Juliana Lott Carvalho

Time-dependent migration of systemically delivered bone marrow mesenchymal stem cells to the infarcted heart.

In this study the time course of homing and the body distribution of systemically delivered bone marrow mesenchymal stem cells (BM-MSCs) after myocardial infarction (MI) were evaluated. BM-MSCs were isolated from Wistar rats, expanded in vitro, and their phenotypical characterization was performed by flow cytometer. Rats were randomly divided into three groups: control, sham MI, and MI. BM-MSCs (5 × 106) were labeled with 99mTc-HMPAO and injected through the tail vein 7 days after MI. Gamma camera imaging was performed at 5, 15, 30, and 60 min after cell inoculation. Due to the 99mTc short half-life, cell migration and location were also evaluated in heart sections using DAPI-labeled cells 7 days after transplantation. Phenotypical characterization showed that BM-MSCs were CD90+, CD73+, CD54+, and CD45-. Five minutes after 99mTc-HMPAO-labeled cell injection, they were detected in various tissues. The cells migrated mainly to the lungs (approximately 70%) and, in small amounts, to the heart, kidneys, spleen, and bladder. The number of cells in the heart and lungs decreased after 60 min. MI markedly increased the amount of cells in the heart, but not in the lungs, during the period of observation (4.55 ± 0.32 vs. 6.34 ± 0.67% of uptake in infarcted hearts). No significant differences were observed between control and sham groups. Additionally, 7 days after DAPI-labeled cells injection, they were still detected in the heart but only in infarcted areas. These results suggest that the migration of systemically delivered BM-MSCs to the heart is time dependent and MI specifically increases BM-MSCs homing to injured hearts. However, the systemic delivery is limited by cell entrapment in the lungs.

Differentiation of adipose tissue-derived mesenchymal stem cells into cardiomyocytes

BACKGROUND Cardiomyocytes have small potential for renovation and proliferation in vivo. Consequently, the heart muscle has limited capacity of self-renewal. Mesenchymal stem cells (MSC) therapy, as well as MSC differentiated into cardiomyocytes, has been used in the attempt to minimize the effects of ischemic-hypoxic lesions and those affecting the electrical conduction system of the heart. OBJECTIVE The present study compared three distinct protocols for induced differentiation of MSC into cardiomyocytes aimed at finding a viable method for producing a large number of functional cells expressing cardiomyogenic phenotype. METHODS Mesenchymal stem cells were obtained from the adipose tissue of young transgenic Lewis rats expressing green fluorescent protein (GFP), and submitted to three distinct differentiation-inducing media: 1) Planat-Bérnard, 2) 5-azacytidine, and 3) Planat-Bérnard + 5-azacytidine; further, these cells were identified based on the expression of cardiac cell markers. RESULTS All three protocols detected the expression of sarcomeric-alpha-actinin protein in the exoskeleton of cells, expression of connexin-43 in the nuclear and cytoplasmic membrane, and formation of gap junctions, which are necessary for electrical impulse propagation in the myocardium. However, no spontaneous cell contraction was observed with any of the tested protocols. CONCLUSION Induction with 5-azacytidine provided an effective cadiomyogenic cellular differentiation similar to that obtained with Planat-Bénard media. Therefore, 5-azacytidine was the method of choice for being the simplest, fastest and lowest-cost protocol for cell differentiation.

Priming mesenchymal stem cells boosts stem cell therapy to treat myocardial infarction.

Cardiovascular diseases are the number one cause of death globally and are projected to remain the single leading cause of death. Treatment options abounds, although efficacy is limited. Recent studies attribute discrete and ephemeral benefits to adult stem cell therapies, indicating the urge to improve stem cell based–therapy. In this study, we show that priming mesenchymal stem cells (MSC) towards cardiomyogenic lineage enhances their beneficial effects in vivo as treatment option for acute phase myocardial infarction. MSC were primed using cardiomyogenic media for 4 days, after which peak expression of key cardiomyogenic genes are reached and protein expression of Cx‐43 and sarcomeric α‐actinin are observed. MSC and primed MSC (pMSC) were characterized in vitro and used to treat infarcted rats immediately after left anterior descending (LAD) occlusion. Echocardiography analysis indicated that MSC‐treated myocardium presented discrete improvement in function, but it also showed that pMSC treatment lead to superior beneficial results, compared with undifferentiated MSC. Seven days after cell injection, MSC and pMSC could still be detected in the myocardium. Connexin‐43 expression was quantified through immunoblotting, and was superior in pMSC, indicating that this could be a possible explanation for the superior performance of pMSC therapy.

