Systematic selection of small molecules to promote differentiation of embryonic stem cells and experimental validation for generating cardiomyocytes.

Small molecules are being increasingly used for inducing the targeted differentiation of stem cells to different cell types. However, until now no systematic method for selecting suitable small molecules for this purpose has been presented. In this work, we propose an integrated and general bioinformatics- and cheminformatics-based approach for selecting small molecules which direct cellular differentiation in the desired way. The approach was successfully experimentally validated for differentiating stem cells into cardiomyocytes. All predicted compounds enhanced expression of cardiac progenitor (Gata4, Nkx2-5 and Mef2c) and mature cardiac markers (Actc1, myh6) significantly during and post-cardiac progenitor formation. The best-performing compound, Famotidine, increased the percentage of Myh6-positive cells from 33 to 56%, and enhanced the expression of Nkx2.5 and Tnnt2 cardiac progenitor and cardiac markers in protein level. The approach employed in the study is applicable to all other stem cell differentiation settings where gene expression data are available.YK and AB thank the European Research Council (ERC Starting Grant 2013 to AB) for funding.This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/cddiscovery.2016.

Mouse and human spermatogonial stem cells

Spermatogonial stem cells (SSCs) are in the beginning of a complex process in which they transmit genetic information from generation to generation. Any failure in this process can result in infertility. It has been suggested that transplantation of spermatogonial stem cells, following their maintenance and culturing, may restore fertility in some infertile patients. Because fertility restoration through SSCs transplantation has been successfully achieved in animal experiments, we hope human studies can follow in the near future. The isolation and cultivation of SSCs help us study their biological characteristics and their application in therapeutic approaches. In this review, we studied spermatogenesis in rodents and humans. We also compared markers and different SSC culture systems in both

A cell-free SDKP-conjugated self-assembling peptide hydrogel sufficient for improvement of myocardial infarction

Biomaterials in conjunction with stem cell therapy have recently attracted attention as a new therapeutic approach for myocardial infarction (MI), with the aim to solve the delivery challenges that exist with transplanted cells. Self-assembling peptide (SAP) hydrogels comprise a promising class of synthetic biomaterials with cardiac-compatible properties such as mild gelation, injectability, rehealing ability, and potential for sequence modification. Herein, we developed an SAP hydrogel composed of a self-assembling gel-forming core sequence (RADA) modified with SDKP motif with pro-angiogenic and anti-fibrotic activity to be used as a cardioprotective scaffold. The RADA-SDKP hydrogel was intramyocardially injected into the infarct border zone of a rat model of MI induced by left anterior descending artery (LAD) ligation as a cell-free or a cell-delivering scaffold for bone marrow mesenchymal stem cells (BM-MSCs). The left ventricular ejection fraction (LVEF) was markedly improved after transplantation of either free hydrogel or cell-laden hydrogel. This cardiac functional repair coincided very well with substantially lower fibrotic tissue formation, expanded microvasculature, and lower inflammatory response in the infarct area. Interestingly, BM-MSCs alone or in combination with hydrogel could not surpass the cardiac repair effects of the SDKP-modified SAP hydrogel. Taken together, we suggest that the RADA-SDKP hydrogel can be a promising cell-free construct that has the capability for functional restoration in the instances of acute myocardial infarction (AMI) that might minimize the safety concerns of cardiac cell therapy and facilitate clinical extrapolation. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Construction of expression vectors carrying mouse peroxisomal protein gene (PeP) with GST and Flag labels

The aim of this study was to construct expression vectors carrying mouse peroxisomal protein gene (PEP-cDNA) in prokaryotic and mammalian expression vectors in chimeric cDNA types, encompassingGST and FLAG with PEP-cDNA. PEP-cDNA was sub-cloned in pGEX6p2 prokaryotic expression vector in order to label this gene with GST to purify PEP protein for further biochemical analysis and identifying related proteins thereafter. FLAG-PEP recombinant DNA was produced and sub-cloned inpUcD3 eukaryotic expression vector to express tagged-PEP protein for transient transfection analysis and identifying intracellular localization of PEP protein in future experiments. PEP-cDNA was amplifiedin different PCR reactions using pEGFP-PEP vector and 2 sets of primers introducing specific restriction sites at the ends of PEP. PCR products with BamHI/SalI restriction sites were treated by restriction enzymes and inserted into the pGEX6p2, downstream of GST tag. PEP-cDNA containingBamHI/ApaI restriction sites and FLAG gene (which amplified using pUcD3-FLAG-PEX3 vector) were used as templates in secondary PCR for amplifying FLAG-PEP recombinant DNA. FLAG-PEP fragment was treated by enzymatic digestion and inserted into the pUcD3 eukaryotic expression vector.pGEX6p2-PEP and pUcD3-FLAG-PEP constructed vectors were transformed into the one shot TOP10 and JM105 bacterial competent cells, respectively. Positive colonies were selected for plasmid preparation. Results confirmed correct amplification of the expected products. PEP-cDNA in both PCRreactions encompasses 630 bp. FLAG fragment containing designed sites was 77 bp and FLAG-PEP fragment was 700 bp. Sequencing of constructed vectors confirmed that PEP-cDNA was tagged appropriately and inserted free of mutation and in frame with GST and FLAG

