Bone Marrow-derived Mesenchymal Progenitor Cells Research Articles

Dysregulated transforming growth factor-beta mediates early bone marrow dysfunction in diabetes

Diabetes affects select organs such as the eyes, kidney, heart, and brain. Our recent studies show that diabetes also enhances adipogenesis in the bone marrow and reduces the number of marrow-resident vascular regenerative stem cells. In the current study, we have performed a detailed spatio-temporal examination to identify the early changes that are induced by diabetes in the bone marrow. Here we show that short-term diabetes causes structural and molecular changes in the marrow, including enhanced adipogenesis in tibiae of mice, prior to stem cell depletion. This enhanced adipogenesis was associated with suppressed transforming growth factor-beta (TGFB) signaling. Using human bone marrow-derived mesenchymal progenitor cells, we show that TGFB pathway suppresses adipogenic differentiation through TGFB-activated kinase 1 (TAK1). These findings may inform the development of novel therapeutic targets for patients with diabetes to restore regenerative stem cell function.

Effects of porcine bone marrow-derived platelet-rich plasma on bone marrow-derived mesenchymal stem cells and endothelial progenitor cells

This study investigated the abundance of pro-regenerative growth factors in bone marrow-derived platelet-rich plasma (BM-PRP) and their effects on bone marrow-derived mesenchymal stem cells (BM-MSC) and bone marrow-derived endothelial progenitor cells (BM-EPC). Four 4–5 months-old domestic pigs were included, and each underwent bone marrow aspiration from its humerus bones and processed into bone marrow aspiration concentrate (BMAC) samples. The plasma and cellular portions of BMAC were subsequently separated and collected. The concentration of growth factors including BMP-2, PDGF-BB, TGF-β1 and VEGF in the plasma portion was measured and compared between BM-PRP and bone marrow-derived platelet-poor plasma (BM-PPP). It was found that platelet count was significantly higher in BM-PRP than in BM-PPP, but the concentration of above-mentioned growth factors was not significantly different between BM-PRP and BM-PPP. As most existing literature has indicated the regenerative potency of PRP, this study focused on assessing the effect of BM-PRP treatment on BM-MSC and BM-EPC proliferation, osteogenic differentiation and angiogenesis capacity by comparing samples with 2.5% BM-PRP treatment and samples without BM-PRP treatment (control). In response to BM-PRP treatment, the cellular doubling time increased with culturing time and was significantly shorter in the BM-PRP-treated samples than in control samples. For osteogenic differentiation, BM-PRP-treated BM-MSCs demonstrated a time-dependent increase in alkaline phosphatase (ALP) activity and expression levels of osteogenic differentiation markers. For the expression of angiogenic genes, none of the differences reached statistical significance despite a tendency of stronger expression at day 18 in BM-PRP-treated BM-EPCs. In conclusion, this in vitro study suggests that most BMP-2, PDGF-BB, TGF-β1 and VEGF-A contained in BM-PRP are not platelet-released and BM-PRP may have some stimulation (less than 1-fold) for MSC, EPC proliferation and MSC osteogenic differentiation.

Modeling appendicular skeletal cartilage development with modified high-density micromass cultures of adult human bone marrow-derived mesenchymal progenitor cells

BackgroundAnimal cell-based systems have been critical tools in understanding tissue development and physiology, but they are less successful in more practical tasks, such as predicting human toxicity to pharmacological or environmental factors, in which the congruence between in vitro and clinical outcomes lies on average between 50 and 60%. Emblematic of this problem is the high-density micromass culture of embryonic limb bud mesenchymal cells, derived from chick, mouse, or rat. While estimated predictive value of this model system in toxicological studies is relatively high, important failures prevent its use by international regulatory agencies for toxicity testing and policy development. A likely underlying reason for the poor predictive capacity of animal-based culture models is the small but significant physiological differences between species. This deficiency has inspired investigators to develop more organotypic, 3-dimensional culture system using human cells to model normal tissue development and physiology and assess pharmacological and environmental toxicity.MethodsWe have developed a modified, miniaturized micromass culture model using adult human bone marrow-derived mesenchymal progenitor cells (hBM-MPCs) that is amenable to moderate throughput and high content analysis to study chondrogenesis. The number of cells per culture was reduced, and a methacrylated gelatin (gelMA) overlay was incorporated to normalize the morphology of the cultures.ResultsThese modified human cell-based micromass cultures demonstrated robust chondrogenesis, indicated by increased Alcian blue staining and immunodetectable production of collagen type II and aggrecan, and stage-specific chondrogenic gene expression. In addition, in cultures of hBM-MPCs transduced with a lentiviral collagen type II promoter-driven GFP reporter construct, levels of GFP reporter activity correlated well with changes in endogenous collagen type II transcript levels, indicating the feasibility of non-invasive monitoring of chondrogenesis.ConclusionsThe modified hBM-MPC micromass culture system described here represents a reproducible and controlled model for analyzing mechanisms of human skeletal development that may later be applied to pharmacological and environmental toxicity studies.

