Stem cells under the influence of alcohol: effects of ethanol consumption on stem/progenitor cells.

Stem cells drive embryonic and fetal development. In several adult tissues, they retain the ability to self-renew and differentiate into a variety of specialized cells, thus contributing to tissue homeostasis and repair throughout life span. Alcohol consumption is associated with an increased risk for several diseases and conditions. Growing and developing tissues are particularly vulnerable to alcohol's influence, suggesting that stem- and progenitor-cell function could be affected. Accordingly, recent studies have revealed the possible relevance of alcohol exposure in impairing stem-cell properties, consequently affecting organ development and injury response in different tissues. Here, we review the main studies describing the effects of alcohol on different types of progenitor/stem cells including neuronal, hepatic, intestinal and adventitial progenitor cells, bone-marrow-derived stromal cell, dental pulp, embryonic and hematopoietic stem cells, and tumor-initiating cells. A better understanding of the nature of the cellular damage induced by chronic and episodic heavy (binge) drinking is critical for the improvement of current therapeutic strategies designed to treat patients suffering from alcohol-related disorders.

Current Biomedical Use of Copper Chelation Therapy.

Copper is an essential microelement that plays an important role in a wide variety of biological processes. Copper concentration has to be finely regulated, as any imbalance in its homeostasis can induce abnormalities. In particular, excess copper plays an important role in the etiopathogenesis of the genetic disease Wilson's syndrome, in neurological and neurodegenerative pathologies such as Alzheimer's and Parkinson's diseases, in idiopathic pulmonary fibrosis, in diabetes, and in several forms of cancer. Copper chelating agents are among the most promising tools to keep copper concentration at physiological levels. In this review, we focus on the most relevant compounds experimentally and clinically evaluated for their ability to counteract copper homeostasis deregulation. In particular, we provide a general overview of the main disorders characterized by a pathological increase in copper levels, summarizing the principal copper chelating therapies adopted in clinical trials.

Challenges and Strategies for Improving the Regenerative Effects of Mesenchymal Stromal Cell-Based Therapies.

Cell-based therapies have the potential to revolutionize current treatments for diseases with high prevalence and related economic and social burden. Unfortunately, clinical trials have made only modest improvements in restoring normal function to degenerating tissues. This limitation is due, at least in part, to the death of transplanted cells within a few hours after transplant due to a combination of mechanical, cellular, and host factors. In particular, mechanical stress during implantation, extracellular matrix loss upon delivery, nutrient and oxygen deprivation at the recipient site, and host inflammatory response are detrimental factors limiting long-term transplanted cell survival. The beneficial effect of cell therapy for regenerative medicine ultimately depends on the number of administered cells reaching the target tissue, their viability, and their promotion of tissue regeneration. Therefore, strategies aiming at improving viable cell engraftment are crucial for regenerative medicine. Here we review the major factors that hamper successful cell engraftment and the strategies that have been studied to enhance the beneficial effects of cell therapy. Moreover, we provide a perspective on whether mesenchymal stromal cell-derived extracellular vesicle delivery, as a cell-free regenerative approach, may circumvent current cell therapy limitations.

Promotion of Survival and Engraftment of Transplanted Adipose Tissue-Derived Stromal and Vascular Cells by Overexpression of Manganese Superoxide Dismutase.

Short-term persistence of transplanted cells during early post-implant period limits clinical efficacy of cell therapy. Poor cell survival is mainly due to the harsh hypoxic microenvironment transplanted cells face at the site of implantation and to anoikis, driven by cell adhesion loss. We evaluated the hypothesis that viral-mediated expression of a gene conferring hypoxia resistance to cells before transplant could enhance survival of grafted cells in early stages after implant. We used adipose tissue as cell source because it consistently provides high yields of adipose-tissue-derived stromal and vascular cells (ASCs), suitable for regenerative purposes. Luciferase positive cells were transduced with lentiviral vectors expressing either green fluorescent protein as control or human manganese superoxide dismutase (SOD2). Cells were then exposed in vitro to hypoxic conditions, mimicking cell transplantation into an ischemic site. Cells overexpressing SOD2 displayed survival rates significantly greater compared to mock transduced cells. Similar results were also obtained in vivo after implantation into syngeneic mice and assessment of cell engraftment by in vivo bioluminescent imaging. Taken together, these findings suggest that ex vivo gene transfer of SOD2 into ASCs before implantation confers a cytoprotective effect leading to improved survival and engraftment rates, therefore enhancing cell therapy regenerative potential.

