scholarly journals Contribution of minced muscle graft progenitor cells to muscle fiber formation after volumetric muscle loss injury in wild-type and immune deficient mice

2017 ◽  
Vol 5 (7) ◽  
pp. e13249 ◽  
Author(s):  
Benjamin T. Corona ◽  
Beth E. P. Henderson ◽  
Catherine L. Ward ◽  
Sarah M. Greising
Keyword(s):  
Progenitor Cells ◽  
Muscle Fiber ◽  
Fiber Formation ◽  
Muscle Loss ◽  
Wild Type ◽  
Muscle Graft ◽  
Volumetric Muscle Loss ◽  
Minced Muscle ◽  
Deficient Mice

scholarly journals Co-delivery of a laminin-111 supplemented hyaluronic acid based hydrogel with minced muscle graft in the treatment of volumetric muscle loss injury

PLoS ONE ◽  
2018 ◽  
Vol 13 (1) ◽  
pp. e0191245 ◽  
Author(s):  
Stephen M. Goldman ◽  
Beth E. P. Henderson ◽  
Thomas J. Walters ◽  
Benjamin T. Corona
Keyword(s):  
Hyaluronic Acid ◽  
Muscle Loss ◽  
Muscle Graft ◽  
Volumetric Muscle Loss ◽  
Minced Muscle

scholarly journals Sox4 Is Required for the Formation and Maintenance of Multipotent Progenitors

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1577-1577 ◽  
Author(s):  
Hong Zhang ◽  
Min Ye ◽  
Robert S. Welner ◽  
Daniel G. Tenen
Keyword(s):  
Progenitor Cells ◽  
Absolute Number ◽  
Brdu Incorporation ◽  
Binding Motif ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Genetic Ablation ◽  
Qpcr Analysis ◽  
Multipotent Progenitors ◽  
Deficient Mice

Abstract Introduction Hematopoiesis is maintained by a hierarchical system, whereas aberrant control of hematopoiesis is the underlying cause of many diseases. Within the hematopoietic hierarchy, hematopoietic stem cells (HSCs) give rise to multipotent progenitors that have lost their self-renewal capacity but remain multipotent to differentiate into mature blood cells. However, the precise molecular mechanisms that modulate this transition are not fully understood yet. Results We recently discovered that genetic ablation of SRY sex determining region Y-box 4 gene (Sox4) in the murine hematopoietic system resulted in dramatic loss of multipotent progenitor population (CD48+CD150-Lin-kit+Sca1+, or CD48+CD150-LSK) both relatively (to the total LSK population) and in absolute number. Interestingly, the absolute number of HSCs (CD48-CD150+Lin-kit+Sca1+, or SLAM+LSK) in these conditional Sox4-deficient mice was comparable to their wild-type counterparts. Transcriptional factor Sox4 belongs to the high-mobility group (HMG) domain superfamily which also includes other Sox proteins, TCF-1 (T-cell factor 1) and LEF-1 (lymphoid enhancer factor 1). Sox4 has been implicated in leukemogenesis and may potentially contribute to stem cell properties. Nevertheless, the precise roles of Sox4 in hematopoietic stem/progenitor cells and the underlying mechanisms have not been defined yet. Further analysis of stem/progenitor compartment defined by Flt3 and CD34 expression demonstrated a major loss in lymphoid-primed multipotent progenitors (LMPPs) (CD34+Flt3+LSK) with relatively normal formation of LT-HSCs (CD34-Flt3-LSK) and ST-HSCs (CD34+Flt3-LSK) upon the loss of Sox4, suggesting that Sox4 is essential for the development from HSCs to multipotent progenitors. Such observation is in line with the expression pattern of Sox4. Quantitative PCR (qPCR) analysis of wild-type mice revealed that expression of Sox4 increased from HSCs to multipotent progenitors which expressed Sox4 at the highest level among all the hematopoietic compartments. Studies of biological behaviors further indicateed that disruption of Sox4 had no effect on proliferative capacity of HSCs and multipotent progenitors, as evidenced by BrdU incorporation assay. However, Annexin V/propidium iodide staining revealed an increased frequency of apoptotic multipotent progenitors, but not that of HSCs upon the ablation of Sox4. In a transplantation setting, although Sox4-deficient LSKs homed appropriately to the bone marrow, they exhibited severely impaired ability to give rise to multipotent progenitors, but contributed normally to HSCs compared to the wild-type donors. Among a set of genes crucial to the biological properties of stem/progenitor cells, qPCR analysis revealed that upon the loss of Sox4, only the levels of Ikaros1 and Ikaros2, the two major Ikaros isoforms in stem/progenitor cells, were downregulated specifically in multipotent progenitors, but remained normal in HSCs. Intriguingly, in a reminiscent manner of Sox4-deficient mice, mice lacking both Ikaros 1 and Ikaros 2 proteins, also exhibited disrupted B cell development and selectively impaired LMPPs. Previous study identified an enhancer of Ikaros locus as the only cis-regulatory element that was capable of stimulating reporter expression in the LMPPs. Our sequence analysis revealed a highly conserved Sox4 binding motif within this enhancer, therefore potentially connecting Sox4 with the known regulatory networks that modulate the differentiation of HSCs. Currently, we are working on (1) confirming the direct transcriptional regulation of Ikaros by Sox4; (2) assessing whether Ikaros mediates the functions of Sox4 in the formation or maintenance of the multipotent progenitors population in vivo; and (3) delineating the downstream regulatory network of Sox4 in stem/progenitor cells. Conclusion In summary, out study reveals a novel role for Sox4 gene in early hematopoiesis and brings important insights into the regulatory mechanisms underlying the commitment of HSCs toward multipotent progenitors. Disclosures No relevant conflicts of interest to declare.