Periosteum-derived mesenchymal progenitor cells in engineered implants promote fracture healing in a critical-size defect rat model.

An attractive alternative to bone autografts is the use of autologous mesenchymal progenitor cells (MSCs) in combination with biomaterials. We compared the therapeutic potential of different sources of mesenchymal stem cells in combination with biomaterials in a bone nonunion model. A critical-size defect was created in Sprague-Dawley rats. Animals were divided into six groups, depending on the treatment to be applied: bone defect was left empty (CTL); treated with live bone allograft (LBA); hrBMP-2 in collagen scaffold (CSBMP2 ); acellular polycaprolactone scaffold (PCL group); PCL scaffold containing periosteum-derived MSCs (PCLPMSCs ) and PCL containing bone marrow-derived MSCs (PCLBMSCs ). To facilitate cell tracking, both MSCs and bone graft were isolated from green fluorescent protein (GFP)-transgenic rats. CTL group did not show any signs of healing during the radiological follow-up (n=6). In the LBA group, all the animals showed bone bridging (n=6) whereas in the CSBMP2 group, four out of six animals demonstrated healing. In PCL and PCLPMSCs groups, a reduced number of animals showed radiological healing, whereas no healing was detected in the PCLBMSCs group. Using microcomputed tomography, the bone volume filling the defect was quantified, showing significant new bone formation in the LBA, CSBMP2 , and PCLPMSCs groups when compared with the CTL group. At 10weeks, GFP positive cells were detected only in the LBA group and restricted to the outer cortical bone in close contact with the periosteum. Tracking of cellular implants demonstrated significant survival of the PMSCs when compared with BMSCs. In conclusion, PMSCs improve bone regeneration being suitable for mimetic autograft design.

Paying the Toll for Inflammation

Paying the Toll for Inflammation

Spontaneous In Vivo Chondrogenesis of Bone Marrow-Derived Mesenchymal Progenitor Cells by Blocking Vascular Endothelial Growth Factor Signaling.

Chondrogenic differentiation of mesenchymal stromal/stem cells (MSCs) is typically targeted by morphogen delivery, which is often associated with limited efficiency, stability, and robustness. This article proposes a strategy to engineer MSCs with the capacity to establish specific microenvironmental conditions, supporting their own targeted differentiation program. Sole blockade of angiogenesis mediated by transduction for sFlk-1, without delivery of additional morphogens, is sufficient for inducing MSC chondrogenic differentiation. The findings represent a relevant step forward in the field because the method allowed reducing interdonor variability in MSC differentiation efficiency and, importantly, onset of a stable, nonhypertrophic chondrocyte phenotype.

Adipose-Derived Stem Cell-Seeded Hydrogels Increase Endogenous Progenitor Cell Recruitment and Neovascularization in Wounds.