Two Decades of Global Progress in Authorized Advanced Therapy Medicinal Products: An Emerging Revolution in Therapeutic Strategies

The introduction of advanced therapy medicinal products (ATMPs) to the global pharma market has been revolutionizing the pharmaceutical industry and has opened new routes for treating various types of cancers and incurable diseases. In the past two decades, a noticeable part of clinical practices has been devoting progressively to these products. The first step to develop such an ATMP product is to be familiar with other approved products to obtain a general view about this industry trend. The present paper depicts an overall perspective of approved ATMPs in different countries, while reflecting the degree of their success in a clinical point of view and highlighting their main safety issues and also related market size as a whole. In this regard, published articles regarding safety, efficacy, and market size of approved ATMPs were reviewed using the search engines PubMed, Scopus, and Google Scholar. For some products which the related papers were not available, data on the relevant company website were referenced. In this descriptive study, we have introduced and classified approved cell, gene, and tissue engineering-based products by different regulatory agencies, along with their characteristics, manufacturer, indication, approval date, related regulatory agency, dosage, product description, price and published data about their safety and efficacy. In addition, to gain insights about the commercial situation of each product, we have gathered accessible sale reports and market size information that pertain to some of these products

A Roadmap for the Production of a GMP-Compatible Cell Bank of Allogeneic Bone Marrow-Derived Clonal Mesenchymal Stromal Cells for Cell Therapy Applications

Background: Allogeneic mesenchymal stromal cells (MSCs) have been used extensively in various clinical trials. Nevertheless, there are concerns about their efficacy, attributed mainly to the heterogeneity of the applied populations. Therefore, producing a consistent population of MSCs is crucial to improve their therapeutic efficacy. This study presents a good manufacturing practice (GMP)-compatible and cost-effective protocol for manufacturing, banking, and lot-release of a homogeneous population of human bone marrow-derived clonal MSCs (cMSCs). Methods: Here, cMSCs were isolated based on the subfractionation culturing method. Afterward, isolated clones that could reproduce up to passage three were stored as the seed stock. To select proliferative clones, we used an innovative, cost-effective screening strategy based on lengthy serial passaging. Finally, the selected clones re-cultured from the seed stock to establish the following four-tired cell banking system: initial, master, working, and end of product cell banks (ICB, MCB, WCB, and EoPCB). Results: Through a rigorous screening strategy, three clones were selected from a total of 21 clones that were stored during the clonal isolation process. The selected clones met the identity, quality, and safety assessments criteria. The validated clones were stored in the four-tiered cell bank system under GMP conditions, and certificates of analysis were provided for the three-individual ready-to-release batches. Finally, a stability study validated the EoPCB, release, and transport process of the frozen final products. Conclusion: Collectively, this study presents a technical and translational overview of a GMP-compatible cMSCs manufacturing technology that could lead to the development of similar products for potential therapeutic applications. Graphical Abstract: [Figure not available: see fulltext.

Phase 1 human trial of autologous bone marrow-hematopoietic stem cell transplantation in patients with decompensated cirrhosis

Aim: To evaluate safety and feasibility of autologous bone marrow-enriched CD34+ hematopoietic stem cell Tx through the hepatic artery in patients with decompensated cirrhosis. Methods: Four patients with decompensated cirrhosis were included. Approximately 200 mL of the bone marrow of the patients was aspirated, and CD34+ stem cells were selected. Between 3 to 10 million CD34+ cells were isolated. The cells were slowly infused through the hepatic artery of the patients. Results: Patient 1 showed marginal improvement in serum albumin and no significant changes in other test results. In patient 2 prothrombin time was decreased; however, her total bilirubin, serum creatinine, and Model of End-Stage Liver Disease (MELD) score worsened at the end of follow up. In patient 3 there was improvement in serum albumin, porthrombin time (PT), and MELD score. Patient 4 developed radiocontrast nephropathy after the procedure, and progressed to type 1 hepatorenal syndrome and died of liver failure a few days later. Because of the major side effects seen in the last patient, the trial was prematurely stopped. Conclusion: Infusion of CD34+ stem cells through the hepatic artery is not safe in decompensated cirrhosis. Radiocontrast nephropathy and hepatorenal syndrome could be major side effects. However, this study does not preclude infusion of CD34+ stem cells through other routes. © 2007 The WJG Press. All rights reserved

