Core-shell bioprinting as a strategy to apply differentiation factors in a spatially defined manner inside osteochondral tissue substitutes

One of the key challenges in osteochondral tissue engineering is to define specified zones with varying material properties, cell types and biochemical factors supporting locally adjusted differentiation into the osteogenic and chondrogenic lineage, respectively. Herein, extrusion-based core-shell bioprinting is introduced as a potent tool allowing a spatially defined delivery of cell types and differentiation factors TGF-β3 and BMP-2 in separated compartments of hydrogel strands, and, therefore, a local supply of matching factors for chondrocytes and osteoblasts. Ink development was based on blends of alginate and methylcellulose, in combination with varying concentrations of the nanoclay Laponite whose high affinity binding capacity for various molecules was exploited. Release kinetics of model molecules was successfully tuned by Laponite addition. Core-shell bioprinting was proven to generate well-oriented compartments within one strand as monitored by optical coherence tomography in a non-invasive manner. Chondrocytes and osteoblasts were applied each in the shell while the respective differentiation factors (TGF-β3, BMP-2) were provided by a Laponite-supported core serving as central factor depot within the strand, allowing directed differentiation of cells in close contact to the core. Experiments with bi-zonal constructs, comprising an osteogenic and a chondrogenic zone, revealed that the local delivery of the factors from the core reduces effects of these factors on the cells in the other scaffold zone. These observations prove the general suitability of the suggested system for co-differentiation of different cell types within a zonal construct.

Novel Evidence That By Employing a Two-Step Differentiation Protocol in the Presence of UM171 and Nicotinamide Acid Human Very Small Embryonic like Stem Cells (VSELs) Differentiate into Functional Endothelial Cells (ECs)

Background . Human Very small embryonic-like stem cells (VSELs) are CD133 + CD34 + small dormant stem cells with properties of self-renewal and multipotential ability to differentiate in the three-germ layers (Circulation Research 2019; 124: 208-210) and currently, more than 40 independent groups worldwide who have carefully followed the multicolor-staining cell-sorting strategy described by us (Current Protocols in Cytometry 2010 , 9.29.1-9.29.15). We have previously reported that human bone marrow (BM)-derived VSELs are able to give rise to vessel formation and endothelial differentiation (Thrombosis and Haemostasis 2015 ; 113 : 1084-1094) and several independent groups confirmed our data with human, mouse, or rat VSELs. Thus, VSELs are a promising source in regenerative medicine for the treatment of vascular diseases. Aim of the study. We aimed to develop an in vitro expansion and differentiation protocol of VSELs into endothelial cells (EC-VSELs) that will provide a clinically relevant cell therapy product without ethical problems and undesirable side effects. Materials and Methods. Highly purified by FACS from umbilical cord blood (UCB), VSELs were sorted as very small lineage-negative, CD45 -, CD34 + cells and then cultured and expanded into EC-VSELs in pro-angiogenic medium supplemented with mesodermal differentiation factors followed by stimulation by endothelial differentiation factors in the presence of UM171 and nicotinamide acid. In parallel, for comparison we expanded ECFCs from MNCs isolated from the same UCB units. The endothelial nature of the expanded VSEL-derived ECs (EC-VSELs) was tested by the expression of typical EC markers as well as by in vitro and in vivo functional angiogenic assays. Results. We report here for the first time a multistep differentiation strategy of highly purified UCB-derived VSELsThese cells after isolation by FACS were small and round, then in the presence of GSK3b inhibitor and BMP4 inducing mesodermal differentiation and high VEGF concentration to induce endothelial differentiation, VSELs enlarged and displayed extended morphology and acquired a characteristic cobblestone morphology. Finally, we have obtained a high number of cells with typical morphology of endothelial cells (EC-VSELs). By inhibiting potential mesodermal transition using TGFb inhibitor, EC-VSELs had a comparable morphology to primary human ECFCs and were characterized by tight junctions, caveolae, and Weibel-Palade Bodies, as demonstrated by transmission electron microscopy analysis of cell cultures conducted on fibrin network on the top of pericardial membranes. ECFCs differentiation was confirmed by analyzing the expression of endothelial markers by flow cytometry, and EC-VSELs were positive for PECAM1, VE-cadherin, VEGFR2, and endoglin. EC-VSELs as compared to ECFCs presented the same levels of expression of all these endothelial markers. What is important at the same time, EC-VSELs, as well as ECFCs, were negative for mesodermal marker Thy-1, confirming their endothelial phenotype. Migration properties of EC-VSELs were studied in basal conditions or in pro-angiogenic conditions using two in vitro models: wound healing assay and Trans well migration assay, and in both models EC-VSELs migration properties were similar to those of ECFCs. Next, we compared paracrine activity by evaluating growth factor and cytokine secretion profile of EC-VSELs, and noticed that the cytokine secretion by expanded VSELs was comparable to that of ECFCs. Moreover, the formation of pseudo-tubes was similar with both conditioned media. Finally, we have assessed the angiogenic capacity of EC-VSELs with a 3D in vitro sprouting assay and in vivo Matrigel plug assay. EC-VSELs display angiogenic properties but with lower potential in comparison with ECFCs which could be explained by their more primitive potential and most likely they need more time to be fully specified into the endothelial lineage. Conclusions. Based on our novel intriguing data, showing that highly purified VSELs expand efficient by employing a two-step differentiation protocol in the presence of UM171 and nicotinamide to EC-VSELs and acquire the same endothelial morphology, phenotype, and secretory potential as ECFCs as well as form functional vessels in in vitro and in vivo angiogenic assays, they could become an alternative source of ECFCs to treat vascular diseases. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Neuromedin B promotes chondrocyte differentiation of mesenchymal stromal cells via calcineurin and calcium signaling

