scholarly journals Co-culture of mesenchymal stem cell spheres with hematopoietic stem cells under hypoxia: a cost-effective method to maintain self-renewal and homing marker expression

2021 ◽  
Author(s):  
Fatemeh Amiri ◽  
Ali Asghar Kiani ◽  
Marzie Bahadori ◽  
Mehryar Habibi Roudkenar
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Cost Effective ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cost Effective Method ◽  
Marker Expression

scholarly journals Co-Culture of Mesenchymal Stem Cell Spheres with Hematopoietic Stem Cells Under Hypoxia: A Cost-Effective Method to Maintain Self-Renewal and Homing Marker Expression

2021 ◽  
Author(s):  
Fatemeh Amiri ◽  
Ali Asghar Kiani ◽  
Marzie Bahadori ◽  
mehryar habibi roudkenar
Keyword(s):  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Cost Effective ◽  
Hypoxic Condition ◽  
Endothelial Protein C Receptor ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Cost Effective Method ◽  
Nuclear Factor I

Abstract Background: Hematopoietic stem cell (HSC) transplantation is considered a possible treatment option capable of curing various diseases. The aim of this study was the co-culturing of mesenchymal stem cell (MSC) spheres with HSCs under hypoxic condition to enhance the proliferation, self-renewal, stemness, and homing capacities of HSCs.Methods and results: HSCs were expanded after being subjected to different conditions including cytokines without feeder (Cyto), co-culturing with adherent MSCs (MSC), co-culturing with adherent MSCs+ hypoxia (MSC+ Hyp), co-culturing with MSCs spheres (Sph-MSC), co-culturing with MSCs spheres+ hypoxia (Sph-MSC+ Hyp), co-culturing with MSC spheres+ cytokines (Sph-MSC+Cyto). After 10 days, total nucleated cell (TNC) and CD34+/CD38- cell counts, colony-forming unit assay (CFU), long-term culture initiating cell (LTC-IC), the expression of endothelial protein C receptor (EPCR), nucleostemin (NS), nuclear factor I/X (Nfix) CXCR4, and VLA-4 were evaluated. The TNC, CD34+/CD38- cell count, CFU, and LTC-IC were higher in the Sph-MSC+ Hyp and Sph-MSC+ Cyto groups as compared with those of the MSC+ Hyp group (P<0.001). The expanded HSCs co-cultured with MSC spheres in combination with hypoxia expressed more EPCR, CXCR4, VLA-4, NS, and Nfix mRNA. The protein expression was also more up-regulated in the Sph-MSC+Cyto and Sph-MSC+ Hyp groups.Conclusion: Co-culturing HSCs with MSC spheres under hypoxic condition not only leads to higher cellular yield but also increases the expression of self-renewal and homing genes. Therefore, we suggest this approach as a simple and non-expensive strategy that might improve the transplantation efficiency of HSCs.

scholarly journals Clonal-level lineage commitment pathways of hematopoietic stem cells in vivo

2019 ◽  
Vol 116 (4) ◽  
pp. 1447-1456 ◽  
Author(s):  
Rong Lu ◽  
Agnieszka Czechowicz ◽  
Jun Seita ◽  
Du Jiang ◽  
Irving L. Weissman
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Lineage Commitment ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Different Levels

While the aggregate differentiation of the hematopoietic stem cell (HSC) population has been extensively studied, little is known about the lineage commitment process of individual HSC clones. Here, we provide lineage commitment maps of HSC clones under homeostasis and after perturbations of the endogenous hematopoietic system. Under homeostasis, all donor-derived HSC clones regenerate blood homogeneously throughout all measured stages and lineages of hematopoiesis. In contrast, after the hematopoietic system has been perturbed by irradiation or by an antagonistic anti-ckit antibody, only a small fraction of donor-derived HSC clones differentiate. Some of these clones dominantly expand and exhibit lineage bias. We identified the cellular origins of clonal dominance and lineage bias and uncovered the lineage commitment pathways that lead HSC clones to different levels of self-renewal and blood production under various transplantation conditions. This study reveals surprising alterations in HSC fate decisions directed by conditioning and identifies the key hematopoiesis stages that may be manipulated to control blood production and balance.

