scholarly journals A functional genetic screen identifies Aurora kinase b as a critical regulator of Sox9-positive embryonic lung progenitor cells

Development ◽  
2021 ◽  
Author(s):  
Ah-Cann C ◽  
Wimmer VC ◽  
Weeden CE ◽  
Marceaux C ◽  
Law CW ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Ex Vivo ◽  
Aurora Kinase ◽  
Postnatal Growth ◽  
Branching Morphogenesis ◽  
Genetic Screen ◽  
Conditional Knockout ◽  
Embryonic Lung ◽  
Aurora Kinase B

Development of a branching tree in the embryonic lung is critical for the formation of a fully mature functional lung at birth. Sox9+ cells present at the tip of the primary embryonic lung endoderm are multipotent cells responsible for branch formation and elongation. We performed a genetic screen and identified Aurora kinase b (Aurkb) as a critical regulator of Sox9+ cells ex vivo. In vivo conditional knockout studies confirmed that Aurkb was critical for lung development but was not necessary for postnatal growth and the repair of the adult lung after injury. Deletion of Aurkb in embryonic Sox9+ cells led to the formation of a stunted lung that retained the expression of Sox2 in the proximal airways, as well as Sox9 in the distal tips. While we found no change in cell polarity, we showed that loss of Aurkb or chemical inhibition of Aurkb induced a block of Sox9+ cells in G2/M, likely responsible for the lack of branch bifurcation. This work demonstrates the power of genetic screens in identifying novel regulators of Sox9+ progenitor cells and lung branching morphogenesis.

Abstract 18845: Induced Cardiac Progenitor Cells Differentiate Into Cardiac Lineage Cells in vivo and Improve Survival in Mice post Myocardial Infarction

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Pratik A Lalit ◽  
Max R Salick ◽  
Daryl O Nelson ◽  
Jayne M Squirrell ◽  
Christina M Shafer ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Disease Modeling ◽  
Ex Vivo ◽  
Cardiac Progenitor Cells ◽  
Heart Tube ◽  
Whole Embryo Culture ◽  
Cardiac Actin ◽  
Cardiac Lineage

Several studies have reported reprogramming of fibroblasts (Fibs) to induced cardiomyocytes, and we have recently reprogrammed mouse Fibs to induced cardiac progenitor cells (iCPCs), which may be more favorable for cardiac repair because of their expandability and multipotency. Adult cardiac (AC), lung and tail-tip Fibs from an Nkx2.5-EYFP reporter mouse were reprogrammed using a combination of five defined factors into iCPCs. Transcriptome and immunocytochemistry analysis revealed that iCPCs were cardiac mesoderm-restricted progenitors that expressed CPC markers including Nkx2.5, Gata4, Irx4, Tbx5, Cxcr4, Flk1 etc. iCPCs could be extensively expanded (over 30 passages) while maintaining multipotency to differentiate in vitro into cardiac lineage cells including cardiomyocytes (CMs), smooth muscle cells and endothelial cells. iCPC derived CMs upon co-culture with mESC-derived CMs formed intercellular gap junctions, exhibited calcium transients, and contractions. The purpose of this study was to determine the in vivo potency of iCPCs. Given that the Nkx2.5-EYFP reporter identifies embryonic CPCs, we first tested the embryonic potency of iCPCs using an ex vivo whole embryo culture model injecting cells into the cardiac crescent (CC) of E8.5 mouse embryos and culturing for 24 to 48 hours. GFP labeled AC Fibs were first tested and live imaging revealed that after 24 hours these cells were rejected from the embryo proper and localized to the ecto-placental cone. In contrast, iCPCs reprogrammed from AC Fibs when injected into the CC localized to the developing heart tube and differentiated into MLC2v, αMHC and cardiac actin expressing CMs. Further we injected iCPCs into infarcted adult mouse hearts and determined their regenerative potential after 1-4 wks. The iCPCs significantly improved survival (p<0.01 Mantel-Cox test) in treated animals (75%) as compared to control (11%). Immunohistochemistry revealed that injected iCPCs localized to the scar area and differentiated into cardiac lineage cells including CMs (cardiac actin). These results indicate that lineage reprogramming of adult somatic cells into iCPCs provides a scalable cell source for cardiac regenerative therapy as well as drug discovery and disease modeling.

