Comparison of Harvesting and Processing Technique for Adipose Tissue Graft: Evaluation of Cell Viability

Background: Clinical studies demonstrated the efficacy of therapies based on the autologous grafting of adult mesenchymal stem cells to accelerate the healing and regenerative processes of the skin and mesenchymal tissues; therefore, it is considered a valuable approach in the aesthetic rejuvenation treatment to give volumization and skin regeneration effects. Objective: The aim of the project consisted of the control of cell viability of adipose tissue (AT) harvested using the two types of cannulas having 0.8 mm and 1 mm side port holes. The results were compared with tissue harvested with a standard liposuction technique and processed with a standard procedure consisting of enzymatic digestion (collagenase). Methods: This study was performed on adipose tissues harvested from 7 patients (6 females and 1 male) with an average age of 48.5 years with 3 different techniques. We compared the cell vitality of every sample at T0 and T72. Results: Lipoaspirate tissue-derived by 0.8- and 1 mm cannula from all samples proved to be vital and possess viable cells. The average absorbance was similar immediately after plating (T0) and 72 hours after (T72) for the two cannulas, 0.8- and 1 mm cannula. The two systems proved to equally harvest vital tissue. An increase in cell viability was observed in all samples for each condition (0.8-, 1 mm and enzymatic digestion). Conclusion: This study proved that guided harvested adipose tissue with small cannulas with small side port holes yields a comparable amount of viable cells compared to adipose tissue harvested with a liposuction system and processed with enzymatic digestion (collagenase). This study confirms that the minimally invasive technique and minimal manipulation of the adipose tissue could yield a tissue with a good amount of viable cells. This micro fragmented adipose tissue is a promising source for regenerative treatments.

Extracellular Matrix Expression in Human Induced Pluripotent Stem Cell-Derived Optic Vesicles

Background: Human tissue/organ development is a complex, highly orchestrated process, regulated in part by the surrounding extracellular matrix (ECM). Every complex tissue, including the retina, has a unique ECM configuration that plays a critical role in cellular differentiation, adhesion, migration, and maturation. Aim: To characterize ECM expression of human induced pluripotent stem cell-derived optic vesicles (iPSC-OVs). Methods: A 3- dimensional (3D) in vitro suspension culture system was used to direct differentiation of human induced pluripotent stem cells (iPSCs) into optic vesicles (OVs). Stepwise differentiation of iPSCs into retinal progenitor cells was confirmed by sequential expression of OTX2, SOX1, SIX6, LHX2, PAX6, and CHX10. Expression of ECM genes in iPSC-derived OVs was analyzed by RT2 ProfilerTM PCR Array, whereas immunofluorescence staining was performed to detect ECM proteins in the OVs. Results: A number of cell adhesion molecules (CAMs) previously reported to be abundantly expressed in iPSCs such as E-cadherin, Intercellular adhesion molecule-1 (ICAM1), Integrin-α L, Integrin-α M, Integrin-α 6 were downregulated while neural and retina specific CAMs including neural cell adhesion molecule 1 (NCAM1), neural plakophilin-related armadillo repeat protein (NPRAP), Integrin-α 1 and Integrin-α 4 were upregulated. Several glycoproteins that have been reported to play key roles during retinogenesis, namely CD44, Tenascin C, Tenascin R, Neurocan, Neuroglycan C, Delta 2 Catenin, Vitronectin, and Reelin were also present. Conclusion: We have identified an array of ECM proteins that were expressed during retinogenesis. Further characterization of these proteins will lead to a better understanding of retinal development.