scholarly journals Osteoblast ablation reduces normal long-term hematopoietic stem cell self-renewal but accelerates leukemia development

Blood ◽  
2015 ◽  
Vol 125 (17) ◽  
pp. 2678-2688 ◽  
Author(s):  
Marisa Bowers ◽  
Bin Zhang ◽  
Yinwei Ho ◽  
Puneet Agarwal ◽  
Ching-Cheng Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Key Points ◽  
Leukemia Development

Key Points Bone marrow OB ablation leads to reduced quiescence, long-term engraftment, and self-renewal capacity of hematopoietic stem cells. Significantly accelerated leukemia development and reduced survival are seen in transgenic BCR-ABL mice following OB ablation.

scholarly journals Characterization of a mesenchymal stem cell line that differentiates to bone and provides niches supporting mouse and human hematopoietic stem cells

2012 ◽  
Vol 02 (01) ◽  
pp. 5-14 ◽  
Author(s):  
Sonal R. Tuljapurkar ◽  
John D. Jackson ◽  
Susan K. Brusnahan ◽  
Barbara J. O’Kane ◽  
John G. Sharp
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Cell Line ◽  
Stem Cell Line ◽  
Hematopoietic Stem

Zeb2-Defficiency in the Adult Murine Hematopoietic Precursor Cells Leads to Differentiation Defects in Multiple Hematopoietic Lineages and a Myeloproliferative-Like Phenotype

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1199-1199
Author(s):  
Tamara Riedt ◽  
Steven Goossens ◽  
Ines Gütgemann ◽  
Carmen Carrillo-Garcia ◽  
Hichem D Gallala ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Cells ◽  
Extramedullary Hematopoiesis ◽  
Independent Effect ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Reticular Fibers

Abstract Abstract 1199 The life long replenishment of highly specialized blood cells by a small number of hematopoietic cells (HSC) requires a strict regulation between self-renewal and differentiation in the immature compartment of the bone marrow. Perturbation of this equilibrium can result in stem cell loss or hematologic malignancies. This balance is at least in part controlled by a network of transcription factors. Zeb2 is a transcriptional repressor and plays an important role during the embryonic development as a modulator of the epithelial to mesenchymal transition (EMT) as well as tumor progression and metastasis. We have previously identified the essential role of Zeb2 in murine embryonic hematopoiesis, where selective Zeb2 deficiency in the hematopoietic stem cells resulted in early lethality around day 12.5. The aim of this study was to analyze whether Zeb2 plays a specific role in the regulation of homeostasis in the adult hematopoietic system. Using the Mx1-Cre based inducible Zeb2 conditional knock out mouse model we analyzed the impact of Zeb2 loss on adult hematopoietic stem cell function. Upon the induction of Zeb2 deletion we found a significant decrease in most cell lineages of the peripheral blood, except the neutrophil granulocytes. However, the reduction of mature cells in the blood was not accompanied by reduced bone marrow cellularity, as the cellularity was similar between Zeb2Δ/Δ Mx1-Cre (Zeb2 conditional KO) mice and the control animals (Zeb2+/+Mx1-Cre). However, in the bone marrow of the Zeb2Δ/Δ Mx1-Cre animals the granulocytic lineage was dominating, whereas other lineages e.g. red blood cell precursors and B-lymphoid precursors were drastically reduced. Histological sections of the bone marrow cavity revealed megacaryocytes with abnormal morphology reflecting maturation defects and an increased production of reticular fibers in the BM of Zeb2Δ/Δ Mx1-Cre mice. In addition Zeb2Δ/Δ Mx1-Cre mice displayed a two to three fold increase in spleen size compared to control animals due to an extramedullary hematopoiesis. Analysis of the primitive hematopoietic compartment in the bone marrow and spleens revealed that Zeb2 deletion resulted in a pronounced increase in the most immature hematopoietic cells, defined as Lin-Sca1+cKit+CD48-CD150+ population, and perturbation in different lineage restricted progenitor subpopulations. No difference in cell cycling or apoptotic rate in the stem cell enriched bone marrow population (Lin-Sca1+cKit+CD48-CD150+) was detectable between the genotypes. Upon transplantation into lethally irradiated wild type recipients, Zeb2 deficient stem cells demonstrated significantly reduced ability to differentiate into multiple hematopoietic lineages indicating a niche independent effect of Zeb2 in promoting differentiation of hematopoietic stem cells. On the molecular level, gene expression analysis of hematopoietic stem and progenitor cells using microarray approach revealed increased transcripts of downstream targets of Wnt/ß-Catenin signaling, suggesting increased Wnt signaling activity in absence of Zeb2 in the hematopoietic compartment, which at least in part might be responsible for the observed phenotype. These data indicate that Zeb2 is involved in the regulation of the balance between self-renewal and differentiation at multiple stages of hematopoietic cell maturation. Furthermore the lack of Zeb2 in the hematopoietic compartment leads to a phenotype that resembles the features of human myeloproliferative disorders, especially the early stages of primary myelofibrosis with dominant granulopoiesis, production of reticular fibers in the bone marrow, and morphological abnormalities in megacaryocytes, accompanied by extramedullary hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Molecular Integration Of HoxB4 and STAT3 For Self-Renewal Of Hematopoietic Stem Cells: A Model Of Molecular Convergence For Stemness