scholarly journals Human Immunodeficiency Virus Inhibits Multilineage Hematopoiesis In Vivo

1998 ◽  
Vol 72 (6) ◽  
pp. 5121-5127 ◽  
Author(s):  
Prasad S. Koka ◽  
John K. Fraser ◽  
Yvonne Bryson ◽  
Gregory C. Bristol ◽  
Grace M. Aldrovandi ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Erythroid Differentiation ◽  
Inhibitory Effect ◽  
Immunodeficiency Virus ◽  
The Many ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1)-infected individuals often exhibit multiple hematopoietic abnormalities reaching far beyond loss of CD4+ lymphocytes. We used the SCID-hu (Thy/Liv) mouse (severe combined immunodeficient mouse transplanted with human fetal thymus and liver tissues), which provides an in vivo system whereby human pluripotent hematopoietic progenitor cells can be maintained and undergo T-lymphoid differentiation and wherein HIV-1 infection causes severe depletion of CD4-bearing human thymocytes. Herein we show that HIV-1 infection rapidly and severely decreases the ex vivo recovery of human progenitor cells capable of differentiation into both erythroid and myeloid lineages. However, the total CD34+ cell population is not depleted. Combination antiretroviral therapy administered well after loss of multilineage progenitor activity reverses this inhibitory effect, establishing a causal role of viral replication. Taken together, our results suggest that pluripotent stem cells are not killed by HIV-1; rather, a later stage important in both myeloid and erythroid differentiation is affected. In addition, a primary virus isolated from a patient exhibiting multiple hematopoietic abnormalities preferentially depleted myeloid and erythroid colony-forming activity rather than CD4-bearing thymocytes in this system. Thus, HIV-1 infection perturbs multiple hematopoietic lineages in vivo, which may explain the many hematopoietic defects found in infected patients.

Abstract 2876: Early Retention and Subsequent Engraftment of Transplanted Progenitor Cells is Improved by Delivery within Collagen Matrix

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Yan Zhang ◽  
Marc Lamoureux ◽  
Stephanie Thorn ◽  
Vincent Chan ◽  
Joel Price ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Progenitor Cells ◽  
Pet Imaging ◽  
Ex Vivo ◽  
Collagen Matrix ◽  
Cell Labeling ◽  
Type I ◽  
The Matrix ◽  
Early Retention

Background: To investigate the mechanisms involved in the potentiation of cell therapy by delivery matrices, we evaluated the retention and engraftment of transplanted human circulating progenitor cells (CPCs) injected in a collagen matrix by using in vivo positron emission tomography (PET) imaging, ex vivo biodistribution, and immunohistochemistry. Methods: CPCs were labeled with 18 F-FDG and injected with or without a collagen type I-based matrix in the ischemic hindlimb muscle (IM) of rats (2x10 6 cells; n=15/group). Localization of cells was acquired by PET imaging (15 min) at 150 min post-injection. In addition, radionuclide biodistribution, immunofluorescence, and immunohistochemical examination of transplanted CPCs were performed at up to 14 days. Results: Cell labeling efficiency was CPC-concentration dependent (r=0.61, p <0.001), but not 18 F-FDG-dose dependent. Labeled CPCs exhibited excellent short-term stability and viability. Persistence of 18 F-FDG radioactivity in cells was markedly greater than non-specific retention in the matrix. Wholebody (WB) PET images revealed better CPC retention in the IM and less non-specific leakage to other tissues when CPCs were delivered within the matrix (IM/WB retention ratio of 43.9±8.2%), compared to cells injected alone (22.3±10.4%; p =0.040) and to 18 F-FDG injected with or without the matrix (9.7±5.5% and 11.0±5.5%, respectively; p <0.005). Radioactivity biodistribution confirmed that accumulation was increased (by 92.5%; p =0.024) in the IM and reduced (by 1.1 to 23.8%; p <0.05) in non-specific tissues when cells were injected within the matrix, compared to cells injected alone. Anti-human mitochondria staining showed increased cell retention in the IM with use of matrices (3.0±2.1%) versus cells only (1.9±0.8%; p =0.048). At 14 days the number of CD31 + transplanted human cells was greater (1.6±0.1%) when injected within the matrix than injected alone (0.7±0.1%; p =0.004). Conclusions: Collagen-based delivery matrices improve the early retention of transplanted CPCs, which in turn favors subsequent cell engraftment in the ischemic tissue. This mechanism conferred by the matrix has potential implications for the optimization of cell therapy at the early stages after cell delivery.

