Minh Duong

Marrow Stromal Cells as Universal Donor Cells for Myocardial Regenerative Therapy: Their Unique Immune Tolerance

BACKGROUND Recently rodent and porcine bone marrow stromal cells (MSCs) have been reported to be uniquely immune tolerant. To confirm these findings in human cells, we tested whether human MSCs are also immune tolerant, such that they can be used as universal donor cells for myocardial regenerative therapy. METHODS Immunocompetent female rats underwent coronary ligations (n = 90). In group I, lacZ-labeled male human MSCs were implanted into the peri-infarcted area. In groups II, III, and IV, isogeneic rat MSCs, culture medium, or human fibroblasts were injected, respectively. Echocardiography was carried out to assess cardiac function, and the specimens were examined serially for up to 8 weeks with immunohistochemistry, fluorescent in situ hybridization, and polymerase chain reaction to examine MSCs survival and differentiation. RESULTS Human MSCs survived within the rat myocardium for more than 8 weeks without immunosuppression. Furthermore, the implanted MSCs significantly contributed to the improvement in ventricular function and attenuated left ventricular remodeling. No cellular infiltration characteristic of immune rejection was noted in contrast to group IV. CONCLUSIONS Human MSCs survived within this xenogeneic environment, and contributed to the improvement in cardiac function. Our findings support the feasibility of using these cells as universal donor cells for xenogeneic or allogeneic cell therapy, as they can be prepared and stored well in advance for urgent use. Allogeneic MSCs from healthy donors may be particularly useful for severely ill or elderly patients whose own MSCs could be dysfunctional.

Hyaluronic acid-based hydrogel induces neovascularization and improves cardiac function in a rat model of myocardial infarction

OBJECTIVES The use of stem cells in cardiac regeneration is still limited due to low cellular integration and engraftment rates. Consequently, there has been a spurt in research on developing alternative regenerative therapies. Hyaluronic acid (HA) is a major component of the extracellular matrix that is non-immunogenic, and has been implicated in various wound-healing functions such as angiogenesis and inflammation modulation, making it an ideal candidate for regenerative biomaterials. In this study, we examine the potential of acellular hyaluronic acid-based hydrogel in improving cardiac function post-myocardial infarction in a rat model. METHODS Hyaluronic acid-based hydrogel was injected into the peri-infarct region post-myocardial infarction induction in Lewis rats. Cardiac function in control (n = 10) and gel-injected groups (n = 10) was evaluated up to 4 weeks post-myocardial infarction. Evaluation of cardiac function was conducted using transthoracic echocardiography. Histological analysis of scar area was evaluated via haematoxylin and eosin (H & E), and Sirius red staining. Neovascularization was detected using vascular endothelial growth factor (VEGF) staining. RESULTS Evaluation of cardiac function using transthoracic echocardiography revealed a 18.2% (P < 0.01) increase in ejection fraction in gel-injected groups when compared with the control group, almost returning the ejection fraction to baseline levels (preop). Histological analysis of scar area by haematoxylin and eosin (H&E), and Sirius red staining demonstrated decreased scarring, and a 22.6% (P < 0.01) decrease in collagen deposition in the gel-injected group compared with the control group. VEGF staining indicated a significant increase in novel vasculature formation in hydrogel-injected groups when compared with control. CONCLUSIONS Due to its regenerative potential, hyaluronic acid-based hydrogel provides a promising novel therapy to be used alone, or as a scaffold delivering a variety of drugs or cells to combat heart disease in a multifaceted approach.

Marrow stromal cells for cell-based therapy: the role of antiinflammatory cytokines in cellular cardiomyoplasty.