Neuroprotective effects of mesenchymal stem cells on spinal motoneurons following ventral root axotomy: Synapse stability and axonal regeneration

Compression of spinal roots is an important medical problem, which may arise from intervertebral disc herniation, tumor growth or as a result of high energy accidents. Differently from avulsion, root crushing maintains the central/peripheral nervous system (CNS/PNS) connection, although the axons are axotomized and motoneurons degenerate. Such neuronal death may decrease and delay motor function recovery. In the present study we have investigated the neuroprotective effects of mesenchymal stem cell (MSC) therapy following such proximal lesions. Motor recovery and synaptic stabilization were analyzed by the use of morphological and functional approaches. For that, crushing the ventral roots at L4, L5 and L6 was unilaterally performed in Lewis rats. Four weeks after injury, an increased motoneuron survival was observed in the MSC-treated group, coupled with a smaller decrease of inputs at the motoneuron surface and nearby neuropil, seen by synaptophysin and synapsin immunolabeling and decreased astrogliosis, seen by GFAP immunolabeling. In this sense, MSC-treated group displayed a significant preservation of GABAergic terminals, indicating a possible neuroprotection to glutamate excitotoxicity. Motor function recovery was acutely improved in MSC-treated group as compared to Dulbecos modified eagle medium (DMEM)-treated. Overall, we provide evidence that ventral root crushing (VRC), although milder than avulsion, results in significant loss of motoneurons (~51%) that can be reduced by MSC administration within the spinal cord. Such treatment also improves the number of synapses immunoreactive against molecules present in inhibitory inputs. Also, an increased number of regenerated axons was obtained in the MSC-treated group, in comparison to the DMEM-treated control. Overall, MSC therapy acutely improved limb strength and gait coordination, indicating a possible clinical application of such treatment following proximal lesions at the CNS/PNS interface.

Mesenchymal stem cells engrafted in a fibrin scaffold stimulate Schwann cell reactivity and axonal regeneration following sciatic nerve tubulization

The present study investigated the effectiveness of mesenchymal stem cells (MSCs) associated with a fibrin scaffold (FS) for the peripheral regenerative process after nerve tubulization. Adult female Lewis rats received a unilateral sciatic nerve transection followed by repair with a polycaprolactone (PCL)-based tubular prosthesis. Sixty days after injury, the regenerated nerves were studied by immunohistochemistry. Anti-p75NTR immunostaining was used to investigate the reactivity of the MSCs. Basal labeling, which was upregulated during the regenerative process, was detected in uninjured nerves and was significantly greater in the MSC-treated group. The presence of GFP-positive MSCs was detected in the nerves, indicating the long term survival of such cells. Moreover, there was co-localization between MSCs and BNDF immunoreactivity, showing a possible mechanism by which MSCs improve the reactivity of SCs. Myelinated axon counting and morphometric analyses showed that MSC engrafting led to a higher degree of fiber compaction combined with a trend of increased myelin sheath thickness, when compared with other groups. The functional result of MSC engrafting was that the animals showed higher motor function recovery at the seventh and eighth week after lesion. The findings herein show that MSC+FS therapy improves the nerve regeneration process by positively modulating the reactivity of SCs.

Doxorubicin has in vivo toxicological effects on ex vivo cultured mesenchymal stem cells.

Doxorubicin (dox) is an effective chemotherapeutic agent that leads to cardiotoxicity. An alternative treatment for dox-cardiotoxicity is autologous mesenchymal stem cells (MSCs) transplantation. It remains unclear if dox has deleterious effects on MSCs from subjects under chemotherapy, therefore this study aimed to evaluate dox in vivo toxicological effects on ex vivo cultured MSCs, inferring whether autologous transplantation may be an alternative treatment in patients who are exposed to the drug. Wistar rats received either dox or saline. Following treatments, animals were sacrificed and bone marrow MSCs were isolated, characterized for cell surface markers and assessed according to their viability, alkaline phosphatase production, and proliferation kinetics. Moreover, MSCs were primed to cardiac differentiation and troponin T and connexin 43 expressions were evaluated. Compared to control, undifferentiated MSCs from dox group kept the pattern for surface marker and had similar viability results. In contrast, they showed lower alkaline phosphatase production, proliferation rate, and connexin 43 expression. Primed MSCs from dox group showed lower troponin T levels. It was demonstrated a toxic effect of dox in host MSCs. This result renders the possibility of autologous MSCs transplantation to treat dox-cardiotoxicity, which could be a non-suitable option for subjects receiving such antineoplastic agent.

