Viability of Fat Cells in Frozen Fat Tissue in Relation to Thawing Technique.

Damage of frozen fat, which will be used for retransplantation, is inevitable. Reuse of frozen fat requires a thawing process. No standardized method has yet been established for thawing frozen fat. Methods: Microscopic analysis of count and viability of frozen fat of 21 patients. Two fat samples from each patient were harvested and frozen at -20°C in a common commercial refrigerator for different freezing durations. Thawing of fat samples was done. There was one (3 mL) sample for each thawing technique; technique A included natural thawing at 25°C for 15 minutes, while rapid thawing at 37°C for 10 minutes in a water bath was included in technique B. Survival rates of adipocytes were assessed with trypan blue staining. Culturing of adipose-derived stem cells to assess their ability to divide was done. Relating survival rate of frozen fat to patients' age and to duration of freezing was done. Results were statistically analyzed. Results: The count of viable adipocytes is higher in technique A. Adipose-derived stem cells of frozen fat do not have the ability to divide in culture media. Viable adipocytes were higher in younger ages and in shorter freezing duration. Conclusion: Natural thawing is better in maintaining frozen adipocyte viability. Younger patients will benefit from frozen fat more than older ones. Duration of freezing should not exceed 7 months.

Effects of Mesenchymal Stem Cells Therapy on Cardiovascular Risk Factors in Experimental Diabetic Kidney Disease.

BACKGROUND: Diabetic kidney disease (DKD) is a progressive kidney disease and a leading cause of end-stage renal disease (ESRD). Diabetic kidney disease has been strongly associated with increased risk of cardiovascular morbidity and mortality. Despite their susceptibility to cardiovascular diseases (CVDs), patients with DKD are less likely to receive appropriate cardiovascular risk modification as they are generally excluded from major cardiovascular trials. Awareness of vulnerability of these patients necessitates investigating potential interventions that would lessen their risk of adverse outcomes. OBJECTIVES: This study aimed to explore the effect of bone marrow-derived mesenchymal stem cells (MSCs) in modulating cardiovascular risk factors that develop with the progression of DKD. METHODS: A total of 60 adult female albino rats were allocated into 3 groups: control group, untreated DKD group, and mesenchymal stem cells-treated diabetic kidney disease (MSCs-DKD) group. Blood pressure, blood glucose level, lipid profile, and atherogenic index were used to assess cardiovascular risk. All rats were killed and subjected to in vitro aortic reactivity studies 8 weeks after induction of diabetes. The MSCs-DKD rats received a single intravenous injection of MSCs 4 weeks after diabetes induction. RESULTS: Mesenchymal stem cells injection significantly decreased blood pressure, atherogenic index, and blood glucose compared with untreated rats. The MSCs-DKD aorta also exhibited significant enhancement of vascular reactivity parameters despite absence of improvement in kidney function. These findings conformed to tracked MSCs, which were found residing in aortic and pancreatic tissues and absent in kidneys. CONCLUSIONS: Mesenchymal stem cells hold hope of improving cardiovascular risk and mortality in patients with DKD, particularly those deteriorating to ESRD.