BACKGROUND The mechanism by which marrow stromal cells (MSCs) improve cardiac function after myocardial infarction (MI) is still unclear. Because MI patients with lower circulating proinflammatory/antiinflammatory cytokine ratios have been reported to have a better prognosis and in vitro studies showed that MSCs express antiinflammatory cytokines, we hypothesized that changes in cytokine ratios in the infarct microenvironment after MSC therapy may play a role in improving early cardiac function after MI. METHODS Sixty-three rats that survived left coronary artery ligations were injected with culture media (group M) or MSCs (group C). Cardiac functional changes were assessed with echocardiography. Cytokine gene expressions of interleukin (IL)-1beta, IL-6, IL-8, (proinflammatory) and IL-10 (antiinflammatory) were quantified by real-time polymerase chain reaction. Extracellular matrix deposition, injury score, and the matrix metallopeptidase 2/tissue inhibitor of metallopeptidase 1 ratio were also analyzed. RESULTS The ratio of proinflammatory/antiinflammatory cytokine gene expression was decreased in group C at various times, particularly in the early postoperative period. In group C, the matrix metallopeptidase 2/tissue inhibitor of metallopeptidase 1 gene expression ratio was significantly lower than group M at the early phase (12 hours), which in group C was translated into significantly lower extracellular matrix deposition at 24 hours, 1, and 2 weeks. Functional recovery was also significantly better in cell therapy group C. CONCLUSIONS Our data demonstrate that MSC therapy decreases the proinflammatory/antiinflammatory cytokine ratio in the microenvironment early after MI. This is associated with subsequent less scar formation and improved cardiac function.

The fat and the bad: Mature adipocytes, key actors in tumor progression and resistance

Growing evidence has raised the important roles of adipocytes as an active player in the tumor microenvironment. In many tumors adipocytes are in close contact with cancer cells. They secrete various factors that can mediate local and systemic effects. The adipocyte-cancer cell crosstalk leads to phenotypical and functional changes of both cell types, which can further enhance tumor progression. Moreover, obesity, which is associated with an increase in adipose mass and an alteration of adipose tissue, has been established as a risk factor for cancer incidence and cancer-related mortality. In this review, we summarize the mechanisms of the adipocyte-cancer cell crosstalk in both obese and lean conditions as well as its impact on cancer cell growth, local invasion, metastatic spread and resistance to treatments. Better characterization of cancer-associated adipocytes and the key molecular events in the adipocyte-cancer cell crosstalk will provide insights into tumor biology and suggest efficient therapeutic opportunities.Growing evidence has raised the important roles of adipocytes as an active player in the tumor microenvironment. In many tumors adipocytes are in close contact with cancer cells. They secrete various factors that can mediate local and systemic effects. The adipocyte-cancer cell crosstalk leads to phenotypical and functional changes of both cell types, which can further enhance tumor progression. Moreover, obesity, which is associated with an increase in adipose mass and an alteration of adipose tissue, has been established as a risk factor for cancer incidence and cancer-related mortality. In this review, we summarize the mechanisms of the adipocyte-cancer cell crosstalk in both obese and lean conditions as well as its impact on cancer cell growth, local invasion, metastatic spread and resistance to treatments. Better characterization of cancer-associated adipocytes and the key molecular events in the adipocyte-cancer cell crosstalk will provide insights into tumor biology and suggest efficient therapeutic opportunities.

Advances in Bispecific Antibodies Engineering: Novel Concepts for Immunotherapies

Bispecific antibodies are emerging as novel approach for immunotherapies by combining two antigen-recognizing elements into a single construct that is able to simultaneously bind to two distinct targets. Bispecific antibodies can be applied to recruit immunological effector cells for killing of tumor cells or to simultaneously block two signaling pathways or cytokines. Subsequently, they have prompted significant interest for a number of therapeutic applications, both in cancer and in other indications. The growing interest in therapeutic antibodies along with the rapid progress in antibody engineering have yield a multitude of bispecific antibodies formats and derived molecules that differ in size, shape and function. Presently, two major classes of bispecific antibodies are most widely studied: immunoglobulin-like and small single chain Fv (scFv)-based bispecific antibodies. This review summarizes selected key methods used to generate bispecific antibodies, reports new concepts developed for immunotherapy and discusses their potential development and therapeutic benefits.

