for LSC remain poorly characterized. In the present study, we examined alterations in CXCL12-producing niche cells in the CML BMM, and their role in regulating LSC growth, using the SCL-tTA BCR-ABL mouse model of CML. Our previous studies indicated that CXCL12 levels are reduced in CML compared to normal BM (Zhang et al. Cancer Cell. 2012; 21(4):577). To evaluate the effect of leukemia development on specific CXCL12-expressing cell populations in the BMM, we crossed CXCL12GFP mice (GFP reporter knocked into the CXCL12 locus) with SCL-tTA-BCR-ABL mice to generate CXCL12GFP-SCL-tTA-BCR-ABL mice. CXCL12-expressing cells in the BM were identified by GFP expression. Mice developing CML after BCR-ABL induction by tet-withdrawal demonstrated significantly increased numbers of GFP+ endothelial cells (CD45-Ter119-CD31+) and reduced numbers of GFP+ BM stromal cells (CD45-Ter119-31-) compared to WT mice. Within the stromal population, the number of GFP+ MSC (PDGFRα+Sca-1+) were decreased. To evaluate the contribution of CXCL12-expressing populations towards LSC regulation, we crossed CXCL12f/f mice (loxP sites flanking exon 2) with Tek-Cre, Prx1-Cre, OCN-Cre and OSX-Cre transgenic lines. CML BM cells (CD45.1/2+; 2*106/mouse) were transplanted into lethally irradiated (8Gy) WT (CD45.2) Cre- or Cre+ CXCL12f/f knockout animals, and followed for CML development. When compared to WT mice, CXCL12f/f-Tek-Cre+ animals exhibited significantly reduced engraftment of CML cells (CD45.1/2+) in the BM, with associated reduction in CML myeloid cells (Gr-1+Mac-1+), MEP (CD16/32- CD34-), CMP (CD16/32lowCD34+), MPP (LSK+CD48+) and LTHSC (CD150+CD48-) numbers. No changes in splenic engraftment were seen. To evaluate long-term reconstitution, BM cells from primary transplanted WT or knockout animals were transplanted into lethally irradiated (8Gy) WT secondary recipients. CML engraftment in secondary mice receiving BM from Tek-Cre+ and WT animals was similiar at 12 weeks, indicating that residual LTHSC retained repopulating potential. In contrast, CXCL12f/f-OCN-Cre and CXCL12f/f-Osx-Cre mice did not demonstrate significant differences in total CML cell or CML LTHSC engraftment, but showed increased LMPP engraftment in the BM. Interestingly CXCL12f/f-Prx1-Cre+ animals exhibited significantly increased leukocytosis and BM cellularity, and increased MEP, CMP, LMPP, MPP, STHSC and LTHSC numbers in the BM compared to WT mice. Increased numbers of CML myeloid cells and LSK were seen in the peripheral blood, but no change in splenic engraftment was seen. CML engraftment in secondary mice receiving BM from Prx1-Cre+ animals was significantly increased at 12 weeks compared to WT animals, indicating that the expanded LTHSC population maintained repopulating potential. These results suggest that loss of endothelial cell expressed CXCL12 reduced CML LTHSC in BM, whereas loss of MSC/CAR cell expressed CXCL12 enhances CML LTHSC numbers in BM, in association with increased mobilization to PB. Collectively, these results reveal important and distinct niche functions for CXCL12 expressing BM endothelial cell and MSC/CAR cells in CML, and indicate significant differences in niche regulation of CML LSC compared with normal HSC. We expect that improved characterization of BM niches in CML will facilitate further dissection of key niche interactions underlying LSC maintenance and expansion. Disclosures No relevant conflicts of interest to declare.

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

2020 ◽  
Vol 15 ◽  
Author(s):  
Fatima Aerts-Kaya
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Stress Conditions ◽  
Stress Factors ◽  
Hematopoietic Stem ◽  
Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

scholarly journals Long-term lymphoid progenitors independently sustain naïve T and NK cell production in humans

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Natalia Izotova ◽  
Christine Rivat ◽  
Cristina Baricordi ◽  
Elena Blanco ◽  
Danilo Pellin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Nk Cell ◽  
Integration Site ◽  
Hematopoietic Stem ◽  
Cell Production ◽  
Lymphoid Progenitors ◽  
Clinical Gene Therapy ◽  
Cancer Immunotherapies

AbstractOur mathematical model of integration site data in clinical gene therapy supported the existence of long-term lymphoid progenitors capable of surviving independently from hematopoietic stem cells. To date, no experimental setting has been available to validate this prediction. We here report evidence of a population of lymphoid progenitors capable of independently maintaining T and NK cell production for 15 years in humans. The gene therapy patients of this study lack vector-positive myeloid/B cells indicating absence of engineered stem cells but retain gene marking in both T and NK. Decades after treatment, we can still detect and analyse transduced naïve T cells whose production is likely maintained by a population of long-term lymphoid progenitors. By tracking insertional clonal markers overtime, we suggest that these progenitors can support both T and NK cell production. Identification of these long-term lymphoid progenitors could be utilised for the development of next generation gene- and cancer-immunotherapies.

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