Promotion of cardiac microtissue assembly within G-CSF-enriched collagen I-cardiogel hybrid hydrogel.

Tissue engineering as an interdisciplinary field of biomedical sciences has raised many hopes in the treatment of cardiovascular diseases as well as development of in vitro three-dimensional (3D) cardiac models. This study aimed to engineer a cardiac microtissue using a natural hybrid hydrogel enriched by granulocyte colony-stimulating factor (G-CSF), a bone marrow-derived growth factor. Cardiac ECM hydrogel (Cardiogel: CG) was mixed with collagen type I (ColI) to form the hybrid hydrogel, which was tested for mechanical and biological properties. Three cell types (cardiac progenitor cells, endothelial cells and cardiac fibroblasts) were co-cultured in the G-CSF-enriched hybrid hydrogel to form a 3D microtissue. ColI markedly improved the mechanical properties of CG in the hybrid form with a ratio of 1:1. The hybrid hydrogel demonstrated acceptable biocompatibility and improved retention of encapsulated human foreskin fibroblasts. Co-culture of three cell types in G-CSF enriched hybrid hydrogel, resulted in a faster 3D structure shaping and a well-cellularized microtissue with higher angiogenesis compared to growth factor-free hybrid hydrogel (control). Immunostaining confirmed the presence of CD31+ tube-like structures as well as vimentin+ cardiac fibroblasts and cTNT+ human pluripotent stem cells-derived cardiomyocytes. Bioinformatics analysis of signaling pathways related to the G-CSF receptor in cardiovascular lineage cells, identified target molecules. The in silico-identified STAT3, as one of the major molecules involved in G-CSF signaling of cardiac tissue, was upregulated in G-CSF compared to control. The G-CSF-enriched hybrid hydrogel could be a promising candidate for cardiac tissue engineering, as it facilitates tissue formation and angiogenesis.

Conditioned media from human pluripotent stem cell-derived cardiomyocytes inhibit the growth and migration of lung cancer cells.

Conditioned media (CM) from various cell types contain significant levels of paracrine factors. Recently, therapeutic properties of CM derived from stem cells have been revealed. Based on the fact that heart cancer is extremely rarely, we hypothesized that the CM obtained from human pluripotent stem cell-derived cardiomyocytes might inhibit cancer cell growth and survival. To this end, lung cancer cell line A549 along with human foreskin fibroblasts (HFF) were treated with serial concentrations of cardiomyocyte CM (CCM) or fibroblast CM (FCM). We found that CCM markedly reduced the viability of lung cancer cells, while FCM did not compromise the viability of neither cancer cells nor HFF cells. Furthermore, we determined an optimized CCM concentration, 30 mg/mL, at which the growth, clonogenicity, and migration of A549 and Calu6 lung cancer cell lines were substantially impaired, whereas FCM did not influence these properties. Moreover, lung cancer cells exhibited cell cycle regulation upon treatment with CCM and the rate of apoptosis was markedly increased by cardiomyocyte CM in both lung cancer cell lines tested. Finally, in response to CCM treatment, A549 and Calu6 cells expressed lower levels of antiapoptotic and stemness genes, but higher levels of proapoptotic genes. In conclusion, this study provides cellular and molecular evidence for the antitumor ability of secretome obtained from stem cell-derived cardiomyocytes.

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.

An Easy and Fast Method for Production of Chinese Hamster Ovary Cell Line Expressing and Secreting Human Recombinant Activin A.

OBJECTIVE: Growth factors are key elements of embryonic stem cell (ESC) research. Cell line development in eukaryotes is a time-consuming procedure which usually takes 12-18 months. Here, we report an easy and fast method with which production of Chinese hamster ovary (CHO) cells that express and secrete recombinant Activin A, as a major growth factor in endo/mesoderm differentiation of embryonic stem cells is achieved within 3-4 weeks. MATERIALS AND METHODS: In this experimental study, we cloned human Activin A into the pDONR/Zeo gateway entry vector using the BP reaction. Activin A was subcloned next into the pLIX_403 and pLenti6.3/TO/V5-DEST destination vectors by the LR reaction. The result was the production of constructs with which 293T cells were finally transfected for virus production. CHO cells were transduced using viral particles to produce a cell line that secretes the His6- Activin A fusion protein. RESULTS: We developed a quick protocol which saves up to 3-4 weeks of time for producing recombinant proteins in CHO cells. The recombinant cell line produced 90 mg/L of functional Activin A measured in human ESC line Royan H5 (RH5), during in vitro differentiation into meso-endoderm and definitive endoderm. CONCLUSION: Our results showed no significant differences in functionality between commercial Activin A and the one produced using our novel protocol. This approach can be easily used for producing recombinant proteins in CHO.