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2444-2444
Author(s):  
Il-Hoan Oh ◽  
Kim Tae-Min ◽  
Jae-Seung Shim
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Functional Integration ◽  
The Self ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell State ◽  
Stem Cell State

Abstract Multiple transcription factors (TFs) that regulate the self-renewal/stem cell state of hematopoietic stem cells (HSCs) have been identified, but understanding the molecular interplay of these TFs for their functional coordination remains a challenging issue. In this study, we investigated the functional integration and transcriptional coordination of STAT3 and HoxB4, which are TFs known to have similar effects on the self-renewal of HSCs. We found that while STAT3 (STAT3-C) or HoxB4 similarly enhanced the in vitro self-renewal and in vivo repopulating activities of HSCs, simultaneous transduction of both STAT3-C and HoxB4 did not have any additive enhancing effects. In contrast, the overexpression of HoxB4 caused a ligand-independent Tyr-phosphorylation in STAT3, and the inhibition of the STAT3 activity in HoxB4-overexpressing bone marrow cells significantly abrogated the enhancing effects of HoxB4 on both the bone marrow repopulation and maintenance of the undifferentiated state, revealing a molecular integration of these two TFs for HSC self-renewal. Expression microarray analysis revealed a significant overlap of the transcriptomes regulated by STAT3 and HoxB4 in undifferentiated hematopoietic cells. Moreover, a gene set enrichment analysis (GSEA) for TFs that can recapitulate the transcriptional changes induced by HoxB4 or STAT3 showed significant overlap in the candidate TFs. Interestingly, among these identified TFs were the puripotency-related genes, Oct-4 and Nanog. These results indicate the functional integration of tissue-specific TFs for HSC self-renewal and provide insights into the functional convergence of various TFs towards a conserved transcription program for the stem cell state. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The PI3K/Akt1 pathway enhances steady-state levels of FANCL

2013 ◽  
Vol 24 (16) ◽  
pp. 2582-2592 ◽  
Author(s):  
Kim-Hien T. Dao ◽  
Michael D. Rotelli ◽  
Brieanna R. Brown ◽  
Jane E. Yates ◽  
Juha Rantala ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Fanconi Anemia ◽  
Cell Function ◽  
Glycogen Synthase ◽  
Glycogen Synthase Kinase ◽  
Glycogen Synthase Kinase 3Β ◽  
Self Renewal ◽  
Hematopoietic Stem