Conditioned medium of H9c2 triggers VEGF dependent angiogenesis by activation of p38/pSTAT3 pathways in placenta derived stem cells for cardiac repair

AIMS Cardiomyocytes are understood to possess a limited regenerative capacity. Any myocardial insult leads to an irreversible injury. Mesenchymal stem cell differentiation into cardiomyocyte-like cells stands as one of the leading experimental therapies. However, a candidate cell source has yet to be defined. Here, we examined the in vitro and in vivo cardiac differentiation potential of human placenta derived stem cells (hPDSCs); a unique, abundant, and non-immunogenic cell source. MAIN METHODS H9c2 cell culture medium was applied to hPDSCs at different ratios for a period of 4weeks. In parallel, hPDSCs, human bone marrow stem cells, or cell free culture medium was injected in peri-infarcted regions induced in rat hearts. KEY FINDINGS In vitro, hPDSCs pre-conditioned with H9c2 cell culture medium proportionally over-expressed alpha sarcoplasmic actinin and displaced connexin 43 from the cytoplasm to the cell membrane. Additionally, pre-conditioning promoted hPDSCs survival and triggered vascular endothelial growth factor (VEGF) dependent angiogenesis by activating the pAkt and p38MAPK/pSTAT3 pathways. In vivo, echocardiography analysis showed a significant improvement in cardiac parameters in the rats injected with hPDSCs, similar to the human bone marrow stem cells injected group. Moreover, hPDSCs detected within rat cardiac tissues expressed troponin I and myosin heavy chain. In accordance with the pre-conditioning findings, VEGF positive neovessels were observed in hearts injected with hPDSCs. SIGNIFICANCE hPDSCs have the potential to differentiate into cardiac-like cells and induce angiogenesis via paracrine effects. With the advantages of easy availability and young age, these cells could be more suitable for clinical translation.

The Immature Heart: The Roles of Bone Marrow Stromal Stem Cells in Growth and Myocardial Repair

Studies have shown that adult bone marrow derived stem cells (MSCs) can participate in repair of myocardial injury in adult hearts, as well as in cardiac growth during fetal development in utero. Yet, no studies have evaluated the role of MSCs with respect to normal growth or tissue repair in immature hearts after birth. The present study examines whether MSCs may participate in the myocardial growth and injury in the post-natal immature hearts. MSCs were isolated from adult Lewis rats and labeled with Lac-Z gene using retroviral vectors. These MSCs were injected systemically into groups of neonatal (NB=2days-old), immature (B=30days-old) and adult (A=>3months-old) isogeneic Lewis rats. Additionally, left coronary artery ligation was carried out in subgroups of immature (BL) and adult (AL) rats one week after MSCs injection. The hearts were harvested serially from 2-days to 6-weeks, stained with X-Gal for labeled MSCs. Cardiomyocyte phenotypic expression was evaluated by immunohistological staining for Troponin I-C and Connexin-43. Labeled MSCs were found to home into the bone marrow in all rats of different developmental stages. They could be recruited from bone marrow into the infarcted site of myocardium only in groups AL and BL. They were also capable of differentiating into cardiomyocyte phenotype after myocardial injury. In contrast to that reported in the developing fetus, MSCs did not appear to contribute to the growth of non-injured hearts after birth. However, they can be recruited from the bone marrow and regenerate damaged myocardium both in the adult and in the immature hearts.Studies have shown that adult bone marrow derived stem cells (MSCs) can participate in repair of myocardial injury in adult hearts, as well as in cardiac growth during fetal development in utero. Yet, no studies have evaluated the role of MSCs with respect to normal growth or tissue repair in immature hearts after birth. The present study examines whether MSCs may participate in the myocardial growth and injury in the post-natal immature hearts. MSCs were isolated from adult Lewis rats and labeled with Lac-Z gene using retroviral vectors. These MSCs were injected systemically into groups of neonatal (NB=2days-old), immature (B=30days-old) and adult (A=>3months-old) isogeneic Lewis rats. Additionally, left coronary artery ligation was carried out in subgroups of immature (BL) and adult (AL) rats one week after MSCs injection. The hearts were harvested serially from 2-days to 6-weeks, stained with X-Gal for labeled MSCs. Cardiomyocyte phenotypic expression was evaluated by immunohistological staining for Troponin I-C and Connexin-43. Labeled MSCs were found to home into the bone marrow in all rats of different developmental stages. They could be recruited from bone marrow into the infarcted site of myocardium only in groups AL and BL. They were also capable of differentiating into cardiomyocyte phenotype after myocardial injury. In contrast to that reported in the developing fetus, MSCs did not appear to contribute to the growth of non-injured hearts after birth. However, they can be recruited from the bone marrow and regenerate damaged myocardium both in the adult and in the immature hearts.