Adipose-derived mesenchymal stem cells (ASCs) are appealing for cell-based wound therapies because of their accessibility and ease of harvest, but their utility is limited by poor cell survival within the harsh wound microenvironment. In prior work, our laboratory has demonstrated that seeding ASCs within a soft pullulan-collagen hydrogel enhances ASC survival and improves wound healing. To more fully understand the mechanism of this therapy, we examined whether ASC-seeded hydrogels were able to modulate the recruitment and/or functionality of endogenous progenitor cells. Employing a parabiosis model and fluorescence-activated cell sorting analysis, we demonstrate that application of ASC-seeded hydrogels to wounds, when compared with injected ASCs or a noncell control, increased the recruitment of provascular circulating bone marrow-derived mesenchymal progenitor cells (BM-MPCs). BM-MPCs comprised 23.0% of recruited circulating progenitor cells in wounds treated with ASC-seeded hydrogels versus 8.4% and 2.1% in those treated with controls, p < 0.05. Exploring the potential for functional modulation of BM-MPCs, we demonstrate a statistically significant increase in BM-MPC migration, proliferation, and tubulization when exposed to hydrogel-seeded ASC-conditioned medium versus control ASC-conditioned medium (73.8% vs. 51.4% scratch assay closure; 9.1% vs. 1.4% proliferation rate; 10.2 vs. 5.5 tubules/HPF; p < 0.05 for all assays). BM-MPC expression of genes related to cell stemness and angiogenesis was also significantly increased following exposure to hydrogel-seeded ASC-conditioned medium (p < 0.05). These data suggest that ASC-seeded hydrogels improve both progenitor cell recruitment and functionality to effect greater neovascularization.

Circulating fibrocytes correlate with the asthma control test score

BackgroundBronchial asthma is characterised by airway inflammation and remodelling with a decline of lung function. Fibrocytes are bone marrow-derived mesenchymal progenitor cells that play important roles in the pathogenesis of airway remodelling. Several clinical parameters are currently being used in routine clinical practice to assess outcome of therapy in asthma including frequency of rescue with short-acting β2-agonist and the asthma control test. In this study, we hypothesised that asthma control test is associated with circulating levels of fibrocytes in bronchial asthma. MethodsThere were 20 patients with asthma and seven healthy controls. The number of CD45+Collagen I+ circulating fibrocytes was assessed in the peripheral blood by flow cytometry. ResultsThe number of circulating fibrocytes was significantly increased in asthma patients with moderate and severe disease compared to controls, and it was inversely correlated with % forced expiratory volume in one second and % forced vital capacity (%FVC). The frequency of inhalation of short-acting β2 agonist and the asthma control test score was significantly and inversely correlated with the number of circulating fibrocytes. ConclusionThe results of this study showed that the number of circulating fibrocytes is inversely correlated with clinical asthma control parameters, further supporting the relevance of measuring circulating fibrocytes as a marker of clinical control in bronchial asthma.

Scaffold-cell bone engineering in a validated preclinical animal model: precursors vs differentiated cell source.

The properties of osteoblasts (OBs) isolated from the axial skeleton (tOBs) differ from OBs of the orofacial skeleton (mOBs) due to the different embryological origins of the bones. The aim of the study was to assess and compare the regenerative potential of allogenic bone marrow-derived mesenchymal progenitor cells with allogenic tOBs and allogenic mOBs in combination with a mPCL-TCP scaffold in critical-sized segmental bone defects in sheep tibiae. After 6 months, the tibiae were explanted and underwent biomechanical testing, micro-computed tomography (microCT) and histological and immunohistochemical analyses. Allogenic MPCs demonstrated a trend towards a better outcome in biomechanical testing and the mean values of newly formed bone. Biomechanical, microCT and histological analysis showed no significant differences in the bone regeneration potential of tOBs and mOBs in our in vitro study, as well as in the bone regeneration potential of different cell types in vivo. Copyright © 2015 John Wiley & Sons, Ltd.

