Beatriz Pelacho

Transplantation of adipose derived stromal cells is associated with functional improvement in a rat model of chronic myocardial infarction

To determine the effect of transplantation of undifferentiated and cardiac pre‐differentiated adipose stem cells compared with bone marrow mononuclear cells (BM‐MNC) in a chronic model of myocardial infarction.

Efficient Transfection of Embryonic and Adult Stem Cells

The ability of embryonic stem cells and adult stem cells to differentiate into specific cell types holds immense potential for therapeutic use in cell and gene therapy. Realization of this potential depends on efficient and optimized protocols for genetic manipulation of stem cells. In the study reported here, we demonstrate the use of nucleofection as a method to introduce plasmid DNA into embryonic and adult stem cells with significantly greater efficiency than electroporation or lipid‐based transfection methods have. Using enhanced green fluorescent protein (eGFP) as a reporter gene, mouse embryonic stem cells were transfected both transiently and stably at a rate nearly 10‐fold higher than conventional methods. The transfected cells retained their stem cell properties, including continued expression of the stem cell markers SSEA1, Oct4, and Rex1; formation of embryoid bodies; differentiation into cardiomyocytes in the presence of appropriate inducers; and, when injected into developing blastocysts, contribution to chimeras. Higher levels of transfection were also obtained with human embryonic carcinoma and human embryonic stem cells. Particularly hard‐to‐transfect adult stem cells, including bone marrow and multipotent adult progenitor cells, were also transfected efficiently by the method of nucleofection. Based on our results, we conclude that nucleofection is superior to currently available methods for introducing plasmid DNA into a variety of embryonic and adult stem cells. The high levels of transfection achieved by nucleofection will enable its use as a rapid screening tool to evaluate the effect of ectopically expressed transcription factors on tissue‐specific differentiation of stem cells.

Sustained release of VEGF through PLGA microparticles improves vasculogenesis and tissue remodeling in an acute myocardial ischemia-reperfusion model.

The use of pro-angiogenic growth factors in ischemia models has been associated with limited success in the clinical setting, in part owing to the short lived effect of the injected cytokine. The use of a microparticle system could allow localized and sustained cytokine release and consequently a prolonged biological effect with induction of tissue revascularization. To assess the potential of VEGF(165) administered as continuous release in ischemic disease, we compared the effect of delivery of poly(lactic-co-glycolic acid) (PLGA) microparticles (MP) loaded with VEGF(165) with free-VEGF or control empty microparticles in a rat model of ischemia-reperfusion. VEGF(165) loaded microparticles could be detected in the myocardium of the infarcted animals for more than a month after transplant and provided sustained delivery of active protein in vitro and in vivo. One month after treatment, an increase in angiogenesis (small caliber caveolin-1 positive vessels) and arteriogenesis (α-SMA-positive vessels) was observed in animals treated with VEGF microparticles (p<0.05), but not in the empty microparticles or free-VEGF groups. Correlating with this data, a positive remodeling of the heart was also detected in the VEGF-microparticle group with a significantly greater LV wall thickness (p<0.01). In conclusion, PLGA microparticle is a feasible and promising cytokine delivery system for treatment of myocardial ischemia. This strategy could be scaled up and explored in pre-clinical and clinical studies.

Characterization of the paracrine effects of human skeletal myoblasts transplanted in infarcted myocardium

The discrepancy between the functional improvements yielded experimentally by skeletal myoblasts (SM) transplanted in infarcted myocardium and the paucity of their long‐term engraftment has raised the hypothesis of cell‐mediated paracrine mechanisms.

Hematopoietic reconstitution by multipotent adult progenitor cells: precursors to long-term hematopoietic stem cells

For decades, in vitro expansion of transplantable hematopoietic stem cells (HSCs) has been an elusive goal. Here, we demonstrate that multipotent adult progenitor cells (MAPCs), isolated from green fluorescent protein (GFP)-transgenic mice and expanded in vitro for >40–80 population doublings, are capable of multilineage hematopoietic engraftment of immunodeficient mice. Among MAPC-derived GFP+CD45.2+ cells in the bone marrow of engrafted mice, HSCs were present that could radioprotect and reconstitute multilineage hematopoiesis in secondary and tertiary recipients, as well as myeloid and lymphoid hematopoietic progenitor subsets and functional GFP+ MAPC-derived lymphocytes that were functional. Although hematopoietic contribution by MAPCs was comparable to control KTLS HSCs, approximately 103-fold more MAPCs were required for efficient engraftment. Because GFP+ host-derived CD45.1+ cells were not observed, fusion is not likely to account for the generation of HSCs by MAPCs.

Multipotent adult progenitor cells sustain function of ischemic limbs in mice.

Despite progress in cardiovascular research, a cure for peripheral vascular disease has not been found. We compared the vascularization and tissue regeneration potential of murine and human undifferentiated multipotent adult progenitor cells (mMAPC-U and hMAPC-U), murine MAPC-derived vascular progenitors (mMAPC-VP), and unselected murine BM cells (mBMCs) in mice with moderate limb ischemia, reminiscent of intermittent claudication in human patients. mMAPC-U durably restored blood flow and muscle function and stimulated muscle regeneration, by direct and trophic contribution to vascular and skeletal muscle growth. This was in contrast to mBMCs and mMAPC-VP, which did not affect muscle regeneration and provided only limited and transient improvement. Moreover, mBMCs participated in a sustained inflammatory response in the lower limb, associated with progressive deterioration in muscle function. Importantly, mMAPC-U and hMAPC-U also remedied vascular and muscular deficiency in severe limb ischemia, representative of critical limb ischemia in humans. Thus, unlike BMCs or vascular-committed progenitors, undifferentiated multipotent adult progenitor cells offer the potential to durably repair ischemic damage in peripheral vascular disease patients.