Abstract 3783: Adipocytes inhibit trastuzumab-mediated ADCC via induction of GDF15

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background : Trastuzumab is a monoclonal antibody that has been approved in the treatment of HER2-expressing breast cancer. Despite its efficacy, resistance often occurs. Little is known about the role of the tumor microenvironment in resistance to trastuzumab. Recent studies have shown rising role of adipose tissue in promoting cancer progression and inducing resistance to chemotherapy. Given the abundance of adipose tissue in breast and its proximity to cancer cells, we investigated the impact of adipocytes on antibody-dependent cellular cytotoxicity (ADCC), one of the main mechanisms of action of trastuzumab. Methods: We set up a co-culture system in the presence of undifferentiated or differentiated human multi-potent adipose-derived stem (hMADS) cells, HER2-expressing human breast cancer cell lines (BT474, MDA-453, SKBR3, MDA-MB-361), and human natural killer NK-92 expressing the Fc receptor CD16. Trastuzumab-mediated cytotoxicity was assessed by calcein-release assay. Morphological changes on cancer cells and NK-92-CD16 cells were investigated by microscopy and flow cytometry. Results: We found that adipocytes, as well as pre-adipocytes, inhibited trastuzumab-mediated ADCC by approximately 30%. Notably, this inhibition was enhanced in hypoxic conditions with 1% O2 (up to 60% inhibition). The inhibition of ADCC by adipose cells was not due to a titration or degradation of the antibody, but via factors that are secreted by adipose cells into the conditioned media. Analysis of cell phenotype did not reveal any modification of NK cell receptors (CD16, NKG2D, NKG2A, NKp30, NKp44) or NK cell activation markers (CD25, CD69, CD107a), nor of HER2 levels in the presence of adipocytes. Pre-incubation of NK cells with the conditioned medium from adipocytes did not alter NK cytotoxicity. Conversely, pre-incubation of cancer cells with adipocyte-conditioned medium reduced sensitivity of cancer cells to ADCC. Using transcriptomic approach, we found GDF15 to be rapidly up-regulated in cancer cells exposed to adipocyte-conditioned medium. Down-regulation of GDF15 by siRNA reversed the adipocyte-induced inhibition of ADCC. Conclusion: Adipocytes inhibit antitumor activity of trastuzumab in HER2-expressing breast cancer cells via secretion of factors that reduce cancer cell sensitivity to ADCC. Our findings underline the importance of adipose tissue in the resistance to trastuzumab, and suggest that development of approaches targeting GDF15 may sensitize cancer cells to trastuzumab-based therapy. Citation Format: Minh Ngoc Duong, Aurore Cleret, Eva-Laure Matera, Kamel Chettab, Doriane Poloni, Sandrine Valsesia-Wittmann, Beatrice Clemenceau, Charles Dumontet. Adipocytes inhibit trastuzumab-mediated ADCC via induction of GDF15. [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 3783. doi:10.1158/1538-7445.AM2014-3783