Monitoring the Response of Hyperbilirubinemia in the Mouse Brain by In Vivo Bioluminescence Imaging.

Increased levels of unconjugated bilirubin are neurotoxic, but the mechanism leading to neurological damage has not been completely elucidated. Innovative strategies of investigation are needed to more precisely define this pathological process. By longitudinal in vivo bioluminescence imaging, we noninvasively visualized the brain response to hyperbilirubinemia in the MITO-Luc mouse, in which light emission is restricted to the regions of active cell proliferation. We assessed that acute hyperbilirubinemia promotes bioluminescence in the brain region, indicating an increment in the cell proliferation rate. Immunohistochemical detection in brain sections of cells positive for both luciferase and the microglial marker allograft inflammatory factor 1 suggests proliferation of microglial cells. In addition, we demonstrated that brain induction of bioluminescence was altered by pharmacological displacement of bilirubin from its albumin binding sites and by modulation of the blood-brain barrier permeability, all pivotal factors in the development of bilirubin-induced neurologic dysfunction. We also determined that treatment with minocycline, an antibiotic with anti-inflammatory and neuroprotective properties, or administration of bevacizumab, an anti-vascular endothelial growth factor antibody, blunts bilirubin-induced bioluminescence. Overall the study supports the use of the MITO-Luc mouse as a valuable tool for the rapid response monitoring of drugs aiming at preventing acute bilirubin-induced neurological dysfunction.

Harvest of superficial layers of fat with a microcannula and isolation of adipose tissue-derived stromal and vascular cells.

BACKGROUND: Adipose tissue is a source of stromal and vascular cells suitable for regenerative medical applications. Cell recovery depends on several factors, including the characteristics of the cannula used to harvest tissue. OBJECTIVES: The authors assess whether aspiration of superficial layers of adipose tissue performed with a microcannula, rather than a standard cannula, allows for improved isolation of stromal and vascular cells, and they evaluate the angiogenic potential of the isolated cells in vitro and in vivo. METHODS: Adipose-derived stromal and stem cells (ADSC) were collected from the lipoaspirate of the abdomen and hip regions of 6 healthy female donors. For adipose tissue harvest, several options were compared: (1) a rounded-tip cannula with a length of 170 mm, a diameter of 3 mm, and a single elliptic suction port on the side near its distal end (port diameter: 3 × 9 mm) or (2) a rounded-tip infiltration cannula with a length of 170 mm, a diameter of 2 mm, and 5 round ports placed spirally along the sides of the distal cannula shaft (each port diameter: 1 mm) (Shipper Medical Technologies Corporation, Centennial, Colorado). Isolated cells were characterized for (1) expression of the endothelial specific marker CD31 by immunohistochemical and cytofluorimetric analyses and (2) tubular-like structure formation using a 3-dimensional angiogenesis assay on Matrigel. Human ADSC were transduced to express firefly luciferase as a marker suitable for bioluminescent tracking and transplantation studies into immunosuppressed mice were performed. RESULTS: ADSC yield was determined to be significantly higher in samples collected with the microcannula (P = .04). Moreover, isolated cells acquired typical endothelial-like morphology in vitro, formed capillary-like structures, and expressed the distinctive endothelial cell marker CD31. Cells implanted into immunosuppressed mice persisted for several weeks in areas undergoing neovascularization. CONCLUSIONS: These results suggest that aspiration of adipose tissue with a microcannula can be a minimally invasive method to obtain clinically relevant numbers of stromal and vascular cells useful for autologous transplant procedures and for promoting tissue regeneration and neovascularization.

Diabetes impairs adipose tissue-derived stem cell function and efficiency in promoting wound healing.