Fanconi anemia hematopoietic stem cells display poor self-renewal capacity when subjected to a variety of cellular stress. This phenotype raises the question of whether the Fanconi anemia proteins are stabilized or recruited as part of a stress response and protect against stem cell loss. Here we provide evidence that FANCL, the E3 ubiquitin ligase of the Fanconi anemia pathway, is constitutively targeted for degradation by the proteasome. We confirm biochemically that FANCL is polyubiquitinated with Lys-48–linked chains. Evaluation of a series of N-terminal–deletion mutants showed that FANCL's E2-like fold may direct ubiquitination. In addition, our studies showed that FANCL is stabilized in a complex with axin1 when glycogen synthase kinase-3β is overexpressed. This result leads us to investigate the potential regulation of FANCL by upstream signaling pathways known to regulate glycogen synthase kinase-3β. We report that constitutively active, myristoylated-Akt increases FANCL protein level by reducing polyubiquitination of FANCL. Two-dimensional PAGE analysis shows that acidic forms of FANCL, some of which are phospho-FANCL, are not subject to polyubiquitination. These results indicate that a signal transduction pathway involved in self-renewal and survival of hematopoietic stem cells also functions to stabilize FANCL and suggests that FANCL participates directly in support of stem cell function.

scholarly journals PEDF Can Rescue the Inhibition of Injured Endothelial Cells on Hematopoietic Stem Cell Expansion Ex Vivo

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5008-5008
Author(s):  
Lingyu Zeng ◽  
Wenyi Lu ◽  
Lan Ding ◽  
Wen Ju ◽  
Jianlin Qiao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Mesenchymal Transition ◽  
Endothelial To Mesenchymal Transition

Introduction: Endothelial cells (ECs) provide a fertile niche for hematopoietic stem cell (HSC) maintenance, differentiation, and migration.Several studies have indicated that bone marrow (BM) vascular niche was impaired after HSC transplantation and severely inhibited hematopoietic reconstruction. Pigment epithelium-derived factor (PEDF) is an important potential cytoprotection and therapeutic agent for injured cells. The direct role of the injured endothelial cells on hematopoietic stem cells and whether PEDF has protective effect in this system remain unknown. This study aims to observe the influence of enjured ECs on HSCs and to explore the role of PEDF in endothelial-HSC coculture system. Methods: Injury of Endothelial cells by two important preparative regimenconditioning radiation and Busulfan respectively was evaluated with CCK8 assay. The expression of endothelial tight junctions(TJs),adherent junctions related molecules and endothelial to Mesenchymal Transition molecules such as ZO-1, Occludin,VE-cadherin, ICAM, α-SMA, CD31 and VCAM were detected by RT-qPCR, flow cytometry, immunofluorescence and western blot. The effects of injured endothelial cells on HSC self-renewal, differentiation, cell cycle and apoptosis were evaluated by flow cytometry, photography, viable cell count and clone formation assay. Hematopoiesis regulation factors SCF, IL-6, TGF-β and TNF-α were detected by ELISA. The protective effect of PEDF was also explored. Results: Both radiation and Busulfan could decrease cell viability of endothelial cells. The expression level of ZO-1, Occludin, VE-cadherin, ICAM, CD31 and VCAM were decreased and α-SMA was increased when EC exposed to radiation or Busulfan suggesting endothelial activation, impaired EC permeability and endothelial to Mesenchymal Transition after EC injured. Compared with normal endothelial cells and hematopoietic stem cell co-culture group, the HSC% of injured endothelial cells and hematopoietic stem cells co-cultured group were significantly decreased, the cell colony formation ability was decreased, the proportion of mature cells increased, and the damage of endothelial cells could not maintain the characteristics of HSC, weakened the self-renewal and multidirectional differentiation potential of HSC and promoted the maturation of HSC. After the administration of PEDF, endothelial to Mesenchymal Transition of EC was suppressed and the EC permeability was improved. Most importantly, the proportion of HSC was significantly increased, and the proportion of mature cells decreased in the coculture system. Conclusion: Injured endothelial cells can inhibit proliferation of hematopoietic stem cells, self-renewal and promote HSC differentiation. PEDF could ameliorate endothelial injury and promote HSC expansion by suppressing endothelial-mesenchymal transition and protecting TJs and AJs. Disclosures No relevant conflicts of interest to declare.