Myeloid Progenitor Cells from Gadd45a and Gadd45b-Deficient Mice Are Sensitized to Genotoxic-Stress Induced Apoptosis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4239-4239
Author(s):  
Mamta Gupta ◽  
Shiv K. Gupta ◽  
Arthur G. Balliet ◽  
Barbara Hoffman ◽  
Dan A. Lieberman
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Genotoxic Stress ◽  
Wild Type ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells ◽  
Induced Apoptosis ◽  
Deficient Mice ◽  
Marrow Cells

Abstract GADD45 (Growth arrest and DNA damge) regulates cell growth following exposure to diverse stimuli. It has been shown that, mice lacking the gadd45a gene exhibit genomic instability and increased carcinogenesis, but the exact role of the gadd45 family genes still remains unclear. In this study we have aimed at determining the effect of gadd45a or gadd45b deficiency on the response of bone marrow derived myeloid cells to genotoxic stress agents by using gadd45a or gadd45b null mice. We have found that myeloid progenitor cells from gadd45a or gadd45b-null mice are more sensitive to ultraviolet-radiation (UV), VP-16 or daunorubicin induced apoptosis. Introduction of wild-type gadd45 into gadd45-deficient bone marrow cells restored the wild-type apoptotic phenotype. In-vitro colony formation following stress responses has shown that bone marrow cells from gadd45a or gadd45b-deficient mice have a decreased ability to form haematopoetic colonies. Gadd45a or gadd45b-deficient bone marrow cells also displayed defective G2/M cell cycle checkpoint following exposure to either UV and V-16 but were still able to undergo G2/M arrest following exposure to daunorubicin, indicating the existence of different G2/M checkpoints in response to these anticancer agents. Taken together these findings identify gadd45a or gadd45b as anti-apoptotic gene(s), and suggests that the absence of gadd45a or gadd45b results in higher susceptibility of haematopoetic cells to UV radiation and certain anticancer drugs.

Role of the Cell Cycle Regulator Cdh1 in Physiology and Pathology of Hematopoiesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2419-2419
Author(s):  
Jo Ishizawa ◽  
Eiji Sugihara ◽  
Norisato Hashimoto ◽  
Shinji Kuninaka ◽  
Shinichiro Okamoto ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cell Differentiation ◽  
Progenitor Cells ◽  
Genotoxic Stress ◽  
Absolute Number ◽  
Wild Type ◽  
Deficient Mice ◽  
Oncogenic Stress