Quantitative Proteomic Analysis of Human Embryonic Stem Cell Differentiation by 8-Plex iTRAQ Labelling

Analysis of gene expression to define molecular mechanisms and pathways involved in human embryonic stem cells (hESCs) proliferation and differentiations has allowed for further deciphering of the self-renewal and pluripotency characteristics of hESC. Proteins associated with hESCs were discovered through isobaric tags for relative and absolute quantification (iTRAQ). Undifferentiated hESCs and hESCs in different stages of spontaneous differentiation by embryoid body (EB) formation were analyzed. Using the iTRAQ approach, we identified 156 differentially expressed proteins involved in cell proliferation, apoptosis, transcription, translation, mRNA processing, and protein synthesis. Proteins involved in nucleic acid binding, protein synthesis, and integrin signaling were downregulated during differentiation, whereas cytoskeleton proteins were upregulated. The present findings added insight to our understanding of the mechanisms involved in hESC proliferation and differentiation

Co-transplantation of Human Embryonic Stem Cell-derived Neural Progenitors and Schwann Cells in a Rat Spinal Cord Contusion Injury Model Elicits a Distinct Neurogenesis and Functional Recovery

Co-transplantation of neural progenitors (NPs) with Schwann cells (SCs) might be a way to overcome low rate of neuronal differentiation of NPs following transplantation in spinal cord injury (SCI) and the improvement of locomotor recovery. In this study, we initially generated NPs from human embryonic stem cells (hESCs) and investigated their potential for neuronal differentiation and functional recovery when co-cultured with SCs in vitro and co-transplanted in a rat acute model of contused SCI. Co-cultivation results revealed that the presence of SCs provided a consistent status for hESC-NPs and recharged their neural differentiation toward a predominantly neuronal fate. Following transplantation, a significant functional recovery was observed in all engrafted groups (NPs, SCs, NPs+SCs) relative to the vehicle and control groups. We also observed that animals receiving co-transplants established a better state as assessed with the BBB functional test. Immunohistofluorescence evaluation five weeks after transplantation showed invigorated neuronal differentiation and limited proliferation in the co-transplanted group when compared to the individual hESC-NPs grafted group. These findings have demonstrated that the co-transplantation of SCs with hESC-NPs could offer a synergistic effect, promoting neuronal differentiation and functional recovery

Ancient Migratory Events in the Middle East: New Clues from the Y-Chromosome Variation of Modern Iranians

Knowledge of high resolution Y-chromosome haplogroup diversification within Iran provides important geographic context regarding the spread and compartmentalization of male lineages in the Middle East and southwestern Asia. At present, the Iranian population is characterized by an extraordinary mix of different ethnic groups speaking a variety of Indo-Iranian, Semitic and Turkic languages. Despite these features, only few studies have investigated the multiethnic components of the Iranian gene pool. In this survey 938 Iranian male DNAs belonging to 15 ethnic groups from 14 Iranian provinces were analyzed for 84 Y-chromosome biallelic markers and 10 STRs. The results show an autochthonous but non-homogeneous ancient background mainly composed by J2a sub-clades with different external contributions. The phylogeography of the main haplogroups allowed identifying post-glacial and Neolithic expansions toward western Eurasia but also recent movements towards the Iranian region from western Eurasia (R1b-L23), Central Asia (Q-M25), Asia Minor (J2a-M92) and southern Mesopotamia (J1-Page08). In spite of the presence of important geographic barriers (Zagros and Alborz mountain ranges, and the Dasht-e Kavir and Dash-e Lut deserts) which may have limited gene flow, AMOVA analysis revealed that language, in addition to geography, has played an important role in shaping the nowadays Iranian gene pool. Overall, this study provides a portrait of the Y-chromosomal variation in Iran, useful for depicting a more comprehensive history of the peoples of this area as well as for reconstructing ancient migration routes. In addition, our results evidence the important role of the Iranian plateau as source and recipient of gene flow between culturally and genetically distinct population