A Phase II Dose Escalation Study of Intraarterial (Hepatic) Adult Human Bone Marrow Derived, Cultured, Pooled, Allogeneic Mesenchymal Stromal Cells (Stempeucel®) in Patients with Alcoholic Liver Cirrhosis

Background: Alcoholic liver cirrhosis is an end-stage alcoholic liver disease with a poor prognosis. The definitive treatment of alcoholic liver cirrhosis is orthotopic liver transplantation, which is expensive, requires long-term immunosuppression and is limited by the supply of organs. Being an unmet medical need, cell therapy is under investigation for alcoholic liver cirrhosis. Aims: This study was designed primarily for assessing the safety and feasibility of administering stempeucel® through the hepatic artery in alcoholic liver cirrhosis and secondarily to assess possible efficacy and dose-response. Methods: Sixty patients with alcoholic cirrhosis (18-65 years/Child-Pugh class B or C/Model for End-Stage Liver Disease score of minimum 10) were planned to be included in 6 groups: 2.5 million cells/kg Body Weight (2.5M Cell) and respective control (2.5M Control); 5 million cells/kg Body Weight (5M Cell) and respective control (5M Control); 7.5 million cells/kg Body Weight (5M Cell) and respective control (7.5M Control) with 10 patients in each group. Cell groups received stempeucel® administered via hepatic artery by catheterization through the femoral artery (Seldinger technique) and Standard Protocol of Care. The control group received Standard Protocol of Care. Patients were followed up at 1 week, 1 month, 3 months and 6 months. Efficacy evaluations included liver function test, Model for End-Stage Liver Disease score, Child-Pugh score, Short Form-36 questionnaire, liver stiffness using Fibroscan (Transient Elastography), and liver volume using Computerized Tomography scan. Results: Stempeucel® injection was well tolerated. Common treatment-emergent adverse events were gastrointestinal disorders, general disorders and administration site conditions and infections and infestations. Most of the treatment-emergent adverse events were unrelated / remotely related to stempeucel®. Thirty serious adverse events occurred in 10 patients (3 in 2.5M Cell, 5 in 5M Cell and one each in control groups). Three patients died due to SAEs: Two in 2.5M and one in 5M Cell group, none were related to stempeucel®. Statistically significant improvement was seen in 2.5M group compared to the control group in Short Form-36 score: bodily pain, mental component summary, vitality and social functioning. Conclusion: Stempeucel® was safe, well-tolerated and subjective improvement in Short Form-36 (bodily pain, mental component summary, vitality and social functioning and mental health) score was seen in the 2.5M cell group.

Infranatant Portion of Microfragmented Adipose Tissue: A Promising Source of SVF for the Management of Androgenetic Alopecia

Aim: The purpose of this article is to prove the possibility to transfer a good amount of cells of the SVF (and ADSCs) in the infranatant portion of microfragmented adipose tissue. Method: The adipose tissue harvesting procedure is performed under local anaesthesia. The adipose tissue was harvested with a 2 mm diameter microperforated cannula with 1 mm side port holes, mounted inside the special patented guide. Both cannula and guide are included in the SEFFIHAIR™ medical device. Once the adipose tissue is harvested, it is gently washed, and it was divided in two specimens: (EMU) the tissue was emulsified with 20 passages from one syringe to another and (CTRL) the tissue didn’t undergo any emulsification. Results: The emulsification procedure liberated alive and proliferating cells and we observed that the specimens derived with a 1 mm side port hole cannula and then emulsified (EMU) showed a higher number of cells in the infranatant part compared to the one derived from the control tissue without any (1 EMU vs. 1EMU infra). Conclusion: The use of microcannulas, in combination with a mechanical digestion by an emulsification procedure and centrifuge, could ease SVF cells isolation for regenerative treatment and could also be performed in a medical facility.