In vivo blockade of pemphigus vulgaris acantholysis by inhibition of intracellular signal transduction cascades.

Background  Pemphigus vulgaris (PV) is an autoimmune disease characterized by mucocutaneous intraepithelial blisters and pathogenic autoantibodies against desmoglein 3. The mechanism of blister formation in pemphigus has not been defined; however, in vitro data suggest a role for activation of intracellular signalling cascades.

Interacting Resident Epicardium-Derived Fibroblasts and Recruited Bone Marrow Cells Form Myocardial Infarction Scar

BACKGROUND Although efforts continue to find new therapies to regenerate infarcted heart tissue, knowledge of the cellular and molecular mechanisms involved remains poor. OBJECTIVES This study sought to identify the origin of cardiac fibroblasts (CFs) in the infarcted heart to better understand the pathophysiology of ventricular remodeling following myocardial infarction (MI). METHODS Permanent genetic tracing of epicardium-derived cell (EPDC) and bone marrow-derived blood cell (BMC) lineages was established using Cre/LoxP technology. In vivo gene and protein expression studies, as well as in vitro cell culture assays, were developed to characterize EPDC and BMC interaction and properties. RESULTS EPDCs, which colonize the cardiac interstitium during embryogenesis, massively differentiate into CFs after MI. This response is disease-specific, because angiotensin II-induced pressure overload does not trigger significant EPDC fibroblastic differentiation. The expansion of epicardial-derived CFs follows BMC infiltration into the infarct site; the number of EPDCs equals that of BMCs 1 week post-infarction. BMC-EPDC interaction leads to cell polarization, packing, massive collagen deposition, and scar formation. Moreover, epicardium-derived CFs display stromal properties with respect to BMCs, contributing to the sustained recruitment of circulating cells to the damaged zone and the cardiac persistence of hematopoietic progenitors/stem cells after MI. CONCLUSIONS EPDCs, but not BMCs, are the main origin of CFs in the ischemic heart. Adult resident EPDC contribution to the CF compartment is time- and disease-dependent. Our findings are relevant to the understanding of post-MI ventricular remodeling and may contribute to the development of new therapies to treat this disease.

Controlled delivery of fibroblast growth factor-1 and neuregulin-1 from biodegradable microparticles promotes cardiac repair in a rat myocardial infarction model through activation of endogenous regeneration.

Acidic fibroblast growth factor (FGF1) and neuregulin-1 (NRG1) are growth factors involved in cardiac development and regeneration. Microparticles (MPs) mediate cytokine sustained release, and can be utilized to overcome issues related to the limited therapeutic protein stability during systemic administration. We sought to examine whether the administration of microparticles (MPs) containing FGF1 and NRG1 could promote cardiac regeneration in a myocardial infarction (MI) rat model. We investigated the possible underlying mechanisms contributing to the beneficial effects of this therapy, especially those linked to endogenous regeneration. FGF1- and NRG1-loaded MPs were prepared using a multiple emulsion solvent evaporation technique. Seventy-three female Sprague-Dawley rats underwent permanent left anterior descending coronary artery occlusion, and MPs were intramyocardially injected in the peri-infarcted zone four days later. Cardiac function, heart tissue remodeling, revascularization, apoptosis, cardiomyocyte proliferation, and stem cell homing were evaluated one week and three months after treatment. MPs were shown to efficiently encapsulate FGF1 and NRG1, releasing the bioactive proteins in a sustained manner. Three months after treatment, a statistically significant improvement in cardiac function was detected in rats treated with growth factor-loaded MPs (FGF1, NRG1, or FGF1/NRG1). The therapy led to inhibition of cardiac remodeling with smaller infarct size, a lower fibrosis degree and induction of tissue revascularization. Cardiomyocyte proliferation and progenitor cell recruitment were detected. Our data support the therapeutic benefit of NRG1 and FGF1 when combined with protein delivery systems for cardiac regeneration. This approach could be scaled up for use in pre-clinical and clinical studies.

Pemphigus vulgaris autoantibodies induce apoptosis in HaCaT keratinocytes.

Pemphigus vulgaris (PV) is an autoimmune disease characterized by binding of IgG autoantibodies to epidermal keratinocyte desmosomes. IgG autoantibodies obtained from a patient with mucocutaneous PV reacted with plakoglobin (Plkg) in addition to desmoglein‐3 (Dsg3) and Dsg1. Immunofluorescence analysis confirmed that IgG autoantibodies, unlike antibodies from a healthy volunteer, caused disruption of cell–cell contacts in HaCaT keratinocytes. Moreover, apoptosis was enhanced in cells treated with autoantibodies compared to those treated with normal antibodies. The apoptotic process induced by IgG autoantibodies was characterized by caspase‐3 activation, Bcl‐2 depletion and Bax expression. The present report demonstrates that PV IgG autoantibodies promote apoptosis in HaCaT keratinocytes.