Adipose tissue-derived stem cells (ASCs) are gaining increasing consideration in tissue repair therapeutic application. Recent evidence indicates that ASCs enhance skin repair in animal models of impaired wound healing. To assess the therapeutic activity of autologous vs. allogeneic ASCs in the treatment of diabetic ulcers, we functionally characterized diabetic ASCs and investigated their potential to promote wound healing with respect to nondiabetic ones. Adipose tissue-derived cells from streptozotocin-induced type 1 diabetic mice were analyzed either freshly isolated as stromal vascular fraction (SVF), or following a single passage of culture (ASCs). Diabetic ASCs showed decreased proliferative potential and migration. Expression of surface markers was altered in diabetic SVF and cultured ASCs, with a reduction in stem cell marker-positive cells. ASCs from diabetic mice released lower amounts of hepatocyte growth factor, vascular endothelial growth factor (VEGF)-A, and insulin-like growth factor-1, growth factors playing important roles in skin repair. Accordingly, the supernatant of diabetic ASCs manifested reduced capability to promote keratinocyte and fibroblast proliferation and migration. Therapeutic potential of diabetic SVF administered to wounds of diabetic mice was blunted as compared with cells isolated from nondiabetic mice. Our data indicate that diabetes alters ASC intrinsic properties and impairs their function, thus affecting therapeutic potential in the autologous treatment for diabetic ulcers.

Differences in microparticle release in patients with acute coronary syndrome and stable angina.

BACKGROUND: Microparticles (MP) are vesicles released from activated or apoptotic cells. Endothelial MP (EMP) are derived from injured endothelium, platelet MP (PMP) from activated platelets, and Annexin V positive MP (AMP) from apoptotic endothelial cells. The aim was to assess the release of MP and its association with inflammation and atherosclerotic burden. METHODS AND RESULTS: AMP, EMP and PMP were measured on admission (Day 0) in 33 patients with stable angina (SA) and 43 patients with acute coronary syndrome (ACS) undergoing percutaneous coronary interventions (PCI). In SA, peripheral artery disease (PAD) was assessed by ultrasound examination. In 30 of the 76 patients (20 ACS and 10 SA), MP, high-sensitivity-C-reactive protein (hs-CRP), and troponin T (TnT) levels were also assessed 24h (Day 1) and 48 h (Day 2) after PCI. AMP, EMP, and PMP were higher in ACS than in SA (all P<0.01). In the SA group, AMP, PMP, and EMP were similar in patients with or without PAD. In the ACS group, AMP increased until Day 2 (P=0.001), while EMP and PMP peaked on Day 1 (P<0.01) then decreased to baseline values. Day 2 AMP correlated with Day 2 TnT levels (r=0.43, P=0.01) while Day 1 EMP and PMP correlated with Day 1 hs-CRP (r=0.37, P=0.04 and r=0.33, P=0.05; respectively). CONCLUSIONS: Higher MP levels were observed in ACS than in SA. Atherosclerotic burden did not affect MP levels in stable patients.

HIPK2 Cooperates with KRAS Signaling and Associates with Colorectal Cancer Progression.

Homeodomain-interacting protein kinase 2 (HIPK2) is an evolutionary conserved kinase that has gained attention as a fine tuner of multiple signaling pathways, among which those commonly altered in colorectal cancer. The aim of this study was to evaluate the relationship of HIPK2 expression with progression markers and mutational pattern and gain insights into the contribution of HIPK2 activity in colorectal cancer. We evaluated a retrospective cohort of colorectal cancer samples by IHC for HIPK2 expression and by next-generation sequencing (NGS) for the detection of mutations of cancer associated genes. We show that the percentage of HIPK2-positive cells increases with tumor progression, significantly correlates with tumor-node-metastasis (TNM) staging and associates with a worse outcome. In addition, we observed that high HIPK2 expression significantly associates with KRAS mutations but not with other cancer-related genes. Functional characterization of the link between HIPK2 and KRAS show that activation of the RAS pathway either due to KRAS mutation or via upstream receptor stimulation, increases HIPK2 expression at the protein level. Of note, HIPK2 physically participates in the active RAS complex while HIPK2 depletion impairs ERK phosphorylation and the growth of tumors derived from KRAS mutated colorectal cancer cells. Overall, this study identifies HIPK2 as a prognostic biomarker candidate in patients with colorectal cancer and underscores a previously unknown functional link between HIPK2 and the KRAS signaling pathway. IMPLICATIONS: Our data indicate HIPK2 as a new player in the complex picture of the KRAS signaling network, providing rationales for future clinical studies and new treatment strategies for KRAS mutated colorectal cancer.