Hematopoietic Stem Cell Expansion by HOXB4 Is Greatly Enhanced in p21 Deficient Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1688-1688 ◽  
Author(s):  
Noriko Miyake ◽  
Ann C.M. Brun ◽  
Mattias Magnusson ◽  
David T. Scadden ◽  
Stefan Karlsson
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Hox transcription factors have emerged as important regulators of hematopoiesis. In particular, enforced expression of HOXB4 is a potent stimulus for murine hematopoietic stem cell (HSC) self-renewal. Murine HSCs engineered to overexpress HoxB4 expand significantly more than control cells in vivo and ex vivo while maintaining a normal differentiation program. HSCs are regulated by the cell proliferation machinery that is intrinsically controlled by cyclin-dependent kinase inhibitors such as p21Cip1/Waf1(p21) and p27Kip1 (p27). The p21 protein restricts cell cycling of the hematopoietic stem cell pool and maintains hematopoietic stem cell quiescence. In order to ask whether enhanced proliferation due to HOXB4 overexpression is mediated through suppression of p21 we overexpressed HOXB4 in hematopoietic cells from p21−/− mice. First, we investigated whether human HOXB4 enhances in vitro expansion of BM cells from p21−/− mice compared to p21+/+ mice. 5FU treated BM cells from p21−/− or p21+/+ mice were pre-stimulated with SCF, IL-6, IL-3 for 2 days followed by transduction using retroviral vector expressing HOXB4 together with GFP (MIGB4) or the control GFP vector (MIG). The cells were maintained in suspension cultures for 13 days and analyzed for GFP positive cells by flow-cytometry. Compared to MIG transduced BM cells from p21+/+ mice (MIG/p21+), the numbers of GFP positive cells were increased 1.1-fold in MIG/p21−, 3.2-fold in MIGB4/p21+ and 10.0-fold in MIGB4/p21− respectively (n=5). CFU assays were performed after 13 days of culture. The numbers of CFU were increased 4.8-fold in MIG/p21−, 19.5-fold in MIG/p21+ and 33.9 -fold in MIGB4/p21− respectively. Next, we evaluated level of HSCs expansion by bone marrow repopulation assays. After 12-days of culture, 1.5 x 105 MIGB4 or MIG-transduced cells (Ly5.2) were transplanted into lethally irradiated mice in combination with 8 x 105 fresh Ly5.1 bone marrow cells. Sixteen weeks after transplantation, no Ly5.2 cells could be detected in MIG/p21+ or MIG/p21− transplanted mice (n=6). In contrast, Ly5.2 positive cells were detected in both MIGB4/p21+/+ and MIGB4/p21−/− cells. The % of Ly5.2 positive cells in MIGB4/p21− transplanted mice was 9.9-fold higher than MIGB4/p21+ transplanted mice. (38.4 % vs 3.9 %, P<0.02, n=5). These Ly5.2 positive cells differentiated into all lineages, as determined by proportions of Mac-1, B-220, CD3 and Ter119 positive populations. Currently, we are enumerating the expansion of HOXB4 transduced HSCs in p21 deficient BM cells using the CRU assay. Our findings suggest that HOXB4 increases the self-renewal of hematopoietic stem cells by a mechanism that is independent of p21. In addition, the findings demonstrate that deficiency of p21 in combination with enforced expression of HOXB4 can be used to rapidly and effectively expand hematopoietic stem cells.

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