CONTEXTE: La néphropathie diabétique (ND) est une maladie rénale évolutive constituant une des principales causes d'insuffisance rénale terminale (IRT). Il existe une forte corrélation entre la ND et un risque accru de morbidité et de mortalité cardiovasculaire. Malgré leur vulnérabilité, les patients atteints d'IRT sont moins susceptibles de bénéficier d'une modification appropriée des risques cardiovasculaires puisqu'ils sont souvent exclus des essais portant sur les maladies cardiovasculaires. Prendre conscience de leur vulnérabilité nécessite d'étudier les interventions potentielles susceptibles de réduire le risque d'effets indésirables chez ces patients. OBJECTIF: Cette étude visait à explorer l'effet des cellules souches mésenchymateuses (CSM) dérivées de la moelle osseuse dans la modulation des facteurs de risques cardiovasculaires qui se développent avec la progression de la néphropathie diabétique. MÉTHODOLOGIE: Des rates albinos adultes (n=60) ont été réparties en trois groupes: un groupe témoin, un groupe non traité atteint de ND, et un groupe atteint de ND traité aux CSM. Le risque de maladies cardiovasculaires a été évalué selon le bilan lipidique, l'indice d'athérogénicité et les valeurs de pression artérielle et de glycémie. Huit semaines après l'induction du diabète, toutes les rates ont été sacrifiées et soumises à des études in vitro de réactivité aortique. Les rates du groupe traité avaient reçu une dose unique de CSM par intraveineuse quatre semaines après l'induction du diabète. RÉSULTATS: L'injection de CSM a réduit l'indice d'athérogénicité et les valeurs de pression artérielle et de glycémie de façon significative chez les rates traitées comparativement au groupe non traité. L'étude de réactivité aortique des rates traitées aux CSM a également montré une amélioration significative des paramètres de réactivité vasculaire malgré l'absence d'amélioration de la fonction rénale. Ces résultats étaient conformes aux CSM suivies, retrouvées dans les tissus aortiques et pancréatiques et absentes des reins. CONCLUSION: Les cellules souches mésenchymateuses offrent un espoir pour la réduction des risques de maladies et de mortalité cardiovasculaires chez les patients atteints de néphropathie diabétique, particulièrement chez ceux dont l'état évolue vers l'insuffisance rénale terminale.

In Vitro Comparative Study of Emulsified Fat Grafts.

Introduction: Fat grafting is considered one of the most precious armamentarium in the hand of plastic surgeons. The fat grafts consist of 2 components, adipocytes and stromal cells. The adipose tissue is a reserve of stem cells. Aim: The aim of this study was to compare the adipocyte and stem cell viability in both mechanically processed and enzymatically digested fats. Patients and Methods: This in vitro study was conducted using 40 specimens collected from 20 patients who underwent liposuction procedures. Twenty specimens were mechanically processed (group A), whereas the remaining specimens were processed enzymetically (group B). Results: There were no statistically significant differences between fat cell viability between the 2 groups. On the contrary, there was statistically significant increase in stem cells in mechanically processed fat specimens (P = .001). Conclusion: Both the mechanically and chemically processed fat techniques are reliable techniques that provide fat and stem cells. Mechanical processing is easier and provides more stem cells.

Immune-Modulatory Changes After Transplantation Therapy of Insulin Producing Cells Derived from Wharton's Jelly Human Umbilical Cord-Mesenchymal Stem Cells in Diabetes Induced Rats.

Diabetes Mellitus (D.M.) is a disease with a high and increasing prevalence. The Insulin- producing Cells (IPCs) derived from the Wharton's jelly of human umbilical cord transplantation was thought to be the most promising strategy for treating Diabetes. This study aimed to evaluate IPCs immune modulatory changes occurred after transplanted through two different routes and the effect of these changes on their therapeutic efficiency in relation to transplantation microenvironment. Insulin Producing Cells was induced to differentiate from human Umbilical Cord-Mesenchymal Stem Cells and characterized by morphology under phase contrast inverted microscope and staining of secretory granules by DTZ (diphenylthiocarbonazone) stain, then therapeutic effect was evaluated both in vitro and in vivo through glucose challenge test and hyperglycemia correction in STZ (streptozotocin)- induced diabetic rats. Immune-modulatory changes evaluated by cell- mediated lysis assay and Syber green quantification of immune inflammatory cytokines (IFN- , TGF- ß and IL-10) gene expression by real-time PCR. We observed that in spite of the weak immunogenicity of induced IPCs derived from HUC-MSCs in vitro, but when transplanted in vivo especially through the intra portal vein they could induce an immune response when interact with the disease microenvironment resulting in different degree of inflammatory response. Therefore, the relationship between disease microenvironment and immune alteration should be examined before transplantation therapy.

Evaluation of the diagnostic and therapeutic roles of non-coding RNA and cell proliferation related gene association in hepatocellular carcinoma.