Reduction of Cell Proliferation by Acute C2H6O Exposure.

Endogenous acetaldehyde production from the metabolism of ingested alcohol exposes hematopoietic progenitor cells to increased genotoxic risk. To develop possible therapeutic strategies to prevent or reverse alcohol abuse effects, it would be critical to determine the temporal progression of acute ethanol toxicity on progenitor cell numbers and proliferative status. We followed the variation of the cell proliferation rate in bone marrow and spleen in response to acute ethanol intoxication in the MITO-Luc mouse, in which NF-Y-dependent cell proliferation can be assessed in vivo by non-invasive bioluminescent imaging. One week after ethanol administration, bioluminescent signals in bone marrow and spleen decreased below the level corresponding to physiological proliferation, and they progressively resumed to pre-treatment values in approximately 4 weeks. Boosting acetaldehyde catabolism by administration of an aldehyde dehydrogenase activity activator or administration of polyphenols with antioxidant activity partially restored bone marrow cells' physiological proliferation. These results indicate that in this mouse model, bioluminescent alteration reflects the reduction of the physiological proliferation rate of bone marrow progenitor cells due to the toxic effect of aldehydes generated by alcohol oxidation. In summary, this study presents a novel view of the impact of acute alcohol intake on bone marrow cell proliferation in vivo.

HIPK2 is a potential predictive marker of a favorable response for adjuvant chemotherapy in stage II colorectal cancer.

Colorectal cancer (CRC) is the third most frequently diagnosed type of cancer worldwide. Stage II CRC accounts for ~25% all CRC cases and their management after surgical resection remains a clinical dilemma due to the lack of reliable criteria for identifying patients who may benefit from adjuvant chemotherapy. Homeodomain­interacting protein kinase 2 (HIPK2), a multifunctional kinase involved in numerous signaling pathways, serves several key roles in cell response to different types of stresses, including chemotherapy­induced genotoxic damage. In the present study, immunohistochemistry was performed for HIPK2 on a tissue microarray of primary human tumor samples from 84 patients with stage II CRC, treated (30 patients) or not treated (54 patients) with adjuvant chemotherapy, and sequenced for the TP53 gene, a key HIPK2 target in genotoxic damage response. It was observed that, regardless of the TP53 gene status, a high percentage of HIPK2+ cells was associated with therapeutic vulnerability in stage II CRC, suggesting a contribution of HIPK2 to drug­response in vivo. For the in vitro characterization, HIPK2 was depleted in human CRC cells by CRISPR/Cas9 or RNA interference. HIPK2­proficient and HIPK2­defective cells were evaluated for their response to 5­fluorouracil (5­FU) and oxaliplatin (OXA). The results revealed that HIPK2 depletion induced resistance to 5­FU and OXA, and that this resistance was not overcome by brusatol, an inhibitor of the antioxidant response regulator nuclear factor erythroid 2­related factor 2 (NRF2), which is frequently overexpressed in CRC. By contrast, cell sensitivity to 5­FU and OXA was further induced by brusatol supplementation in HIPK2­proficient cells, further supporting the contribution of HIPK2 in chemotherapy response. Overall, the present results suggested that HIPK2 may be a potential predictive marker for adjuvant­treated stage II CRC and for prospective therapy with NRF2 modulators.

Regenerative medicine approaches for the management of respiratory tract fistulas.

Respiratory tract fistulas (or fistulae) are abnormal communications between the respiratory system and the digestive tract or the adjacent organs. The origin can be congenital or, more frequently, iatrogenic and the clinical presentation is heterogeneous. Respiratory tract fistulas can lead to severely reduced health-related quality of life and short survival. Therapy mainly relies on endoscopic surgical interventions but patients often require prolonged hospitalization and may develop complications. Therefore, more conservative regenerative medicine approaches, mainly based on lipotransfer, have also been investigated. Adipose tissue can be delivered either as unprocessed tissue, or after enzymatic treatment to derive the cellular stromal vascular fraction. In the current narrative review, we provide an overview of the main tissue/cell-based clinical studies for the management of various types of respiratory tract fistulas or injuries. Clinical experience is limited, as most of the studies were performed on a small number of patients. Albeit a conclusive proof of efficacy cannot be drawn, the reviewed studies suggest that grafting of adipose tissue-derived material may represent a minimally invasive and conservative treatment option, alternative to more aggressive surgical procedures. Knowledge on safety and tolerability acquired in prior studies can lead to the design of future, larger trials that may exploit innovative procedures for tissue processing to further improve the clinical outcome.

