Inhibition of galectin-3 post-infarction impedes progressive fibrosis by regulating inflammatory profibrotic cascades.

AIMS: Acute myocardial infarction (MI) causes inflammation, collagen deposition, and reparative fibrosis in response to myocyte death and, subsequently, a pathological myocardial remodelling process characterized by excessive interstitial fibrosis, driving heart failure (HF). Nonetheless, how or when to limit excessive fibrosis for therapeutic purposes remains uncertain. Galectin-3, a major mediator of organ fibrosis, promotes cardiac fibrosis and remodelling. We performed a preclinical assessment of a protein inhibitor of galectin-3 (its C-terminal domain, Gal-3C) to limit excessive fibrosis resulting from MI and prevent ventricular enlargement and HF. METHODS AND RESULTS: Gal-3C was produced by enzymatic cleavage of full-length galectin-3 or by direct expression of the truncated form in Escherichia coli. Gal-3C was intravenously administered for 7 days in acute MI models of young and aged rats, starting either pre-MI or 4 days post-MI. Echocardiography, haemodynamics, histology, and molecular and cellular analyses were performed to assess post-MI cardiac functionality and pathological fibrotic progression. Gal-3C profoundly benefitted left ventricular ejection fraction, end-systolic and end-diastolic volumes, haemodynamic parameters, infarct scar size, and interstitial fibrosis, with better therapeutic efficacy than losartan and spironolactone monotherapies over the 56-day study. Gal-3C therapy in post-MI aged rats substantially improved pump function and attenuated ventricular dilation, preventing progressive HF. Gal-3C in vitro treatment of M2-polarized macrophage-like cells reduced their M2-phenotypic expression of arginase-1 and interleukin-10. Gal-3C inhibited M2 polarization of cardiac macrophages during reparative response post-MI. Gal-3C impeded progressive fibrosis post-MI by down-regulating galectin-3-mediated profibrotic signalling cascades including a reduction in endogenous arginase-1 and inducible nitric oxide synthase (iNOS). CONCLUSION: Gal-3C treatment improved long-term cardiac function post-MI by reduction in the wound-healing response, and inhibition of inflammatory fibrogenic signalling to avert an augmentation of fibrosis in the periinfarct region. Thus, Gal-3C treatment prevented the infarcted heart from extensive fibrosis that accelerates the development of HF, providing a potential targeted therapy.

Methods and Strategies for Procurement, Isolation, Characterization, and Assessment of Senescence of Human Mesenchymal Stem Cells from Adipose Tissue.

Human adipose-derived mesenchymal stem (stromal) cells (hADSC) represent an attractive source of the cells for numerous therapeutic applications in regenerative medicine. These cells are also an efficient model to study biological pathways of stem cell action, tissue injury and disease. Like any other primary somatic cells in culture, industrial-scale expansion of mesenchymal stromal cells (MSC) leads to the replicative exhaustion/senescence as defined by the "Hayflick limit." The senescence is not only greatly effecting in vivo potency of the stem cell cultures but also might be the cause and the source of clinical inconsistency arising from infused cell preparations. In this light, the characterization of hADSC replicative and stressor-induced senescence phenotypes is of great interest.This chapter summarizes some of the essential protocols and assays used at our laboratories and clinic for the human fat procurement, isolation, culture, differentiation, and characterization of mesenchymal stem cells from adipose tissue and the stromal vascular fraction. Additionally, we provide manuals for characterization of hADSC senescence in a culture based on stem cells immunophenotype, proliferation rate, migration potential, and numerous other well-accepted markers of cellular senescence. Such methodological framework will be immensely helpful to design standards and surrogate measures for hADSC-based therapeutic applications.

Isolation of human testicular cells and co-culture with embryonic stem cells.