Inclusion Body Expression and Refolding of Recombinant Bone Morphogenetic Protein-2.

BACKGROUND: Bone Morphogenetic Protein-2 (BMP-2) is a cysteine rich growth factor expressed in homodimeric form and has a pivotal role in osteochondral development and fracture healing. Recent studies have benefited more from recombinant BMP-2 in osteochondral tissue engineering. Cost-effective and easy production at large scale makes Escherichia coli (E. coli) the first choice for recombinant protein expression programs. However, inclusion body aggregation and refolding process limits production and purification of recombinant BMP-2 in bacterial systems. METHODS: BMP-2 encoded gene was optimized for expression in bacterial expression system and synthesized with proper restriction sites. The optimized sequence was then cloned in a pET28a expression vector and expressed in Origami™ E. coli strain. The aggregated and monomeric BMP-2 was refolded and purified comparing two oxidoreductase systems and refolding methods as well as different purification techniques. The biological activity of recombinant protein was investigated by increasing alkaline phosphatase activity (ALK) of ATDC-5 cell line. RESULTS: No difference was observed between oxidoreductase systems in improving the efficiency of protein refolding. However, comparisons between two refolding methods showed that pooling monomeric BMP-2 that was refolded under mild condition with equal volume of it refolded under severe oxidoreductase condition resulted in production of more active dimeric protein. CONCLUSION: A new method for production of biologically active dimeric form of BMP-2 in E. coli expression system was established in this study.

Improved Protocol for Chondrogenic Differentiation of Bone Marrow Derived Mesenchymal Stem Cells -Effect of PTHrP and FGF-2 on TGFß1/BMP2-Induced Chondrocytes Hypertrophy.

Growth factors have a pivotal role in chondrogenic differentiation of stem cells. The differential effects of known growth factors involved in the maintenance and homeostasis of cartilage tissue have been previously studied in vitro. However, there are few reported researches about the interactional effects of growth factors on chondrogenic differentiation of stem cells. The aim of this study is to examine the combined effects of four key growth factors on chondrogenic differentiation of mesenchymal stem cells (MSCs). Isolated and expanded rabbit bone marrow-derived MSCs underwent chondrogenic differentiation in a micromass cell culture system that used a combination of the following growth factors: transforming growth factor beta 1 (TGF-ß1), bone morphogenetic protein 2 (BMP2), parathyroid hormone related protein (PTHrP), and fibroblast growth factor 2 (FGF2) according to a defined program. The chondrogenic differentiation program was analyzed by histochemistry methods, quantitative RT-PCR (qRT-PCR), and measurement of matrix deposition of sulfated glycosaminoglycan (sGAG) and collagen content at days 16, 23, and 30. The results showed that the short-term combination of TGF-ß1 and BMP-2 increased sGAG and collagen content, Alkaline phosphates (ALP) activity, and type X collagen (COL X) expression. Application of either PTHrP or FGF2 simultaneously decreased TGF-ß1/BMP-2 induced hypertrophy and chondrogenic markers (at least for FGF2). However, successive application of PTHrP and FGF2 dramatically maintained the synergistic effects of TGF-ß1/BMP-2 on the chondrogenic differentiation potential of MSCs and decreased unwanted hypertrophic markers. This new method can be used effectively in chondrogenic differentiation programs.

Cloning, expression, and functional characterization of in-house prepared human leukemia inhibitory factor.

OBJECTIVE: Leukemia inhibitory factor (LIF) plays important roles in cellular proliferation, growth promotion and differentiation of various types of target cells. In addition, LIF influences bone metabolism, cachexia, neural development, embryogenesis and inflammation. Human LIF (hLIF) is an essential growth factor for the maintenance of mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in a pluripotent, undifferentiated state. MATERIALS AND METHODS: In this experimental study, we cloned hLIF into the pENTR-D/ TOPO entry vector by a TOPO reaction. Next, hLIF was subcloned into the pDEST17 destination vector by the LR reaction, which resulted in the production of a construct that was transferred into E. coli strain Rosetta-gami™ 2(DE3) pLacI competent cells to produce the His6-hLIF fusion protein. RESULTS: This straightforward method produced a biologically active recombinant hLIF protein in E. coli that has long-term storage ability. This procedure has provided rapid, cost effective purification of a soluble hLIF protein that is biologically active and functional as measured in mouse ESCs and iPSCs in vitro. CONCLUSION: Our results showed no significant differences in function between laboratory produced and commercialized hLIF.