Abstract Abstract 2419 Various key molecules for cell cycle, especially G0/G1 regulators, have effects not only on cell proliferation but also on cell differentiation. Cdh1, one of the co-activators for anaphase-promoting complex/cyclosome, plays a crucial role in the mitotic phase, but has recently been identified as a G0/G1 regulator, suggesting that the role of Cdh1 in cell differentiation. Because there are only few reports about Cdh1 from this point of view, we focused on Cdh1 functions on the hematopoietic system, in which distinct populations of cells can be precisely identified by their cell surface markers, in physiology and pathology. For this purpose, we generated Cdh1 conditional gene-trap (GT) mice, by overcoming the embryonic lethality of Cdh1 homozygous GT mice. We introduced the Cdh1 cDNA replacing vector into ES cells derived from Cdh1 heterozygous GT mice. The resulted construct contains the floxed Cdh1 cDNA allele which is cleaved under the existence of Cre recombinases. We crossed mice carrying this Cdh1 transgene in homozygous (Cdh1f/f) with Mx1-Cre transgenic mice to obtain Mx1-Cre (+) / Cdh1f/f mice, in which Cre recombinases are induced in vivo by administration of pIpC. In this system, we found that the Cdh1-deficient mice 4 months after pIpC treatment, compared to Cdh1-intact mice (Mx1-Cre (-) / Cdh1f/f mice), exhibited a subtle but significant decrease in absolute number of mature lineage progenitor cells (4.3 ± 0.31 × 107 vs 3.2 ± 0.10 × 107 /femurs and tibiae; p=0.009). Furthermore, this phenomenon was conspicuous by irradiation as short as 7 days after pIpC treatment. In 48 hours post-irradiation, the absolute number of mature lineage progenitor cells decreased markedly in the Cdh1-deficient mice (7.4 ± 0.82 × 106 vs 3.6 ± 0.46 × 106; p=0.0023) and in addition, both of CD34+ and CD34- LSK cells were also decreased (absolute number of CD34- cells: 905 ± 194 vs 344 ± 223; p= 0.03). These results indicate that the loss of Cdh1 induces genotoxic fragility especially in these two subpopulations, the mature lineage progenitors and the stem cells. We also confirmed that the increased cell loss induced by irradiation in Cdh1-deficient mice is the result of mitotic catastrophe following G2/M checkpoint slippage due to loss of Cdh1 by DNA content analysis. We next focused on how oncogenic stress, as another genotoxic stress, effects on the cell fragility by Cdh1 loss. We performed retroviral transduction of N-myc into Cdh1-intact and Cdh1-deficient bone marrow mononuclear cells (BM-MNCs) and transplanted those into irradiated wild type mice. In this system, which our laboratory has established recently, the transplanted mice develop precursor B cell lymphoblastic leukemia (pre-B ALL) phenotype in high frequency (more than 80%) when wild type BM-MNCs were used as cell source. Our hypothesis at that time was that oncogenic stress due to N-myc induces the loss of stem/progenitor cell function, and in result, that Cdh1 loss reveals negative effects on leukemogenesis or changes its lineage phenotype by affecting pseudodifferentiation due to N-myc. However, against our speculation, 70% (7 out of 10) of mice transplanted with N-myc transduced Cdh1-deficient BM-MNCs developed pre-B ALL, which was the same frequency and the same phenotype as in Cdh1-intact cell sources. Of note, Cdh1 loss did not have a great impact on the prognosis of these pre-B ALL mice (median survival: 80 days in Cdh1-intact group vs 95 days in Cdh1-deficient group; p= 0.049). In conclusion, our results suggest that Cdh1 regulates the pool sizes of the hematopoietic stem cells and mature lineage progenitor cells both physiologically and pathologically; especially under irradiation stress. In contrast, Cdh1 is dispensable for B cell leukemogenesis and does not have a great impact on the natural prognosis of non-treated pre-B ALL. It is interesting that oncomine mRNA microarray database and other few reports indicate that human pre-B ALL cases are also divided into two groups according to the expression level of Cdh1, and it is the matter remained to be solved whether Cdh1 expression level affects the prognosis of treated patients. We propose that our Cdh1-deficient pre-B ALL mice have a potential as promising mouse model in order to assess this proposition and to prove that Cdh1 affects the sensitivity of pre-B ALL to treatments which causes the genotoxic stress, such as radiotherapy and genotoxic agents. Disclosures: Saya: Kyowa Hakko Kirin, Co., Ltd.: Research Funding.

A Porcine Urinary Bladder Matrix Does Not Recapitulate the Spatiotemporal Macrophage Response of Muscle Regeneration after Volumetric Muscle Loss Injury

2016 ◽  
Vol 202 (3-4) ◽  
pp. 189-201 ◽  
Author(s):  
Amit Aurora ◽  
Benjamin T. Corona ◽  
Thomas J. Walters
Keyword(s):  
Urinary Bladder ◽  
Muscle Fiber ◽  
Muscle Regeneration ◽  
De Novo ◽  
Muscle Loss ◽  
Macrophage Response ◽  
Urinary Bladder Matrix ◽  
Volumetric Muscle Loss ◽  
Fibrotic Scar ◽  
Muscle Fiber Regeneration