scholarly journals Regulation of cilia abundance in multiciliated cells

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Rashmi Nanjundappa ◽  
Dong Kong ◽  
Kyuhwan Shim ◽  
Tim Stearns ◽  
Steven L Brody ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Surface Area ◽  
Ex Vivo ◽  
Compensatory Mechanism ◽  
Multiciliated Cells ◽  
Culture Model ◽  
A Cell ◽  
Dependent Mechanism

Multiciliated cells (MCC) contain hundreds of motile cilia used to propel fluid over their surface. To template these cilia, each MCC produces between 100-600 centrioles by a process termed centriole amplification. Yet, how MCC regulate the precise number of centrioles and cilia remains unknown. Airway progenitor cells contain two parental centrioles (PC) and form structures called deuterosomes that nucleate centrioles during amplification. Using an ex vivo airway culture model, we show that ablation of PC does not perturb deuterosome formation and centriole amplification. In contrast, loss of PC caused an increase in deuterosome and centriole abundance, highlighting the presence of a compensatory mechanism. Quantification of centriole abundance in vitro and in vivo identified a linear relationship between surface area and centriole number. By manipulating cell size, we discovered that centriole number scales with surface area. Our results demonstrate that a cell-intrinsic surface area-dependent mechanism controls centriole and cilia abundance in multiciliated cells.

scholarly journals Exosomes derived from induced vascular progenitor cells promote angiogenesis in vitro and in an in vivo rat hindlimb ischemia model

2019 ◽  
Vol 317 (4) ◽  
pp. H765-H776 ◽  
Author(s):  
Takerra K. Johnson ◽  
Lina Zhao ◽  
Dihan Zhu ◽  
Yang Wang ◽  
Yan Xiao ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Aortic Ring ◽  
Size Exclusion ◽  
Hindlimb Ischemia ◽  
Vascular Progenitor Cells ◽  
Angiogenic Effect

Induced vascular progenitor cells (iVPCs) were created as an ideal cell type for regenerative medicine and have been reported to positively promote collateral blood flow and improve cardiac function in a rat model of myocardial ischemia. Exosomes have emerged as a novel biomedicine that mimics the function of the donor cells. We investigated the angiogenic activity of exosomes from iPVCs (iVPC-Exo) as a cell-free therapeutic approach for ischemia. Exosomes from iVPCs and rat aortic endothelial cells (RAECs) were isolated using a combination of ultrafiltration and size-exclusion chromatography. Nanoparticle tracking analysis revealed that exosome isolates fell within the exosomal diameter (<150 nm). These exosomes contained known markers Alix and TSG101, and their morphology was validated using transmission electron microscopy. When compared with RAECs, iVPCs significantly increased the secretion of exosomes. Cardiac microvascular endothelial cells and aortic ring explants were pretreated with RAEC-Exo or iVPC-Exo, and basal medium was used as a control. iVPC-Exo exerted an in vitro angiogenic effect on the proliferation, tube formation, and migration of endothelial cells and stimulated microvessel sprouting in an ex vivo aortic ring assay. Additionally, iVPC-Exo increased blood perfusion in a hindlimb ischemia model. Proangiogenic proteins (pentraxin-3 and insulin-like growth factor-binding protein-3) and microRNAs (-143-3p, -291b, and -20b-5p) were found to be enriched in iVPC-Exo, which may mediate iVPC-Exo induced vascular growth. Our findings demonstrate that treatment with iVPC-Exo promotes angiogenesis in vitro, ex vivo, and in vivo. Collectively, these findings indicate a novel cell-free approach for therapeutic angiogenesis. NEW & NOTEWORTHY The results of this work demonstrate exosomes as a novel physiological mechanism by which induced vascular progenitor cells exert their angiogenic effect. Moreover, angiogenic cargo of proteins and microRNAs may define the biological contributors in activating endothelial cells to form a new capillary plexus for ischemic vascular diseases. Listen to this article's corresponding podcast at https://ajpheart.podbean.com/e/angiogenic-exosomes-from-vascular-progenitor-cells/ .