MICRORNA-301A TARGETS DICER TO ATTENTUATE PRIMARY C-KIT+ HUMAN ATRIAL CELL DIFFERENTIATION

Cardiac fibroblasts (CFs) and bone marrow-derived mesenchymal progenitor cells (BM-MPCs) both contribute to cardiac fibrosis by differentiating into pro-fibrotic cell types. These pro-fibrotic cells can exacerbate fibrosis by producing excess extracellular matrix proteins that cause the scar to infiltrate viable cardiac muscle that can lead to congestive heart failure. We have previously identified microRNAs (miR) as key regulators of differentiation. These 22-25 nucleotide long non-coding segments of RNA target multiple mRNA transcripts simultaneously to prevent translation of their respective proteins. We found that miR-301a in particular is influential in attenuating the differentiation of both primary CFs isolated from atrial tissue, and primary BM-MPCs, both isolated from patients undergoing open heart surgery. One of the potential targets of miR-301a is Dicer, a protein responsible for processing miRNA and facilitating its inhibitory action. We sought to determine if miR-301a had a similar effect on the recently discovered c-kit+ adult cardiac stem cells (CSCs). C-kit+ adult cardiac stem cells (CSCs) were isolated from atrial appendages isolated from patients undergoing open heart surgery. Atrial tissue was digested using collagenase in SMEM media, and then cells were isolated and co-incubated for 2 hrs at 37°C with Dynabeads coated with c-kit antibody. These magnetic beads were washed to remove c-kit- cells, and then remaining c-kit+ cells were plated. At 70% confluency, cells were transfected with miR-301a or scrambled negative control, and then protein and RNA was collected 48hrs later. We performed qRT-PCR and Western blot analysis to investigate Dicer expression and expression of pro-fibrotic markers. miR-301a transfection of c-kit+ CSCs significantly reduced expression of both Dicer1a and Dicer 1b. In addition, miR-301a over-expression decreased the mRNA level of myosin heavy chain 9, and in the protein levels of non-muscle myosin IIA and alpha-smooth muscle actin. These results provide insight into a potential cellular mechanism that influences the differentiation of AFs, BM-MPCs, and c-kit+ CSCs which could be caused by changes in Dicer, the key protein in activating micro-RNAs.

Mouse aorta-derived mesenchymal progenitor cells contribute to and enhance the immune response of macrophage cells under inflammatory conditions.

IntroductionMesenchymal progenitor cells interact with immune cells and modulate inflammatory responses. The cellular characteristics required for this modulation are under fervent investigation. Upon interaction with macrophage cells, they can contribute to or suppress an inflammatory response. Current studies have focused on mesenchymal progenitors derived from bone marrow, adipose, and placenta. However, the arterial wall contains many mesenchymal progenitor cells, which during vascular disease progression have the potential to interact with macrophage cells. To examine the consequence of vascular-tissue progenitor cell-macrophage cell interactions in an inflammatory environment, we used a recently established mesenchymal progenitor cell line derived from the mouse aorta.MethodsMouse bone marrow-derived macrophage (MΦ) cells and mouse aorta-derived mesenchymal progenitor (mAo) cells were cultured alone or co-cultured directly and indirectly. Cells were treated with oxidized low-density lipoprotein (ox-LDL) or exposed to the inflammatory mediators lipopolysaccharide (LPS) and interferon-gamma (IFNγ) or both. A Toll-like receptor-4 (TLR4)-deficient macrophage cell line was used to determine the role of the mAo cells. To monitor inflammation, nitric oxide (NO), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNFα) secretions were measured.ResultsMesenchymal progenitor cells isolated from aorta and cloned by high proliferative capacity (mAo) can differentiate into multiple mesenchymal lineages and are positive for several commonly used mouse mesenchymal stem cell markers (that is, CD29, CD44, CD105, CD106, and Sca-1) but are negative for CD73 and ecto-5′-nucleotidase. In co-culture with MΦ cells, they increase MΦ oxidized-LDL uptake by 52.2%. In an inflammatory environment, they synergistically and additively contribute to local production of both NO and IL-6. After exposure to ox-LDL, the inflammatory response of MΦ cells to LPS and LPS/IFNγ is muted. However, when lipid-laden MΦ cells are co-cultured with mAo cell progenitors, the muted response is recovered and the contribution by the mAo cell progenitor is dependent upon cell contact.ConclusionsThe resident mesenchymal progenitor cell is a potential contributor to vascular inflammation when in contact with inflamed and lipid-laden MΦ cells. This interaction represents an additional target in vascular disease treatment. The potential for resident cells to contribute to the local immune response should be considered when designing therapeutics targeting inflammatory vascular disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0071-8) contains supplementary material, which is available to authorized users.

Membrane-type 1 matrix metalloproteinase controls osteo- and chondrogenesis by a proteolysis-independent mechanism mediated by its cytoplasmic tail