Innovative Strategies for Tissue Engineering

Unmet need. It is beyond dispute that human population is ageing. For the first time in history, people age 65 and over will outnumber children under age 5. This trend is emerging around the globe, and will bring several challenges for health technologies. For instance, in a few decades, the loss of health and life worldwide will be greater from chronic diseases than from infectious diseases and accidents [4].

Characterization of Decellularized Heart Matrices as Biomaterials for Regular and Whole Organ Tissue Engineering and Initial In-vitro Recellularization with Ips Cells

Tissue engineering strategies, based on solid/porous scaffolds, suffer from several limitations, such as ineffective vascularization, poor cell distribution and organization within scaffold, in addition to low final cell density, among others. Therefore, the search for other tissue engineering approaches constitutes an active area of investigation. Decellularized matrices (DM) present major advantages compared to solid scaffolds, such as ideal chemical composition, the preservation of vascularization structure and perfect three-dimensional structure. In the present study, we aimed to characterize and investigate murine heart decellularized matrices as biomaterials for regular and whole organ tissue engineering. Heart decellularized matrices were characterized according to: 1. DNA content, through DNA quantificationo and PCR of isolated genomic DNA; 2. Histological structure, assessed after Hematoxylin and Eosin, as well as Massons Trichrome stainings; 3. Surface nanostructure analysis, performed, using SEM. Those essays allowed us to conclude that DM was indeed decellularized, with preserved extracellular matrix structure. Following characterization, decellularized heart slices were seeded with induced Pluripotent Stem cells (iPS). As expected, but - to the best of our knowledge - never shown before, decellularization of murine heart matrices maintained matrix biocompatibility, as iPS cells rapidly attached to the surface of the material and proliferated. Strikingly though, heart DM presented a differentiation induction effect over those cells, which lost their pluripotency markers after 7 days of culture in the DM. Such loss of differentiation markers was observed, even though bFGF containing media mTSR was used during such period. Gene expression of iPS cells cultured on DM will be further analyzed, in order to assess the effects of culturing pluripotent stem cells in decellularized heart matrices.

Bacterial Cellulose Membranes Constitute Biocompatible Biomaterials for Mesenchymal and Induced Pluripotent Stem Cell Culture and Tissue Engineering

In animal models of bone injury, for instance, up to 105-106 cells may be used in tissue engineering strategies [2]. Considering the translation of such techniques to human proportions, the number of cells required for tissue engineering may reach up to a hundred million cells. Currently, adult stem cells are being used for such situations, mostly in clinical trial settings, and results are encouraging. Unfortunately, though, many limitations still hinder adult stem cell therapy.

Production of Human Endothelial Cells Free from Soluble Xenogeneic Antigens for Bioartificial Small Diameter Vascular Graft Endothelization

Arterial bypass graft implantation remains the primary therapy for patients with advanced cardiovascular disease, but most lack adequate saphenous vein or other conduits for bypass procedures and would benefit from a bioartificial conduit. This study aimed to produce human endothelial cells (hECs) in large scale, free from xenogeneic antigens, to develop a small diameter, compatible vessel for potential use as a vascular graft. Human adipose-derived stromal cells (hASCs) were isolated, cultured, and differentiated in the presence of human serum and used for the reendothelization of a decellularized rat aorta. hASC derived ECs (hASC-ECs) expressed VEGFR2, vWf and CD31 endothelial cell markers, the latter in higher levels than hASCs and HUVECs, and were shown to be functional. Decellularization protocol yielded aortas devoid of cell nuclei, with preserved structure, including a preserved basement membrane. When seeded with hASC-ECs, the decellularized aorta was completely reendothelized, and the hASC-ECs maintained their phenotype in this new condition. hASCs can be differentiated into functional hECs without the use of animal supplements and are capable of reendothelizing a decellularized rat aorta while maintaining their phenotype. The preservation of the basement membrane following decellularization supported the complete reendothelization of the scaffold with no cell migration towards other layers. This approach is potentially useful for rapid obtention of compatible, xenogeneic-free conduit.