Our overall goal is to create a three-dimensional human cell-based testicular model for toxicological and spermatogenesis studies. Methods to purify the major somatic testicular cells, namely Leydig cells (LCs), peritubular myoid cells (PCs) and Sertoli cells (SCs), from rats, mice and guinea pigs have been reported. In humans, the isolation of populations enriched for primary LCs, PCs or SCs also have described. One objective of this study was to determine if populations of cells enriched for all three of these cell types can be isolated from testes of single human donors, and we were successful in doing so from testes of three donors. Testes tissues were enzymatically digested, gravity sedimented and Percoll filtered to isolate populations enriched for LCs, PCs and SCs. LCs and PCs were identified by colorimetric detection of the expression of prototypical enzymes. Division of PCs and SCs in culture has been reported. We observed that primary human LCs could divide in culture by incorporation of 5-ethynyl-2'-deoxyuridine. SCs were identified and their functionality was demonstrated by the formation of tight junctions as shown by the expression of tight junction proteins, increased transepithelial electrical resistance, polarized secretion of biomolecules and inhibition of lucifer yellow penetration. Furthermore, we found that human SC feeder layers could facilitate germ cell progression of human embryonic stem cells (hESCs) by microarray analysis of gene expression.

Opposing activities of oncogenic MIR17HG and tumor suppressive MIR100HG clusters and their gene targets regulate replicative senescence in human adult stem cells.

Growing evidence suggests that many diseases of aging, including diseases associated with robust changes and adipose deports, may be caused by resident adult stem cell exhaustion due to the process called cellular senescence. Understanding how microRNA pathways can regulate cellular senescence is crucial for the development of novel diagnostic and therapeutic strategies to combat these pathologies. Herein, using integrated transcriptomic and semi-quantitative proteomic analysis, we provide a system level view of the regulation of human adipose-derived stem cell senescence by a subset of mature microRNAs (termed senescence-associated-microRNAs) produced by biogenesis of oncogenic MIR17HG and tumor-suppressive MIR100HG clusters. We demonstrate functional significance of these mature senescence-associated-microRNAs in the process of replicative senescence of human adipose-derived stem cells ex-vivo and define a set of senescence-associated-microRNA gene targets that are able to elicit, modulate and, most importantly, balance intimate connections between oncogenic and senescent events.

Mesenchymal Stem Cells from Adipose Tissue in Clinical Applications for Dermatological Indications and Skin Aging.

Operating at multiple levels of control, mesenchymal stem cells from adipose tissue (ADSCs) communicate with organ systems to adjust immune response, provide signals for differentiation, migration, enzymatic reactions, and to equilibrate the regenerative demands of balanced tissue homeostasis. The identification of the mechanisms by which ADSCs accomplish these functions for dermatological rejuvenation and wound healing has great potential to identify novel targets for the treatment of disorders and combat aging. Herein, we review new insights into the role of adipose-derived stem cells in the maintenance of dermal and epidermal homeostasis, and recent advances in clinical applications of ADSCs related to dermatology.

Mesenchymal Stem Cells Secretory Responses: Senescence Messaging Secretome and Immunomodulation Perspective.

Mesenchymal stem/stromal cells (MSC) have been tested in a significant number of clinical trials, where they exhibit regenerative and repair properties directly through their differentiation into the cells of the mesenchymal origin or by modulation of the tissue/organ microenvironment. Despite various clinical effects upon transplantation, the functional properties of these cells in natural settings and their role in tissue regeneration in vivo is not yet fully understood. The omnipresence of MSC throughout vascularized organs equates to a reservoir of potentially therapeutic regenerative depots throughout the body. However, these reservoirs could be subjected to cellular senescence. In this review, we will discuss current progress and challenges in the understanding of different biological pathways leading to senescence. We set out to highlight the seemingly paradoxical property of cellular senescence: its beneficial role in the development and tissue repair and detrimental impact of this process on tissue homeostasis in aging and disease. Taking into account the lessons from the different cell systems, this review elucidates how autocrine and paracrine properties of senescent MSC might impose an additional layer of complexity on the regulation of the immune system in development and disease. New findings that have emerged in the last few years could shed light on sometimes seemingly controversial results obtained from MSC therapeutic applications.