Remdesivir for the Treatment of Severe SARS-CoV-2 (COVID-19): A Systematic Review and Meta-Analysis

Background: Coronavirus Disease in 2019 (COVID-19) is a pandemic caused by SARS-CoV-2 infection. Over 53 million people have been infected with over 1.3 million deaths. However, there is no standard treatment or vaccines to date. Recently, several randomized controlled trials and cohort studies have demonstrated the efficacy of remdesivir for the treatment of severe COVID-19 patients. This is a systematic review and meta-analysis to define its efficacy. Methods: A systematic review was done on databases (PubMed, Embase, Medline, Cochrane) on 9 Nov 2020. Search keywords were remdesivir, COVID-19, SARS-CoV-2, randomized controlled trials and cohort studies. Studies with high-evidence values were selected to evaluate its clinical efficacy in terms of risk ratio, time to clinical improvement, and mortality risk. Subgroup analysis was performed based on baseline hospitalization status, age and ethnicity. Results: Of the 1328 studies, 6 studies were selected and pooled for meta-analysis. Remdesivir was associated with clinical improvement (risk ratio 1.14, 95% CI 1.02-1.28, p=0.02). It shortened the mean time of clinical improvement by 3.32 days (95% CI -4.37 to -2.28, p<0.001). However, its use was not associated with reduced mortality risk (risk ratio 0.75, 95% CI 0.40–1.40). In subgroup analysis, remdesivir was associated with clinical improvement in patients without the need of invasive ventilation (risk ratio 1.90, 95% CI 1.58-2.29, p<0.001; hazard ratio 2.22, 95% CI, 1.64-3.02), and age less than 70 years (risk ratio 2.14, 95% CI 1.39-3.28, p<0.001). Conclusion: Remdesivir is effective in the treatment of severe COVID-19 patients, in particular those without invasive ventilation

Maintaining Optimum Health Requires Longer-Term Stable Vitamin D Concentrations

Humans are constantly invaded by environmental microbes. The body is protected from pathogen attacks by the immune defense system. In 99.8% of the time, our innate immune system is capable of getting rid of these organisms without before these can cause harm. Those who are with weaker immune systems constantly get infections and having chronic diseases. Among many factors contributing to maintaining a robust immune system, vitamin D has the highest impact. It has a major protective effect against acute respiratory infections and subduing both communicable and non-communicable diseases. A healthy person with stronger immunity may not manifest clinical signs and symptoms of COVID-19-silent, asymptomatic carriers of the virus and can be infectious. Whereas not all PCR positive persons are infectious. A rapid response occurs through the innate system that is followed by the adaptive response that lasts a longer period. Vitamin D kick starts both systems. However, the protective immune and other functions are damped in the presence of hypovitaminosis and also when the levels are fluctuating. Thus, the importance of maintaining serum 25(OH)D at a steady level above 30 ng/mL. When maintaining such, among all nutrients vitamin D has the widest benefits to multiple body systems. Thus, this sunshine vitamin (a steroid hormone) has been modulated through evolution to emerge as a key survival mechanism in humans. Nevertheless, vitamin D is not a panacea.

Regenerative Immunology: Pushing Biomaterials Beyond Tissue Repair

In this short review, we discuss how the paradigm in the development of new biomaterials has shifted over the last years and the growing idea that the immune system is of key importance in an effective tissue repair and regeneration. The immune system is currently considered crucial for tissue regeneration, leading to the emerging concept of Regenerative Immunology.

Progesterone Decreased Cell Infiltration in Airways of Systemic Sclerosis Mice Model

Systemic sclerosis (SSc) is the fibrotic autoimmune disease with a higher incidence in women. Lung fibrosis is the most common cause of death in SSc patients. Sex steroids have crucial role in the induction of autoimmune diseases. Progesterone impacts autoimmunity by direct action on parenchymal cells or through its immunomodulatory effect. This study aimed to investigate the effect of progesterone on the cellularity of airways in an animal model of systemic sclerosis. 6 groups of mice were considered in this study. Systemic sclerosis (SSc) was induced in female BALB/c mice by subcutaneous injection of bleomycin for 28 days. For evaluating the effect of Progesterone in SSc model, Progesterone was administered subcutaneously parallel with bleomycin for 28 days or one week after the first administration of bleomycin for 21 days. Further, three control groups were included in this study. On day 29, under lethal anesthesia bronchoalveolar lavage (BAL) was collected and evaluated for cellularity. Our results indicate the increment of cells in BAL of SSc (P<0.0001) mice. Administration of Progesterone for 28 days significantly reduced the infiltrating cells in BALs (P<0.01) of SSc mice. The differential count of BALs indicates that Progesterone reduced the number of lymphocytes (P˂0.05) in SSc mice but did not affect the number of macrophages. Therefore, we conclude that progesterone reduced the inflammatory cells in airways by decreasing the number of lymphocytes.