s / Osteoarthritis and Cartilage 23 (2015) A26eA81 A64 deregulated genes included Has1, ADAMTS4, Tnf, IL6, IL18, Inhba, Cd68, Ngf, Ccr2, Wnt 16, Tnfaip6 and Il1r. Conclusions: Deletion of JNK2 retarded the development of experimental murine OA, showing the importance of the kinase for cartilage homeostasis in vivo and implicating an intracellular signalling pathway in OA for the first time. 68 MEMBRANE-TYPE 1 MATRIX METALLOPROTEINASE CONTROLS OSTEOAND CHONDROGENESIS BY A PROTEOLYSIS-INDEPENDENT MECHANISM MEDIATED BY ITS CYTOPLASMIC TAIL Q. Yang y, M. Attur y, T. Kirsch z, Y.J. Lee z, S. Yakar x, Z. Liu x, S.B. Abramson y, P. Mignatti yk. yNYU Sch. of Med. Dept. of Med., New York, NY, USA; zNYU Sch. of Med. Dept. of Orthopedic Surgery, New York, NY, USA; xNYU Coll. of Dentistry Dept. of Basic Sci. and Craniofacial Biology, New York, NY, USA; kNYU Sch. of Med. Dept. of Cell Biology, New York, NY, USA Purpose: We aimed to understand the mechanism by which membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14) controls bone and cartilage homeostasis. MT1-MMP, a cell-membrane-bound proteinase with an extracellular catalytic site and a 20-amino acid cytoplasmic tail, degrades the extracellular matrix and plays a key role in postnatal bone formation. MT1-MMP is overexpressed in osteoarthritis, and mutation of MT1-MMP in humans causes the multicentric osteolysis and arthritis disease, Winchester syndrome. The genetic deficiency of MT1-MMP (MT1-MMP-/-) in the mouse causes dwarfism, osteopenia and severe arthritis. Conditional deletion of MT1-MMP in bone marrow-derived mesenchymal progenitor cells (BM-MSC) fully recapitulates this phenotype, showing that MT1-MMP controls osteogenic differentiation inMSC. It has been proposed that the phenotype of MT1-MMP-/mice results from the lack of MT1-MMP proteolytic activity. However, mounting evidence shows a variety of proteolysisindependent functions of MT1-MMP. The unique tyrosine (Y573) in the MT1-MMP cytoplasmic tail is fundamental for the control of intracellular signaling. We have shown that Y573 mediates Ras-ERK1/2 signaling, and that Y573 mutation (Y573D) blocks this functionwithout affecting MT1-MMP proteolytic activity. Methods: We generated a mouse with the Y573D mutation in MT1MMP (MT1-MMP Y573D) and characterized its skeletal phenotype by histological and microCT analyses. We isolated BM-MSC and induced them to differentiate into osteoblasts, chondrocytes and adipocytes, using qRT-PCR to analyze gene expression. Mouse C3H10T1/2MSCwere transfected with MT1-MMP cDNA and analyzed for Wnt signaling and Runx2 activation by luciferase reporter assays. Results: Analysis of the long bones of both homozygote and heterozygote MT1-MMP Y573D mice showed increased trabecular bone relative to wt littermates (BV/TV %; wt: 10 4±8; homo: 23.2±2.0; Tb.Th (mm) wt: 20.5±3.8; homo: 40.1±7.0; Tb.N./mm wt: 10.5±4.2; homo: 19.5 4.2; Tb.Sp (mm): 325.2±52.1; 190.6 49.1; p 0.01), a phenotypeopposite to that of MT1-MMP-/mice. MT1-MMP Y573D mice also showed marked thinning of articular cartilage, with disorganized tissue architecture, clustering and cloning of chondrocytes, as well as pronounced decrease in bone marrow-associated and total body fat (fat weight/body weight wt: 0.270±0.0105; homo: 0.0135±0.006; p1⁄40.0029). These findings indicated a defect in MSC differentiation. Therefore, we isolated BM-MSC from wt and homozygous mutant littermates, and induced them to differentiate into osteoblast and chondrocytes, and myeloid precursors were induced to differentiate into osteoclasts. The Y573D mutation dramatically increased MSC expression of osteoblast markers (ALP, COL1, BSP, OCN, RUNX2) and strongly downregulated chondrocyte (ADA4, ADA5, SOX9, MMP13) and osteoclast markers (TRACP, CALCR, CTSK, DC-STAMP, MMP9, iNOS). These findings indicated thatWnt signaling is upregulated inMT1MMP Y573D-expressing MSC. The canonical Wnt pathway is indeed unique, as it exerts opposite effects on osteoblast and chondrocyte differentiation fromMSC. Therefore, we analyzedWnt signaling and Runx2 activation. We transiently transfected C3H10T1/2 MSC cells in osteoblast medium with the cDNAs for wt MT1-MMP and MT1-MMP Y573D. As controls the cellswere transfectedwith the empty vector (pcDNA) orwith MT1-MMP E240A, a mutant devoid of proteolytic activity. MT1-MMP Y573D dramatically upregulated both Wnt signaling (2.8-4.6-fold; p1⁄40.007) and Runx2 activity (8.7-46.5-fold; 1⁄40.009) relative to wt MT1MMP and MT1-MMP E240A. Conclusions: MT1-MMP controls Wnt signaling by a mechanism independent of extracellular proteolysis and mediated by its cytoplasmic tail. MT1-MMP is a bifunctional protein, with an extracellular proteolytic activity that promotes bone formation through ECM remodeling and a cytoplasmic tail that controls osteogenesis by interacting with a key pro-osteogenic signaling pathway. 69 A DUAL ROLE FOR NOTCH SIGNALING IN JOINT CARTILAGE MAINTENANCE AND OSTEOARTHRITIS Z. Liu y, J. Chen z, A.J. Mirando z, C. Wang y, M.J. Zuscik y, R.J. O'Keefe x, M.J. Hilton z. yUniv. of Rochester Med. Ctr., Rochester, NY, USA; zDuke Univ. Sch. of Med., Durham, NC, USA; xWashington Univ. Sch. of Med.,