Volumetric muscle loss (VML) results in irrecoverable loss of muscle tissue making its repair challenging. VML repair with acellular extracellular matrix (ECM) scaffolds devoid of exogenous cells has shown improved muscle function, but limited de novo muscle fiber regeneration. On the other hand, studies using minced autologous and free autologous muscle grafts have reported appreciable muscle regeneration. This raises the fundamental question whether an acellular ECM scaffold can orchestrate the spatiotemporal cellular events necessary for appreciable muscle fiber regeneration. This study compares the macrophage and angiogenic responses including the remodeling outcomes of a commercially available porcine urinary bladder matrix, MatriStem™, and autologous muscle grafts. The early heightened and protracted M1 response of the scaffold indicates that the scaffold does not recapitulate the spatiotemporal macrophage response of the autograft tissue. Additionally, the scaffold only supports limited de novo muscle fiber formation and regressing vessel density. Furthermore, scaffold remodeling is accompanied by increased presence of transforming growth factor and α-smooth muscle actin, which is consistent with remodeling of the scaffold into a fibrotic scar-like tissue. The limited muscle formation and scaffold-mediated fibrosis noted in this study corroborates the findings of recent studies that investigated acellular ECM scaffolds (devoid of myogenic cells) for VML repair. Taken together, acellular ECM scaffolds when used for VML repair will likely remodel into a fibrotic scar-like tissue and support limited de novo muscle fiber regeneration primarily in the proximity of the injured musculature. This is a work of the US Government and is not subject to copyright protection in the USA. Foreign copyrights may apply. Published by S. Karger AG, Basel

Identification of Sox4 As Key Oncogene in Leukemias with Mutated or Silenced C/EBPα

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 114-114
Author(s):  
Hong Zhang ◽  
Min Ye ◽  
Meritxell Alberich-Jordà ◽  
Giovanni Amabile ◽  
Philipp B. Staber ◽  
...  
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Model Systems ◽  
Myeloid Differentiation ◽  
Wild Type ◽  
Down Regulation ◽  
Hematopoietic Stem ◽  
Gene Expression Signatures ◽  
Deficient Mice

Abstract Abstract 114 Transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα) controls cell proliferation and myeloid differentiation. In 7∼10% of patients with acute myeloid leukemia (AML) C/EBPA is either mutated or epigenetically silenced. C/EBPA mutated leukemias differ from C/EBPA silenced leukemias in prognosis and phenotype, yet both leukemias cluster together based on genome wide gene expression signatures, indicating a unifying mechanism of disease. So far, the key molecular downstream events required for C/EBPA loss to trigger leukemogenesis are still unclear. Based on microarray gene expression analysis, we here used a shRNA screening platform to search for mediators of leukemic outgrow of C/EBPα-deficient progenitor cells. In our screen, oncogene Sox4 was identified as a gene that was up-regulated in C/EBPα-deficient hematopoietic stem cells (HSCs, both lineage−c-kit+ScaI+ HSCs and SLAM+ HSCs) and whose down-regulation abrogated aberrant self-renewal ability and restored myeloid differentiation of C/EBPα-deficient stem/progenitor cells, as demonstrated by in vitro serial-replating and differentiation assays. Chromatin immunoprecipitation confirmed the endogenous binding of C/EBPα at the proximal promoter of Sox4 in the stem/progenitor cells enriched population (lineage−c-kit+) and the mature myeloid population (Mac1+Gr1+). In vitro promoter reporter assay demonstrated that wild-type human C/EBPA, but none of the C/EBPA mutants identified from AML patients, repressed Sox4 transcription through its binding to a highly conserved C/EBPα binding site. C/EBPα and Sox4 showed reciprocal expression patterns in both HSCs and various hematopoietic compartments during myeloid maturation of wild type mice. Furthermore, expression of Sox4 was up-regulated in HSCs of C/EBPα-deficient mice as well as in leukemia-initiating cells (LICs) of a murine C/EBPα mutant AML model. To further genetically dissect the role of Sox4 in driving leukemia in the absence of functional CEBPα, we generated Sox4, C/EBPα double deficient mice and observed that loss of Sox4 alleviated the abnormal stem/progenitor cell expansion and defective myeloid programming caused by C/EBPα deficiency. In addition, comparisons of the murine C/EBPα mutant AML model with a Sox4-induced AML model revealed that leukemia initiating cells of both leukemia models were enriched in immunophenotypically similar populations and exhibited comparable gene expression signatures. Similar to the C/EBPα knockout model, down-regulation of Sox4 by shRNA in LICs from murine C/EBPα mutant AML was also sufficient to abolish their augmented serial-replating ability. Importantly, enhanced expression of SOX4 in AML patients with either mutated or epigenetically silenced C/EBPA compared to other AML subtypes confirmed the findings in our mouse model systems. Our data demonstrate that failure to suppress Sox4 expression is the underlying mechanism of leukemias with mutated or silenced C/EBPα. These data also uncover a promising rationale for a therapeutic target in these leukemias. Disclosures: No relevant conflicts of interest to declare.