Treatment with hESC-Derived Myocardial Precursors Improves Cardiac Function after a Myocardial Infarction.

BACKGROUND: We previously reported the generation of a reporter line of human embryonic stem cells (hESCs) with enhanced green fluorescent protein (eGFP) expression driven by the α-myosin heavy chain (αMHC) promoter. The GFP+/αMHC+ cells derived from this cell line behave as multipotent, human myocardial precursors (hMPs) in vitro. In this study, we evaluated the therapeutic effects of GFP+/αMHC+ cells isolated from the reporter line in a mouse model of myocardial infarction (MI). METHODS: MI was generated in immunodeficient mice. hMPs were injected into murine infarcted hearts under ultrasound guidance at 3 days post-MI. Human fetal skin fibroblasts (hFFs) were injected as control. Cardiac function was evaluated by echocardiography. Infarct size, angiogenesis, apoptosis, cell fate, and teratoma formation were analyzed by immunohistochemical staining. RESULTS: Compared with control, hMPs resulted in improvement of cardiac function post-MI with smaller infarct size, induced endogenous angiogenesis, and reduced apoptosis of host cardiomyocytes at the peri-infarct zone at 28 days post-MI. CONCLUSION: Intramyocardial injection of hMPs improved cardiac function post-MI. The engraftment rate of these cells in the myocardium post-MI was low, suggesting that the majority of effect occurs via paracrine mechanisms.

Myocardial improvement with human embryonic stem cell-derived cardiomyocytes enriched by p38MAPK inhibition.

BACKGROUND AIMS: We have shown previously that inhibition of the p38 mitogen-activated protein kinase (p38MAPK) directs the differentiation of human embryonic stem cell (hESC)-derived cardiomyocytes (hCM). We investigated the therapeutic benefits of intramyocardial injection of hCM differentiated from hESC by p38MAPK inhibition using closed-chest ultrasound-guided injection at a clinically relevant time post-myocardial infarction (MI) in a mouse model. METHODS: MI was induced in mice and the animals treated at day 3 with: (a) hCM, (b) human fetal fibroblasts (hFF) as cell control, or (c) medium control (n = 10 animals/group). Left ventricular ejection fraction (LVEF) was evaluated post-MI prior to therapy, and at days 28 and 60 post-cell therapy. Hearts were analyzed at day 60 for infarct size, angiogenesis, cell fate and teratoma formation. RESULTS: LVEF was improved in the hCM-treated animals compared with both hFF and medium control-treated animals at day 28 (39.03 ± 1.79% versus 27.89 ± 1.27%, P < 0.05, versus 32.90 ± 1.46%, P < 0.05, respectively), with sustained benefit until day 60. hCM therapy resulted in significantly smaller scar size, increased capillary bed area, increased number of arterioles, less native cardiomyocyte (CM) apoptosis, and increased CM proliferation compared with the other two groups. These benefits were achieved despite a very low retention rate of the injected cells at day 60, as assessed by immunohistochemistry and quantitative real-time polymerase chain reaction (qPCR). Therapy with hCM did not result in intramyocardial teratoma formation at day 60. CONCLUSIONS: This study demonstrates that hCM derived from p38MAPK-treated hESC have encouraging therapeutic potential.

Timed inhibition of p38MAPK directs accelerated differentiation of human embryonic stem cells into cardiomyocytes.