Abstract 30: Modulating the angiogenic potential of human microvascular endothelial cells by an endogenous zinc finger transcription factor, ZNF24

Abstract Angiogenesis, the formation of new blood vessels from preexisting ones, is an indispensible process under normal physiological conditions such as embryonic development, certain stages of the female reproductive cycle and placenta formation during pregnancy as well as under pathological conditions such as wound healing, diabetic retinopathy, and cancer initiation, progression and metastasis. We have previously identified the molecular mechanism by which a zinc finger transcription factor, ZNF24, functions as a new inhibitor of tumor angiogenesis. ZNF24 represses the transcription of vascular endothelial growth factor (VEGF) in breast cancer cells via direct binding to an 11-bp fragment within the VEGF proximal promoter. In the current study, we have focused on the role of ZNF24 in modulating the angiogenic potential of the endothelial compartment of the tumor microenvironment. Utilizing primary human microvascular endothelial cells, we have found that silencing ZNF24 in these cells leads to significantly decreased endothelial cell proliferation. Quantitative real-time PCR array analyses have identified multiple cell cycle regulators as potential downstream targets of ZNF24, which may be responsible for the decreased proliferation in these cells. Moreover, knockdown of ZNF24 in microvascular endothelial cells leads to significantly decreased cell migration and invasion. Consistently, we found that VEGFR2 signaling and the proteolytic activity of matrix metalloproteinase-2 (MMP2) were significantly downregulated in these cells. We also determined the effect of ZNF24 silencing on the levels of major regulators of MMP2 proteolytic activity such as the tissue inhibitors of metalloproteinases (TIMPs) and other MMPs. Knockdown of ZNF24 does not induce significant changes in levels of these TIMPs and MMPs, suggesting that ZNF24 regulates MMP2 at the transcriptional level. Indeed, the mRNA level of MMP2 is significantly decreased in cells transfected with ZNF24 siRNA, indicating that ZNF24 may be potentiating endothelial cell invasion by positively regulating MMP2 transcription. In a pilot in vivo angiogenesis assay, knockdown of ZNF24 in microvascular endothelial cells leads to the significantly decreased formation of vascular networks when co-implanted with bone marrow-derived mesenchymal progenitor cells in immunodeficient mice. Taken together, these results demonstrate that ZNF24 may be playing an essential role in modulating the angiogenic potential of microvascular endothelial cells by regulating the proliferation and motility of these cells. (Supported by NIH P01 CA045548 and the Breast Cancer Research Foundation) Citation Format: Di Jia, Lan Huang, Joyce Bischoff, Marsha Moses. Modulating the angiogenic potential of human microvascular endothelial cells by an endogenous zinc finger transcription factor, ZNF24. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 30. doi:10.1158/1538-7445.AM2014-30