Designing and Validating a New Method the TUNEL Microwave (TUNEL-MW) for Rapid Quantification of Apoptosis in Islets Following Isolation and Post-Thaw

Background: Among the current quality control assays used in islet transplantation, there is an urgent need for more appropriate assays that measure cell damage via apoptosis that are accurate and rapid. Although the Terminal Uridine Nucleotide End Labeling (TUNEL) is a popular marker for apoptosis, the protocol takes 4 hours to complete. In this regard, microwave assisted histoprocessing, which shortens the time taken for processing, holds promise. Keeping this in mind, a new TUNEL Microwave (TUNEL-MW) method, for rapid quantification of apoptosis, was designed, developed and validated. Method: Two lots of post-thaw isolated human islets cultured for 24 hours, 3 days, 5 days and 7 days i.e. 8 samples, were used for the study. Dewaxed and rehydrated tissues were processed for routine histology, stained with haematoxylin and eosin (H&E) and the conventional TUNEL was carried out as per manufacturer’s instructions. For the TUNEL-MW, kit instructions were modified and microwave-assisted histoprocessing was done. The assessment of apoptotic index (AI%) by light microscopy (LM) was carried out by a pathologist who was completely blinded to the study. Results: The new TUNEL-Microwave (TUNEL-MW) developed by us reduced processing time from 4 hours to 30 minutes (saving 3½ hours). Results were validated by univariate linear regression (r2>0.990), coefficient of variation (<5% between all three methods) and the Bland Altman plot comparing AI% determined by the new TUNEL-MW with the conventional TUNEL and with LM (gold standard). Conclusion: TUNEL Microwave appears to be an ideal method. It is simple and takes just 30 minutes to perform and can therefore be used along with existing quality control measures to rule out or measure apoptosis prior to islet release for islet transplantation.

Olfactory Stem Cells for the Treatment of Spinal Cord Injury: Isolation, Purification and Behaviour in a Plasma Clot Matrix

Cell therapies represent promising strategies to improve neurological functions after spinal cord injury (SCI). Olfactory mucosa (OM) might be an attractive source of multipotent cells for neuroregeneration because olfactory stem cells (OSCs) are resident. The regenerative capacity of OSCs has been demonstrated in animal models and some clinical case reports. Up to now, there are no standard methods for purification, characterization, and delivery of OSCs to the injury site. However, purification and characterization of the grafted cells are prerequisites for clinical use to ensure maximum safety for the patients. In this study, we isolated and purified OSCs from human OM using the neurosphere assay. Subsequently, the cells were characterized, and the behavior of purified OSCs in a plasma clot was investigated. Our study demonstrated that isolated cells from OM form neurospheres, which cells are positive for CD105 (98%) and CD90 (99%) and negative for Epcam (<1%) and MUC5AC (<1%). Purified OSCs were positive for Nestin, CD44 as well as GFAP and showed a lack of CD34 and CD45 expression. OSCs differentiated into neuron-like cells expressing ß-III tubulin. However, differentiation into adipocytes, chondrocytes or osteoblast could not be observed. In addition, OSCs stayed viable and were able to proliferate within the plasma clot. These results highlight OSC as a candidate for autologous transplantation in combination with the plasma clot as a cell carrier in SCI and neurodegenerative disease.