Micro RNA-34a-5p (miR-34a-5p) is an important molecule that can act as a modulator of tumor growth. It controls expression of a plenty of proteins controlling cell cycle, differentiation and apoptosis and opposing processes that favor viability of cancer cells, their metastasis and resistance to chemotherapy. Bioinformatics analysis indicated that minichromosome maintenance protein 2 (MCM2) is a target gene of miR-34a-p. In this study, RT-qPCR was employed to detect the expression of miR-34a-5p and MCM2 in 10 hepatocellular carcinoma (HCC) tissues. The functional role of miR-34a-5p in HCC was investigated and the interaction between miR-34a-5p and MCM2 was explored. Results showed miR-34a-5p expression in HCC tissues was significantly lower than in non HCC liver tissues (P < 0.05), but MCM2 expression in HCC tissues was markedly higher than in non HCC liver tissues (P < 0.05). In addition, miR-34a-5p expression was negatively related to MCM2 expression. To confirm effect of miR-34a-5p on tumor growth and its possible effect on MCM2, miR-34a-5p mimic and inhibitor was transfected into HCC cell lines (HepG2). MTS assay, showed miR-34a-5p over-expression could inhibit the proliferation of HCC cells. RT-qPCR was done to detect the expression of miR-34a-5p and MCM2 in HepG2 cells before and after transfection. Results showed that MCM2 expression in HCC tissues was markedly lower in mimic transfected group than in inhibitor transfected group and control group (P < 0.05) while miR-34a-5p expression in HepG2 cells was significantly higher in mimic transfected group than in inhibitor transfected group and control group (P < 0.05). Thus, miR-34a-5p may inhibit the proliferation of HCC cells via regulating MCM2 expression. These findings provide an evidence for the emerging role of microRNAs as diagnostic markers and therapeutic targets in HCC.

Effect of Bone Marrow-Derived Mesenchymal Stem Cells on Ischaemic-Reperfused Hearts in Adult Rats with Established Chronic Kidney Disease.

BACKGROUND AND OBJECTIVES: Bone marrow-derived mesenchymal stem cells (BM-MSCs) are adult multipotent non-haematopoietic stem cells that have regeneration potential. The current study aimed to detect the ability of BM-MSCs to improve kidney and cardiac functions in adult rats with established chronic kidney disease. METHODS: Rats were divided into sham-operated control, untreated sub totally nephrectomised and treated sub totally nephrectomised groups. Body weight, kidney and cardiac tissue weights, plasma creatinine and urea levels and arterial blood pressure were measured. ECG was recorded, and an in vitro isolated heart study was performed. RESULTS: Stem cell treatment decreased the elevated plasma creatinine and urea levels and decreased systolic, diastolic and mean arterial blood pressure values. These changes were accompanied by a decrease in glomerular hypertrophy with apparent normal renal parenchyma. Additionally, BM-MSCs shortened Q-To and Q-Tc intervals, all time to peak tension values, the half relaxation value at 30 min of reperfusion and the contraction time at 15 and 30 min of reperfusion. Moreover, stem cell treatment significantly increased the heart rate, QRS voltage, the peak tension at the 15- and 30-min reperfusion time points and the peak tension per left ventricle at the 30-min reperfusion time point compared to the pre-ischaemia baseline. BM-MSCs resolve inter muscular oedema and lead to the re-appearance of normal cardiomyocytes. This improvement occurs with the observations of BM-MSCs in renal and heart tissues. CONCLUSIONS: BM-MSCs can attenuate chronic kidney disease progression and the associated cardiac electrophysiological and inotropic dysfunction.

Bone marrow derived-mesenchymal stem cells downregulate IL17A dependent IL6/STAT3 signaling pathway in CCl4-induced rat liver fibrosis.

Therapeutic potential of bone marrow-derived mesenchymal stem cells (BM-MSCs) has been reported in several animal models of liver fibrosis. Interleukin (IL) 17A, IL6 and Stat3 have been described to play crucial roles in chronic liver injury. However, the modulatory effect of MSCs on these markers was controversial in different diseases. BM-MSCs might activate the IL6/STAT3 signaling pathway and promote cell invasion in hepatocellular carcinoma, but the immunomodulatory role of BM-MSCs on IL17A/IL6/STAT3 was not fully elucidated in liver fibrosis. In the present study, we evaluated the capacity of the BM-MSCs in the modulation of cytokines milieu and signal transducers, based on unique inflammatory genes Il17a and Il17f and their receptors Il17rc and their effect on the IL6/STAT3 pathway in CCl4-induced liver fibrosis in rats. A single dose of BM-MSCs was administered to the group with induced liver fibrosis, and the genes and proteins of interest were evaluated along six weeks after treatment. Our results showed a significant downregulation of Il17a, Il17ra, il17f and Il17rc genes. In accordance, BM-MSCs administration declined IL17, IL2 and IL6 serum proteins and downregulated IL17A and IL17RA proteins in liver tissue. Interestingly, BM-MSCs downregulated both Stat3 mRNA expression and p-STAT3, while Stat5a gene was downregulated and p-STAT5 protein was elevated. Also P-SMAD3 and TGFßR2 proteins were downregulated in response to BM-MSCs treatment. Collectively, we suggest that BM-MSCs might play an immunomodulatory role in the treatment of liver fibrosis through downregulation of IL17A affecting IL6/STAT3 signaling pathway.