The Developing Role of Neuregulin1 In Cardiac Regenerative Stem Cell Therapy

Myocardial infarction, heart failure, and chronic ischemic heart disease account for the majority of the cardiovascular burden. The current treatment strategies focus on limiting the progression of disease and preserving cardiac myocardium. The goal of stem cell therapy, on the other hand, is to reverse or replace damaged cardiac tissue. Over the past two decades many studies have been conducted to understand stem cell performance, survival, and the potential for cardiac repair. Neuregulin1, an epidermal growth factor family member, promotes embryonic stem cell differentiation into the cardiac lineage and improves survival in bone marrow-derived mesenchymal stem cell and embryonic endothelial progenitor cells. Current clinical trials are actively pursuing Neuregulin1's therapeutic potential in the areas of heart failure and cardiac ischemia. It is the intent of this paper to review the current knowledge of Neuregulin1 in stem cell biology and discuss the potential of using Neuregulin1 to improve stem cell therapy for cardiac repair.

Diabetes irreversibly depletes bone marrow-derived mesenchymal progenitor cell subpopulations.

Diabetic vascular pathology is largely attributable to impairments in tissue recovery from hypoxia. Circulating progenitor cells have been postulated to play a role in ischemic recovery, and deficiencies in these cells have been well described in diabetic patients. Here, we examine bone marrow–derived mesenchymal progenitor cells (BM-MPCs) that have previously been shown to be important for new blood vessel formation and demonstrate significant deficits in the context of diabetes. Further, we determine that this dysfunction is attributable to intrinsic defects in diabetic BM-MPCs that are not correctable by restoring glucose homeostasis. We identify two transcriptionally distinct subpopulations that are selectively depleted by both type 1 and type 2 diabetes, and these subpopulations have provasculogenic expression profiles, suggesting that they are vascular progenitor cells. These results suggest that the clinically observed deficits in progenitor cells may be attributable to selective and irreversible depletion of progenitor cell subsets in patients with diabetes.

Propranolol inhibits growth of hemangioma-initiating cells but does not induce apoptosis

BackgroundInfantile hemangioma (IH) is the most common tumor of infancy. The first-line therapy for IH is propranolol, a non-selective β-adrenergic receptor antagonist. However, mechanisms for the therapeutic effect of propranolol and regrowth of IH following cessation of treatment in some cases are not clear. We have recently shown that IH arises from multipotent stem cells. Whether IH stem cells are responsive to propranolol and are selectively targeted is unknown, and is the focus of this study.MethodsIH stem cells were exposed to propranolol and assayed for cellular and molecular alterations. We used endothelial cells (ECs) as controls and bone marrow-mesenchymal progenitor cells (bm-MPCs) as normal stem/progenitor counterparts to determine selectivity.ResultsOur results show that propranolol significantly reduced IH stem cell growth but failed to induce caspase-3 activation. Normal bm-MPCs and mature ECs showed maintained or increased caspase-3 activation and significantly reduced cyclin-D1 levels. We further show that IH stem cells may escape apoptosis by inducing anti-apoptotic pathways.ConclusionsThis study reveals that propranolol does not induce apoptosis in IH stem cells, which is in contrast to ECs. Escape from apoptosis in IH stem cells may involve induction of anti-apoptotic pathways.

Fibrocytes in health and disease

Fibrocytes, a group of bone marrow-derived mesenchymal progenitor cells, were first described in 1994 as fibroblast-like, peripheral blood cells that migrate to regions of tissue injury. These cells are unique in their expression of extracellular matrix proteins concomitantly with markers of hematopoietic and monocyte lineage. The involvement of fibrocytes and the specific role they play in the process of wound repair has been a focus of study since their initial description. Fibrocytes contribute to the healing repertoire via several mechanisms; they produce a combination of cytokines, chemokines, and growth factors to create a milieu favorable for repair to occur; they serve as antigen presenting cells (APCs); they contribute to wound closure; and, they promote angiogenesis. Furthermore, regulatory pathways involving serum amyloid P, leukocyte-specific protein 1, and adenosine A2A receptors have emphasized the significant role that fibrocytes have in wound healing and fibrosis. The therapeutic targeting of fibrocytes holds promise for the augmentation of wound repair and the treatment of different fibrosing disorders.