BACKGROUND AIMS: Heart failure therapy with human embryonic stem cell (hESC)-derived cardiomyocytes (hCM) has been limited by the low rate of spontaneous hCM differentiation. As others have shown that p38 mitogen-activated protein kinase (p38MAPK) directs neurogenesis from mouse embryonic stem cells, we investigated whether the p38MAPK inhibitor, SB203580, might influence hCM differentiation. METHODS: We treated differentiating hESC with SB203580 at specific time-points, and used flow cytometry, immunocytochemistry, quantitative real-time (RT)-polymerase chain reaction (PCR), teratoma formation and transmission electron microscopy to evaluate cardiomyocyte formation. RESULTS: We observed that the addition of inhibitor resulted in 2.1-fold enrichment of spontaneously beating human embryoid bodies (hEB) at 21 days of differentiation, and that 25% of treated cells expressed cardiac-specific α-myosin heavy chain. This effect was dependent on the stage of differentiation at which the inhibitor was introduced. Immunostaining and teratoma formation assays demonstrated that the inhibitor did not affect hESC pluripotency; however, treated hESC gave rise to hCM exhibiting increased expression of sarcomeric proteins, including cardiac troponin T, myosin light chain and α-myosin heavy chain. This was consistent with significantly increased numbers of myofibrillar bundles and the appearance of nascent Z-bodies at earlier time-points in treated hCM. Treated hEB also demonstrated a normal karyotype by array comparative genomic hybridization and viability in vivo following injection into mouse myocardium. CONCLUSIONS: These studies demonstrate that p38MAPK inhibition accelerates directed hCM differentiation from hESC, and that this effect is developmental stage-specific. The use of this inhibitor should improve our ability to generate hESC-derived hCM for cell-based therapy.

A method for single-cell sorting and expansion of genetically modified human embryonic stem cells.

Genetic modification of human embryonic stem (hES) cells is essential for studies of gene function and differentiation. The expression of transgenes may direct tissue-specific differentiation and aid in the identification of various differentiated cell types. Stable genomic integration of transgenes is optimal because hES cell differentiation can span several days to weeks and include numerous cell divisions, and establishing homogeneous modified cell lines will facilitate research studies. Herein we provide a method for producing and expanding hES cell lines from single cells that have been isolated by fluorescence-activated cell sorting (FACS) following genetic modification by lentivirus vectors. Using this method, we have established enhanced green fluorescent protein (eGFP)-expressing hES cell lines that are pluripotent, contain a diploid chromosomal content, and stably express eGFP following more than 2 months of routine culture and in vivo differentiation.

Caspase-12: a developmental link between G-protein-coupled receptors and integrin alphaIIbbeta3 activation.

Fibrinogen binding by integrin alphaIIbbeta3 is promoted by platelet agonists that increase the affinity and avidity of alphaIIbbeta3 for fibrinogen through a process called "inside-out" signaling. Having previously demonstrated that inside-out activation of alphaIIbbeta3 is defective in murine megakaryocytes that lack the transcription factor NF-E2, we screened for NF-E2-regulated genes that affect alphaIIbbeta3 activation. Caspase-12 is the most down-regulated gene we identified in NF-E2(-/-) megakaryocytes. Therefore, the role of this protein in alphaIIbbeta3 activation was determined using platelets from caspase-12(-/-) mice. Despite wild-type levels of alphaIIbbeta3, caspase-12(-/-) platelets exhibit reduced fibrinogen binding to alphaIIbbeta3 following stimulation by adenosine diphosphate (ADP) or protease-activated receptor 4 (PAR4) receptor-activating peptide. The defect in alphaIIbbeta3 activation is associated with decreased cytosolic free calcium and inositol triphosphate levels, and with reduced aggregation, despite wild-type phospholipase Cbeta expression levels. In contrast, agonist-induced surface expression of P-selectin, suppression of cAMP levels following ADP stimulation, and spreading on immobilized fibrinogen are unimpaired. Moreover, although caspase-12 is highly expressed in mature megakaryocytes, it is undetectable in platelets. Taken together, these studies establish that caspase-12 expression in murine megakaryocytes is regulated, directly or indirectly, by NF-E2, and suggest that caspase-12 participates in the development of fully functional signaling pathways linking some G-protein-coupled receptors to alphaIIbbeta3 activation.