An Alternative Splice Variant of HIPK2 with Intron Retention Contributes to Cytokinesis.

HIPK2 is a DYRK-like kinase involved in cellular stress response pathways, development, and cell division. Two alternative splice variants of HIPK2, HIPK2-FL and HIPK2-Δe8, have been previously identified as having different protein stability but similar functional activity in the stress response. Here, we describe one additional HIPK2 splice variant with a distinct subcellular distribution and functional activity in cytokinesis. This novel splice variant lacks the last two exons and retains intron13 with a stop codon after 89 bp of the intron, generating a short isoform, HIPK2-S, that is detectable by 2D Western blots. RT-PCR analyses of tissue arrays and tumor samples show that HIPK2-FL and HIPK2-S are expressed in normal human tissues in a tissue-dependent manner and differentially expressed in human colorectal and pancreatic cancers. Gain- and loss-of-function experiments showed that in contrast to HIPK2-FL, HIPK2-S has a diffuse, non-speckled distribution and is not involved in the DNA damage response. Rather, we found that HIPK2-S, but not HIPK2-FL, localizes at the intercellular bridge, where it phosphorylates histone H2B and spastin, both required for faithful cell division. Altogether, these data show that distinct human HIPK2 splice variants are involved in distinct HIPK2-regulated functions like stress response and cytokinesis.

TRF2 and VEGF-A: an unknown relationship with prognostic impact on survival of colorectal cancer patients.

BACKGROUND: Colorectal cancer is one of most common tumors in developed countries and, despite improvements in treatment and diagnosis, mortality rate of patients remains high, evidencing the urgent need of novel biomarkers to properly identify colorectal cancer high-risk patients that would benefit of specific treatments. Recent works have demonstrated that the telomeric protein TRF2 is over-expressed in colorectal cancer and it promotes tumor formation and progression through extra-telomeric functions. Moreover, we and other groups evidenced, both in vitro on established cell lines and in vivo on tumor bearing mice, that TRF2 regulates the vascularization mediated by VEGF-A. In the present paper, our data evidence a tight correlation between TRF2 and VEGF-A with prognostic relevance in colorectal cancer patients. METHODS: For this study we sampled 185 colorectal cancer patients surgically treated and diagnosed at the Regina Elena National Cancer Institute of Rome and investigated the association between the survival outcome and the levels of VEGF-A and TRF2. RESULTS: Tissue microarray immunohistochemical analyses revealed that TRF2 positively correlates with VEGF-A expression in our cohort of patients. Moreover, analysis of patients' survival, confirmed in a larger dataset of patients from TCGA, demonstrated that co-expression of TRF2 and VEGF-A correlate with a poor clinical outcome in stage I-III colorectal cancer patients, regardless the mutational state of driver oncogenes. CONCLUSIONS: Our results permitted to identify the positive correlation between high levels of TRF2 and VEGF-A as a novel prognostic biomarker for identifying the subset of high-risk colorectal cancer patients that could benefit of specific therapeutic regimens.

Extracellular Vesicles-Encapsulated MicroRNA-125b Produced in Genetically Modified Mesenchymal Stromal Cells Inhibits Hepatocellular Carcinoma Cell Proliferation.

Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer and one of the prominent causes of cancer mortality, leading to approximately 780,000 deaths per year worldwide. Down-regulation of microRNA-125b (miR-125b) is a prognostic indicator in HCC patients. Conversely, over-expression of miR-125b in HCC cells induces cell cycle arrest, inhibits proliferation, migration and invasion. Extracellular vesicles (EVs) function as intercellular messengers transferring proteins, RNAs, DNAs, carbohydrates, and lipids. Since EVs protect their cargo from degradation, delivery of therapeutic bioactive molecules, in particular miRNAs, through EVs represents an innovative avenue for cancer therapy. In this study, we evaluated a replacement strategy for the treatment of HCC via delivery of EVs secreted from human adipose tissue-derived mesenchymal stromal/medicinal signaling cells (ASCs) genetically modified with a lentiviral vector expressing miR-125b with a specific ExoMotif sequence tag to enhance the loading into extracellular vesicles. In particular, we determined that the delivery of miR-125b-loaded EVs produced in engineered ASCs specifically reduces HCC cell proliferation in vitro modulating a series of miR-125b targets, which belong to the p53 signaling pathway. This proof-of-concept study supports the development of innovative therapeutic strategies for HCC via EV-mediated miRNA delivery.

Effects of Copper Chelation on BRAFV600E Positive Colon Carcinoma Cells.

High affinity copper binding to mitogen-activated protein kinase kinase 1 (MAP2K1, also known as MEK1) allosterically promotes the kinase activity of MEK1/2 on extracellular signal regulated kinases 1 and 2 (ERK1/2). Consequently, copper-dependent activation of the mitogen-activated (MAP) kinase pathway has a role in promoting tumor growth. Conversely, copper chelation may represent a possible therapeutic approach for a specific subset of tumors characterized by activating mutations in the serine/threonine protein kinase V-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF), such as the V600E, occurring within the kinase domain (BRAFV600E). Tetrathiomolybdate (TM) is a specific copper chelating agent currently used for the treatment of Wilson's disease and in preclinical studies for the management of metastatic cancers owing to its anti-angiogenic and anti-inflammatory properties. We evaluated in vitro and in vivo the effects of copper depletion achieved by pharmacological treatment with TM in human colorectal cells bearing the BRAFV600E mutation in comparison with BRAF wild type cells. We provide evidence that selective copper chelation differentially affects proliferation, survival and migration of colon cancer cells bearing the BRAFV600E mutation compared to BRAFwt acting via differential phosphorylation levels of ERK1/2. Moreover, tetrathiomolybdate treatment was also effective in reducing the clonogenic potential of colon cancer BRAFV600E cells resistant to BRAF pharmacological inhibition. In conclusion, these results support further assessment of copper chelation therapy as an adjuvant therapy for inhibiting the progression of colon cancers containing the BRAFV600E mutation.

p53 mitotic centrosome localization preserves centrosome integrity and works as sensor for the mitotic surveillance pathway.

Centrosomal p53 has been described for three decades but its role is still unclear. We previously reported that, in proliferating human cells, p53 transiently moves to centrosomes at each mitosis. Such p53 mitotic centrosome localization (p53-MCL) occurs independently from DNA damage but requires ATM-mediated p53Ser15 phosphorylation (p53Ser15P) on discrete cytoplasmic p53 foci that, through MT dynamics, move to centrosomes during the mitotic spindle formation. Here, we show that inhibition of p53-MCL, obtained by p53 depletion or selective impairment of p53 centrosomal localization, induces centrosome fragmentation in human nontransformed cells. In contrast, tumor cells or mouse cells tolerate p53 depletion, as expected, and p53-MCL inhibition. Such tumor- and species-specific behavior of centrosomal p53 resembles that of the recently identified sensor of centrosome-loss, whose activation triggers the mitotic surveillance pathway in human nontransformed cells but not in tumor cells or mouse cells. The mitotic surveillance pathway prevents the growth of human cells with increased chance of making mitotic errors and accumulating numeral chromosome defects. Thus, we evaluated whether p53-MCL could work as a centrosome-loss sensor and contribute to the activation of the mitotic surveillance pathway. We provide evidence that centrosome-loss triggered by PLK4 inhibition makes p53 orphan of its mitotic dock and promotes accumulation of discrete p53Ser15P foci. These p53 foci are required for the recruitment of 53BP1, a key effector of the mitotic surveillance pathway. Consistently, cells from patients with constitutive impairment of p53-MCL, such as ATM- and PCNT-mutant carriers, accumulate numeral chromosome defects. These findings indicate that, in nontransformed human cells, centrosomal p53 contributes to safeguard genome integrity by working as sensor for the mitotic surveillance pathway.