Correlation between circulating fibrocytes, and activity and progression of interstitial lung diseases

Interstitial lung diseases (ILD) are characterized by progressive interstitial pulmonary fibrosis and a decline in lung function. Fibrocytes are bone marrow-derived mesenchymal progenitor cells that may play a role in the pathogenesis of pulmonary fibrosis. Circulating fibrocyte numbers have been correlated with the prognosis of patients with idiopathic pulmonary fibrosis. The aim of the present study was to evaluate the relationship between circulating fibrocytes, and parameters of disease activity and progression in several groups of patients with ILD. The study population comprised 41 patients with ILD and seven healthy control subjects. Circulating CD45(+) collagen-I(+) fibrocytes were evaluated by flow cytometry. The number of circulating fibrocytes was significantly increased in all patients with ILD and particularly in patients with idiopathic interstitial pneumonitis and interstitial pneumonitis associated with collagen vascular disease as compared with healthy control subjects. The numbers of circulating fibrocytes were significantly correlated with pulmonary function test parameters and with serum levels of sialylated carbohydrate antigen, a marker of disease activity. Temporal changes in circulating fibrocyte numbers were evaluated in two patients, and the results suggested that these changes correlated with the activity of ILD. The results from this study provide further evidence for the role of circulating fibrocytes in fibrotic lung diseases.

Differentiation potential of mesenchymal progenitor cells following transplantation into calvarial defects

The complexity of stem cell lineage commitment requires studies to investigate the intrinsic and extrinsic regulatory events during differentiation. The objective of this long-term in vivo study was to investigate cellular differentiation and tissue formation of transplanted undifferentiated bone-marrow-derived mesenchymal progenitor cells (BMPCs) in combination with a medical grade polycaprolactone (mPCL) scaffold and to compare them to osteoblasts; a more differentiated cell type in a calvarial defect model.Tissue formation was assessed via histology, mechanical and radiological methods after 3 12, and 24 months. After 3 months our results indicated that transplanted mesenchymal progenitor cells were influenced by the niche of the host environment. Scaffold/BMPCs formed islands of bone tissue inside the defect area. However when the surrounding host calvarium contained a high content of fatty tissue, the fat content in the defect areas was also significantly higher. In contrast, defects repaired with scaffold/cOBs did not show this phenomenon. Analysis after 12 and 24 months confirmed these observations indicating that a predominantly fatty environment leads to adipogenic development in the progenitor group. Biomechanical data revealed that the tissue was less firm in the BMPC group compared to the cOB seeded group. Evaluation of cell plasticity in vivo has important consequences in clinical cell transplantation protocols. This study indicates that cell fate decisions are partially regulated by extrinsic control mechanisms of the immediate environment suggesting that induction of BMPCs into a specific lineage could be beneficial prior transplantation.

Surfactant Protein-D Regulates Effector Cell Function and Fibrotic Lung Remodeling in Response to Bleomycin Injury

Surfactant protein (SP)-D and SP-A have been implicated in immunomodulation in the lung. It has been reported that patients with idiopathic pulmonary fibrosis (IPF) often have elevated serum levels of SP-A and SP-D, although their role in the disease is not known. The goal of this study was to test the hypothesis that SP-D plays an important role in lung fibrosis using a mouse model of fibrosis induced by bleomycin (BLM). Triple transgenic inducible SP-D mice (iSP-D mice), in which rat SP-D is expressed in response to doxycycline (Dox) treatment, were administered BLM (100 U/kg) or saline subcutaneously using miniosmotic pumps. BLM-treated iSP-D mice off Dox (SP-D off) had increased lung fibrosis compared with mice on Dox (SP-D on). SP-D deficiency also increased macrophage-dominant cell infiltration and the expression of profibrotic cytokines (transforming growth factor [TGF]-β1, platelet-derived growth factor-AA). Alveolar macrophages isolated from BLM-treated iSP-D mice off Dox (SP-D off) secreted more TGF-β1. Fibrocytes, which are bone marrow-derived mesenchymal progenitor cells, were increased to a greater extent in the lungs of the BLM-treated iSP-D mice off Dox (SP-D off). Fibrocytes isolated from BLM-treated iSP-D mice off Dox (SP-D off) expressed more of the profibrotic cytokine TGF-β1 and more CXCR4, a chemokine receptor that is important in fibrocyte migration into the lungs. Exogenous SP-D administered intratracheally attenuated BLM-induced lung fibrosis in SP-D(-/-) mice. These data suggest that alveolar SP-D regulates numbers of macrophages and fibrocytes in the lungs, profibrotic cytokine expression, and fibrotic lung remodeling in response to BLM injury.