Comparative Study on Acellular Dermal Graft Versus Propylene Mesh Both Either Loaded or Unloaded with BM-MSCs in Healing of Skull Bone Defect in Rats: Histological and Immunohistochemical Study.

Bone defect occurs as a consequence of many conditions. Diseased bones don't heal properly and defects in face area need proper bone reconstruction to avoid psychological and social problems. Tissue engineering is an emerging new modality of treatment. We thought to study different methods to fill skull bone defect in rats in order to find the most safe and effective method. So, this study was designed to evaluate the efficacy of acellular dermal graft (ADM) versus propylene mesh both either loaded or unloaded with bone marrow derived mesenchymal stem cells (BM-MSCs) in healing of skull bone defect of a 5 mm diameter. The study included 36 adult male Wistar albino rats that were divided into three groups according to the way of filling skull bone defect. Group I: Ia (sham control), Ib (negative control). Group II: IIa (unseeded propylene), IIb (seeded propylene) and Group III: IIIa (unseeded ADM), IIIb (seeded ADM). The trephine operation was done on the left parietal bone. Specimens were collected four weeks postoperative and processed for H&E, osteopontin immunohistochemistry and scanning electron microscope. Morphometric and statistical analysis were also performed. After studying the results of the experiment, we found that propylene mesh and ADM were suitable scaffolds that could support new bone formation in clavarial bone defect. Healing of skull bone defect was better in rats that received seeded scaffolds more than rats with unseeded scaffolds. The seeded ADM showed significant increase in bone forming activity as confirmed by histomorphometric and statistical results.

Immunomodulatory capacity of the local mesenchymal stem cells transplantation after severe skeletal muscle injury in female rats.

CONTEXT: Cell therapy technique with stem cells is a very attractive strategy for the treatment of muscle disorders. OBJECTIVE: The objective of this study was to investigate the mechanism of local transplantation of mesenchymal stem cells (MSCs) which could contribute to skeletal muscle healing. MATERIALS AND METHODS: Female rats were divided into three equal groups as the following: group 1, the negative control group (untreated group), group 2, sham-treated group, rats with muscle injuries involving volumetric muscle loss (VML) of adductor brevis muscle and injected locally with phosphate-buffered saline (PBS) 0.5 ml without stem cells after 7 d of muscle injury, group 3, treated group, rats with VML and injected locally (intramuscular) with 1.5 × 106 bone marrow MSCs suspended in PBS 0.5 ml (1) after 7 d of muscle tissue injury. All animals were sacrificed after 4 weeks of stem cell transplantation. RESULTS: In vitro culture the morphology of MSCs reached confluence and appeared as long spindle in shape on 9-14 d. Most of the cells did not express the hematopoietic cell marker, CD34 and CD45 but expressed MSCs marker CD44, CD90 and CD105. The remarkable increase of proliferating cell nuclear antigen positive nucleus was recorded in MSCs group as compared to PBS group. After 28 d of injection, administration of only PBS into the site of muscle injury caused up-regulation in the levels of interleukins IL-1ß, IL-6, tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-ß1), interferon alpha (IFN-α) and down-regulate the level of IL-10 in muscular tissue comparing to the untreated control. Bone marrow MSCs + PBS injected at the site of muscle injury significantly down-regulate the inflammatory cytokines levels IL-1ß and IL-6 and TNF-α, TGF-ß1 and IFN-α and up-regulate the level of IL-10. Collagen concentrations in the injured skeletal muscle estimated by enzyme-linked immuno sorbent assay and stained with Masson trichrome stain were increased with PBS group and decreased after transplantation of bone marrow MSCs in the site of injury. Muscle sections stained with H&E showed a higher number of centronucleated regenerating myofibers in the stem-cell-treated group than in the (PBS) and untreated control group. Microvasculature of skeletal muscle was decreased as demonstrated by immunostaining technique for CD34 in PBS group from untreated control. The MSCs group showed angiogenesis and marked increase of skeletal muscle microvasculature than PBS group. CONCLUSION: MSCs can modify the local immunological responses and improve muscle regeneration by suppressing of inflammatory cytokines, activating of the anti-inflammatory cytokine, restoration of muscle fibers and angiogenesis. By means of increase in TGF-ß production in response to muscle injury prevent the repair of injured fibers and increase connective tissue production (collagen fibers), thus propagating skeletal muscle weakness and fibrosis whereas MSCs + PBS injected at the site of muscle injury significantly down-regulate (TGF-ß1) and hence the level of collagen (fibrosis or scar areas). MSCs are able to block the fibrotic signaling cascade by declining TGF-ß1 and scar areas in the injured muscle.