Intraoperative Strategies for Minimal Manipulation of Autologous Adipose Tissue for Cell- and Tissue-Based Therapies: Concise Review.

The stromal vascular fraction (SVF) is a heterogeneous population of stem/stromal cells isolated from perivascular and extracellular matrix (ECM) of adipose tissue complex (ATC). Administration of SVF holds a strong therapeutic potential for regenerative and wound healing medicine applications aimed at functional restoration of tissues damaged by injuries or chronic diseases. SVF is commonly divided into cellular stromal vascular fraction (cSVF) and tissue stromal vascular fraction (tSVF). Cellular SVF is obtained from ATC by collagenase digestion, incubation/isolation, and pelletized by centrifugation. Enzymatic disaggregation may alter the relevant biological characteristics of adipose tissue, while providing release of complex, multiattachment of cell-to-cell and cell-to-matrix, effectively eliminating the bioactive ECM and periadventitial attachments. In many countries, the isolation of cellular elements is considered as a "more than minimal" manipulation, and is most often limited to controlled clinical trials and subject to regulatory review. Several alternative, nonenzymatic methods of adipose tissue processing have been developed to obtain via minimal mechanical manipulation an autologous tSVF product intended for delivery, reducing the procedure duration, lowering production costs, decreasing regulatory burden, and shortening the translation into the clinical setting. Ideally, these procedures might allow for the integration of harvesting and processing of adipose tissue for ease of injection, in a single procedure utilizing a nonexpanded cellular product at the point of care, while permitting intraoperative autologous cellular and tissue-based therapies. Here, we review and discuss the options, advantages, and limitations of the major strategies alternative to enzymatic processing currently developed for minimal manipulation of adipose tissue. Stem Cells Translational Medicine 2019;8:1265&1271.

Molecular mechanisms of cardiomyocyte regeneration and therapeutic outlook.

Differently from some lower vertebrates, which can completely regenerate their heart, in higher vertebrates cardiac injury generally leads to progressive failure. Induction of cycle re-entry in terminally differentiated cardiomyocytes and stem-cell transplantation are strategies to increase the regenerative potential of the heart. As experimental and clinical studies progress, demonstrating that adult stem-cell administration has a favorable impact on myocardial function, the identification of cardiac stem cells suggests that some endogenous repair mechanisms actually exist in the mammalian heart. However, a deeper understanding of the mechanism that drives cardiomyocyte proliferation and stem-cell-mediated cardiac repair is required to translate such strategies into effective therapies.

Protein disulfide isomerase as a prosurvival factor in cell therapy for muscular and vascular diseases.

BACKGROUND: Cell therapy for degenerative diseases aims at rescuing tissue damage by delivery of precursor cells. Thus far, this strategy has been mostly unsuccessful due to massive loss of donor cells shortly after transplantation. Several strategies have been applied to increase transplanted cell survival but only with limited success. The endoplasmic reticulum (ER) is an organelle involved in protein folding, calcium homeostasis, and lipid biosynthesis. Protein disulfide isomerase (PDI) is a molecular chaperone induced and activated by ER stress. PDI is induced by hypoxia in neuronal, cardiac, and endothelial cells, supporting increased cell survival to hypoxic stress and protection from apoptosis in response to ischemia. METHODS: We achieved ex vivo PDI gene transfer into luciferase-expressing myoblasts and endothelial cells. We assessed cell engraftment upon intramuscular transplantation into a mouse model of Duchenne muscular dystrophy (mdx mouse) and into a mouse model of ischemic disease. RESULTS: We observed that loss of full-length dystrophin expression in mdx mice muscle leads to an increase of PDI expression, possibly in response to augmented ER protein folding load. Moreover, we determined that overexpression of PDI confers a survival advantage for muscle cells in vitro and in vivo to human myoblasts injected into murine dystrophic muscle and to endothelial cells administered upon hindlimb ischemia damage, improving the therapeutic outcome of the cell therapy treatment. CONCLUSIONS: Collectively, these results suggest that overexpression of PDI may protect transplanted cells from hypoxia and other possibly occurring ER stresses, and consequently enhance their regenerative properties.