Further Evidence That HO-1 Regulates SDF-1 Expression in the Bone Marrow Microenvironment and That HO-1-Deficient Mice Show a Defect in the SDF-1–CXCR4 Retention Axis of Hematopoietic Stem/Progenitor Cells in Bone Marrow and Thus Are Easy Mobilizers - Studies in HO-1 Mutant Mice, Irradiation Chimeras, and the Effect of in Vivo Pharmacological Inhibition of HO-1

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 344-344
Author(s):  
Marcin Wysoczynski ◽  
Janina Ratajczak ◽  
Gregg Rokosh ◽  
Roberto Bolli ◽  
Mariusz Z Ratajczak
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Crucial Role ◽  
Conflicts Of Interest ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Deficient Mice

Abstract Abstract 344 Background: Stromal derived factor-1 (SDF-1), which binds to the CXCR4 receptor expressed on the surface of hematopoietic stem/progenitor cells (HSPCs), plays an important role in the retention of HSPCs in BM niches. Heme oxygenase (HO-1) is a stress-responsive enzyme that catalyzes the degradation of heme and plays an important function in various physiological and pathophysiological states associated with cellular stress, such as ischemic/reperfusion injury, atherosclerosis, and cancer. Interestingly, it has also been reported that HO-1 regulates the expression of SDF-1 in myocardium (J Mol Cell Cardiol. 2008;45:44–55). Aim of study: Since SDF-1 plays a crucial role in retention and survival of HSPCs in BM, we become interested in whether HO-1 is expressed by BM stromal cells and whether deficiency of HO-1 affects normal hematopoiesis and retention of HSPCs in BM. Experimental approach: To address this issue, we employed several complementary strategies to investigate HO-1–/–, HO-1+/–, and wild type (wt) mouse littermates for i) the expression level of SDF-1 in BM, ii) the number of clonogenic progenitors from major hematopoietic lineages in BM, iii) peripheral blood (PB) cell counts, iv) the chemotactic responsiveness of HSPCs to an SDF-1 gradient as well as to other chemoattractants, including sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), and extracellular nucleotiodes (ATP, UTP), iv) the adhesiveness of clonogenic progenitors to immobilized SDF-1 and stroma, v) the number of circulating HSPCs in PB, and vi) the degree of mobilization in response to granulocyte-colony stimulating factor (G-CSF) or AMD3100, assessed by enumerating the number of CD34–SKL cells and clonogeneic progenitors (CFU-GM) circulating in PB. We also exposed mice to the small HO-1 molecular inhibitor tin protoporphyrin IX (SnPP) and studied the effect of this treatment on G-CSF- or AMD3100-induced mobilization of HSPCs. Finally, to prove an environmental HSPC retention defect in HO-1-deficient mice, we created radiation chimeras, wild type mice transplanted with HO-1-deficient BM cells, and, vice versa, HO-1-deficient mice reconstituted with wild type BM cells. Results: Our data indicate that under normal, steady-state conditions, HO-1–/– and HO+/– mice have normal PB cell counts and numbers of circulating CFU-GM, while a lack of HO-1 leads to an increase in the number of erythroid (BFU-E) and megakaryocytic (CFU-GM) progenitors in BM. However, while BMMNCs from HO-1–/– have normal expression of the SDF-1-binding receptor, CXCR4, we observed that the mRNA level for SDF-1 in BM-derived fibroblasts was ∼4 times lower. This corresponded with the observation in vitro that HSPCs from HO-1–/– animals respond more robustly to an SDF-1 gradient, and HO-1–/– animals mobilized a higher number of CD34–SKL cells and CFU-GM progenitors into PB in response to G-CSF and AMD3100. Both G-CSF and AMD3100 mobilization were also significantly enhanced in normal wild type mice after in vivo administration of HO-1 inhibitor. Finally, mobilization studies in irradiation chimeras confirmed the crucial role of the microenvironmental SDF-1-based retention mechanism of HSPCs in BM niches. Conclusions: Our data demonstrate for the first time that HO-1 plays an important and underappreciated role in modulating the SDF-1 level in the BM microenvironment and thus plays a role in retention of HSPCs in BM niches. Furthermore, our recent data showing a mobilization effect by a small non-toxic molecular inhibitor of HO-1 (SnPP), suggest that blockage of HO-1 could be a promising strategy to facilitate mobilization of HSPCs. Further studies are also needed to evaluate the role of HO-1 in homing of HSPCs after transplantation to BM stem cell niches. Disclosures: No relevant conflicts of interest to declare.

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