Therapeutic Potential of Mesenchymal Stem Cells on Early and Late Experimental Hepatic Schistosomiasis Model.

Cell-based therapy is emerging as a promising therapeutic approach for a wide range of liver diseases. This study aimed to investigate the regenerative and antifibrotic therapeutic potential of bone marrow-derived mesenchymal stem cells (BM-MSCs) in an early and late experimental hepatic schistosomiasis model. BM-MSCs were isolated from 6-wk-old BALB/c donor male mice, then grown and propagated in culture until cell count was 5-8 × 10(6)/ml. MSCs were then separated and injected into Schistosoma mansoni -infected female BALB/c mice on their 6, 10, 14, and 18 wk post-infection. Mice were sacrificed on the fourth and eighth week after BM-MSCs transplantation in each group. Homing of BM-MSCs was confirmed by PCR detection of male Y-chromosome gene (sry) in the liver tissue of the recipient female mice. The regenerative and antifibrotic potential of BM-MSCs was assessed by histopathological examination, morphometric analysis, electron microscopy, and liver function tests. Schistosoma-infected mice, which were treated with BM-MSCs, showed a decrease in the granuloma size, percentage and density of the fibrotic area, formation of new hepatocytes, and improvement of the liver function tests. Immunohistochemical examination of alpha-smooth muscle actin revealed a significant decrease in the immunoreactive hepatic stellate cells in mice treated with MSCs. Early granulomas (acute infection) showed better response to MSC injection than did later granulomas (chronic infection). Dosing and timing of MSCs transplantation should undergo more investigations in long-term experiments before application to the clinical field. This study is the first to assess and compare the effect of MSCs treatment on early and late granulomas.

Systematic review of appropriate cognitive assessment instruments used in clinical trials of schizophrenia, major depressive disorder and bipolar disorder.

Cognitive dysfunction is increasingly recognized as a symptom in mental conditions including schizophrenia, major depressive disorder (MDD), and bipolar disorder (BPD). Despite the many available cognitive assessment instruments, consensus is lacking on their appropriate use in clinical trials. We conducted a systematic literature review in Embase, PubMed/Medline and PsychINFO to identify appropriate cognitive function instruments for use in clinical trials of schizophrenia, MDD, and BPD. Instruments were identified from the articles. Instruments and articles were excluded if they did not address schizophrenia, MDD, or BPD. Instrument appropriateness was further assessed by the criteria of the Measurement and Treatment Research to Improve Cognition in Schizophrenia (MATRICS) initiative: test-retest reliability, utility, relationship to functional status, potential changeability to pharmacological agents, and tolerability and practicality for clinical trials. The database search yielded 173 articles describing 150 instruments used to assess cognitive function. Seventeen additional instruments were identified through Google and clinicaltrials.gov. Among all these, only 30 (18%) were deemed appropriate for use in the diseases of interest. Of these, 27 were studied in schizophrenia, one in MDD and two in BPD. These findings suggest the need for careful selection of appropriate cognitive assessment instruments, as not all may be valid in these disorders.