Expansion Culture Of Hematopoietic Stem & Progenitor Cells From Frozen-Thawed, Non-Enriched Human Umbilical Cord Blood In Animal Component– & Serum–Free Medium Enhances Engraftment & Reduces Graft-Versus-Host-Disease

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4460-4460
Author(s):  
Sudipto Bari ◽  
Pat PY Chu ◽  
Andrea Lim ◽  
Xiubo Fan ◽  
Gigi NC Chiu ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Ex Vivo ◽  
Serum Free Medium ◽  
Free Medium ◽  
Hematopoietic Stem ◽  
Serum Free ◽  
Graft Versus Host ◽  
Nsg Mice ◽  
Animal Component

Hematopoietic stem cell transplantation in adults using umbilical cord blood (UCB) is limited by low cell dosage & post-thaw viability. In several clinical trials cytokine supplementation & stromal cell support have been shown to enhance total nucleated cells (TNC). However, clinical safety is compromised due to source inconsistency & population heterogeneity of stromal cells along with animal components of the conventional growth media. In this study, we demonstrate effective use of an animal component– & serum–free growth medium to enhance the viability & ex vivo expansion of SCID repopulating cells (SRC) from frozen-thawed, non-enriched UCB–mononucleated cells (UCB-MNC). UCB-MNC were cultured in a commercially available animal component– & serum–free medium, StemSpanTM–ACF (ACF), while StemSpanTM–SFEM (SFEM), a conventional serum–free medium with human and bovine components served as control. Both media (from STEMCELL Technologies INC. Vancouver, Canada) were supplemented with clinical grade SCF, Flt-3 ligand, TPO, & IGFBP2. The expansion effects were characterized based on cell viability, phenotypic stem & progenitor cells & functional in vitro & in vivo assays. After 3-days of culturing, viability of CD45+ UCB-MNC was maintained at a significantly higher level in ACF (90.7±0.2%) compared to SFEM (75.4±0.1%) (p<0.0001; n=3). Culturing for 11-days significantly (p<0.0001; n=6) increased CD45+CD34+CD38– hematopoietic progenitors in ACF (90.6±13.5 fold) compared to control (4.8±0.4 fold). Further phenotypic study of ACF expanded cells showed significant increases of 4.1-fold for CD45+CD34+C38–CD90+ stem cells (p<0.0001), 2.1-fold for CD45+CD34+CD13+CD33+ myeloid progenitors (p<0.01) and 2.3-fold for CD45+CD34+C38–CD7+(p<0.01) lymphoid progenitors compared to SFEM (n=6). Viable TNC expansions were 4.3±0.2 fold and 5.9±0.7 fold in ACF and SFEM respectively (n=6; p<0.05). Colony forming unit (CFU) assay showed that ACF supported significantly higher expansion of GM progenitors than SFEM (60.1±7.9 vs. 14.6±2.1 fold; p<0.00001; n=16). The numbers of multi-potent progenitors, CFU-GEMM, were maintained in ACF but decreased in SFEM (0.83±0.21 vs. 0.09±0.04 fold relative to non-expanded UCB; p<0.01; n=16). UCB-MNC cultured for 11 days reconstituted the bone marrow (BM) of sub-lethally irradiated NOD/SCID gamma (NSG) mice with human CD45+/71+ cells as measured 16 weeks after transplantation at a dosage of 1x108 cells/kg. The frequency of human cells was higher for UCB expanded in ACF (38.1±15.4%; n=5) than for UCB expanded in SFEM (3.4±2.1; n=14; p<0.01). Human CD34+ progenitors were also detected in BM of the engrafted mice at frequencies of 2.4±1.4% and 0.2±0.1% for ACF and SFEM expanded cells respectively (p<0.05). Human hematopoiesis was multi-lineage with significantly higher numbers of CD45+/71+ & CD15+/66b+ granulopoietic cells (71.4-fold; p<0.001) and CD19+/20+ B-lineage cells (23.1-fold; p<0.001) in mice transplanted with cells expanded in ACF (n=5) as compared to SFEM (n=14). At a transplantation dosage of 2.5x107 cells/kg, non-expanded grafts (n=10) had similar engraftment of CD45+/71+cells compared to ACF expanded grafts (n=5; p=0.14), while engraftment was lower for SFEM expanded grafts (n=12; p<0.01). Limiting dilution analysis revealed that SRC frequencies were increased, on average, 7.9– and 1.2–fold in ACF relative to SFEM expanded & non-expanded grafts respectively. NSG mice transplanted with non-expanded grafts had a significantly lower (p<0.001) survival rate (40.4%, n=47) compared to those transplanted with grafts expanded in ACF (90.9%, n=11) or SFEM (92.3% n=26), or injected with saline only (100%, n=7). The high mortality rate in recipients of non-expanded grafts was due to higher incidence of graft-versus-host-disease (GVHD) associated with significantly (p<0.01; n=6) higher CD45+CD7+T cells in comparison to expanded grafts. In conclusion, expansion of freeze-thawed, non-enriched UCB-MNC in animal component– & serum–free medium improves in vivo repopulation and reduces mortality due to GVHD in a xenotransplantation model. These findings could set the platform for developing safer, cheaper & time efficient clinical transplantation, since no animal components, in the form of serum albumin or stromal cells, are required to achieve desired ex vivo expansion of hematopoietic stem & progenitor cells & pre-clinical outcomes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Functional disruption of α4 integrin mobilizes bone marrow–derived endothelial progenitors and augments ischemic neovascularization

2006 ◽  
Vol 203 (1) ◽  
pp. 153-163 ◽  
Author(s):  
Gangjian Qin ◽  
Masaaki Ii ◽  
Marcy Silver ◽  
Andrea Wecker ◽  
Evelyn Bord ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Critical Role ◽  
Cell Surface Receptor ◽  
Ischemic Disease ◽  
Angiogenic Therapy ◽  
Ischemic Tissue ◽  
Surface Receptor

The cell surface receptor α4 integrin plays a critical role in the homing, engraftment, and maintenance of hematopoietic progenitor cells (HPCs) in the bone marrow (BM). Down-regulation or functional blockade of α4 integrin or its ligand vascular cell adhesion molecule-1 mobilizes long-term HPCs. We investigated the role of α4 integrin in the mobilization and homing of BM endothelial progenitor cells (EPCs). EPCs with endothelial colony-forming activity in the BM are exclusively α4 integrin–expressing cells. In vivo, a single dose of anti–α4 integrin antibody resulted in increased circulating EPC counts for 3 d. In hindlimb ischemia and myocardial infarction, systemically administered anti–α4 integrin antibody increased recruitment and incorporation of BM EPCs in newly formed vasculature and improved functional blood flow recovery and tissue preservation. Interestingly, BM EPCs that had been preblocked with anti–α4 integrin ex vivo or collected from α4 integrin–deficient mice incorporated as well as control cells into the neovasculature in ischemic sites, suggesting that α4 integrin may be dispensable or play a redundant role in EPC homing to ischemic tissue. These data indicate that functional disruption of α4 integrin may represent a potential angiogenic therapy for ischemic disease by increasing the available circulating supply of EPCs.

scholarly journals Extracellular vesicles of stromal origin target and support hematopoietic stem and progenitor cells

2017 ◽  
Vol 216 (7) ◽  
pp. 2217-2230 ◽  
Author(s):  
Gregoire Stik ◽  
Simon Crequit ◽  
Laurence Petit ◽  
Jennifer Durant ◽  
Pierre Charbord ◽  
...  
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Extracellular Vesicles ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Critical Role ◽  
Molecular Signature ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Extracellular vesicles (EVs) have been recently reported as crucial mediators in cell-to-cell communication in development and disease. In this study, we investigate whether mesenchymal stromal cells that constitute a supportive microenvironment for hematopoietic stem and progenitor cells (HSPCs) released EVs that could affect the gene expression and function of HSPCs. By taking advantage of two fetal liver–derived stromal lines with widely differing abilities to maintain HSPCs ex vivo, we demonstrate that stromal EVs play a critical role in the regulation of HSPCs. Both supportive and nonsupportive stromal lines secreted EVs, but only those delivered by the supportive line were taken up by HSPCs ex vivo and in vivo. These EVs harbored a specific molecular signature, modulated the gene expression in HSPCs after uptake, and maintained the survival and clonogenic potential of HSPCs, presumably by preventing apoptosis. In conclusion, our study reveals that EVs are an important component of the HSPC niche, which may have major applications in regenerative medicine.

scholarly journals Angiotensin II AT2 receptor regulates ureteric bud morphogenesis

2010 ◽  
Vol 298 (3) ◽  
pp. F807-F817 ◽  
Author(s):  
Renfang Song ◽  
Melissa Spera ◽  
Colleen Garrett ◽  
Samir S. El-Dahr ◽  
Ihor V. Yosypiv
Keyword(s):  
Cell Proliferation ◽  
Signaling Pathway ◽  
Ex Vivo ◽  
Branching Morphogenesis ◽  
Mrna Levels ◽  
Ureteric Bud ◽  
Embryonic Mouse ◽  
Genetic Inactivation ◽  
The Impact

ANG II AT2 receptor (AT2R)-deficient mice exhibit abnormal ureteric bud (UB) budding, increased incidence of double ureters, and vesicoureteral reflux. However, the role of the AT2R during UB morphogenesis and the mechanisms by which aberrant AT2R signaling disrupts renal collecting system development have not been fully defined. In this study, we mapped the expression of the AT2R during mouse metanephric development, examined the impact of disrupted AT2R signaling on UB branching, cell proliferation, and survival, and investigated the cross talk of the AT2R with the glial-derived neurotrophic factor ( GDNF)/ c-Ret/Wnt11 signaling pathway. Embryonic mouse kidneys express AT2R in the branching UB and the mesenchyme. Treatment of embryonic day 12.5 ( E12.5) metanephroi with the AT2R antagonist PD123319 or genetic inactivation of the AT2R in mice inhibits UB branching, decreasing the number of UB tips compared with control (34 ± 1.0 vs. 43 ± 0.6, P < 0.01; 36 ± 1.8 vs. 48 ± 1.3, P < 0.01, respectively). In contrast, treatment of metanephroi with the AT2R agonist CGP42112 increases the number of UB tips compared with control (48 ± 1.8 vs. 39 ± 12.3, P < 0.05). Using real-time quantitative RT-PCR and whole mount in situ hybridization, we demonstrate that PD123319 downregulates the expression of GDNF, c-Ret, Wnt11, and Spry1 mRNA levels in E12.5 metanephroi grown ex vivo. AT2R blockade or genetic inactivation of AT2R stimulates apoptosis and inhibits proliferation of the UB cells in vivo. We conclude that AT2R performs essential functions during UB branching morphogenesis via control of the GDNF/c-Ret/Wnt11 signaling pathway, UB cell proliferation, and survival.

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