Background Articular cartilage is a complex tissue with poor healing capacities. Current approaches for cartilage repair based on mesenchymal stromal cells (MSCs) are often disappointing because of the lack of relevant differentiation factors that could drive MSC differentiation towards a stable mature chondrocyte phenotype. Results We used a large-scale transcriptomic approach to identify genes that are modulated at early stages of chondrogenic differentiation using the reference cartilage micropellet model. We identified several modulated genes and selected neuromedin B (NMB) as one of the early and transiently modulated genes. We found that the timely regulated increase of NMB was specific for chondrogenesis and not observed during osteogenesis or adipogenesis. Furthermore, NMB expression levels correlated with the differentiation capacity of MSCs and its inhibition resulted in impaired chondrogenic differentiation indicating that NMB is required for chondrogenesis. We further showed that NMB activated the calcineurin activity through a Ca2+-dependent signaling pathway. Conclusion NMB is a newly described chondroinductive bioactive factor that upregulates the key chondrogenic transcription factor Sox9 through the modulation of Ca2+ signaling pathway and calcineurin activity. Graphical abstract

Suppression of Cell Tumorigenicity by Non-neural Pro-differentiation Factors via Inhibition of Neural Property in Tumorigenic Cells

Our studies have demonstrated that cell tumorigenicity and pluripotent differentiation potential stem from neural stemness or a neural ground state, which is defined by a regulatory network of higher levels of machineries for basic cell physiological functions, including cell cycle, ribosome biogenesis, protein translation, spliceosome, epigenetic modification factors, reprogramming factors, etc., in addition to the neural stemness specific factors. These machineries and neural stemness factors mostly play cancer-promoting roles. It can be deduced that differentiation requires the repression of neural ground state and causes the reduction or loss of neural ground state and thus tumorigenicity in tumorigenic cells. Formerly, we showed that neuronal differentiation led to reduced tumorigenicity in tumorigenic cells. In the present study, we show that non-neural pro-differentiation factors, such as GATA3, HNF4A, HHEX, and FOXA3 that specify mesodermal or/and endodermal tissues during vertebrate embryogenesis, suppress tumorigenicity via repression of neural stemness and promotion of non-neural property in tumorigenic cells. Mechanistically, these transcription factors repress the transcription of neural enriched genes and meanwhile activate genes that specify non-neural properties via direct binding to the promoters of these genes. We also show that combined expression of HHEX and FOXA3 suppresses tumorigenesis effectively in the AOM/DSS model of colitis-associated cancer. We suggest that targeting the property of neural stemness could be an effective strategy for cancer therapy.

The Phosphonate Derivative of C60 Fullerene Induces Differentiation towards the Myogenic Lineage in Human Adipose-Derived Mesenchymal Stem Cells

Inductors of myogenic stem cell differentiation attract attention, as they can be used to treat myodystrophies and post-traumatic injuries. Functionalization of fullerenes makes it possible to obtain water-soluble derivatives with targeted biochemical activity. This study examined the effects of the phosphonate C60 fullerene derivatives on the expression of myogenic transcription factors and myogenic differentiation of human mesenchymal stem cells (MSCs). Uptake of the phosphonate C60 fullerene derivatives in human MSCs, intracellular ROS visualization, superoxide scavenging potential, and the expression of myogenic, adipogenic, and osteogenic differentiation genes were studied. The prolonged MSC incubation (within 7–14 days) with the C60 pentaphoshonate potassium salt promoted their differentiation towards the myogenic lineage. The transcription factors and gene expressions determining myogenic differentiation (MYOD1, MYOG, MYF5, and MRF4) increased, while the expression of osteogenic differentiation factors (BMP2, BMP4, RUNX2, SPP1, and OCN) and adipogenic differentiation factors (CEBPB, LPL, and AP2 (FABP4)) was reduced or did not change. The stimulation of autophagy may be one of the factors contributing to the increased expression of myogenic differentiation genes in MSCs. Autophagy may be caused by intracellular alkalosis and/or short-term intracellular oxidative stress.

Methodological pathways to portray superdiversity: A few concluding thoughts

Superdiversity as a term has captured the attention of many researchers since it was first coined in 2007. However, the very characteristics of the concept have made its methodological operationalization insufficiently clear, beyond being bridged by the great migratory movements of the 21st century. As a result, we find a considerable diversity of analytical approaches without being empirically clear what the dimensions and variables are that should accompany those related to migratory flows. In this epilogue, methodological implications are critically discussed, especially those related to social differentiation factors and a hierarchy of predefined variables. Also, future research implications are discussed.

Faktor Diferensiasi Sekolah di Indonesia

Abstrak Diferensiasi sekolah di Indonesia yaitu sekolah swasta dan sekolah negeri milik pemerintah. Perbedaan lainnya yaitu sekolah milik yayasan yang berlatar belakang agama atau tanpa latar belakang agama. Tujuan penelitian ini adalah menemukan faktor diferensiasi sekolah di Indonesia. Metode penelitian yang digunakan yaitu deskriptif analitik. Berdasarkan hasil penelitian, ditemukan sepuluh diferensiasi sekolah di Indonesia, yaitu afiliasi, bahasa yang digunakan, latar belakang budaya, kurikulum, waktu belajar di sekolah, nilai etika, jalur ke perguruan tinggi, fasilitas, dan harga. Kata Kunci Diferensiasi sekolah, sekolah swasta, sekolah negeri Abstract School differentiation in Indonesia is private schools and government-owned schools. Another difference is that schools belong to foundations with religious or religious backgrounds. The purpose of this study is to find the differentiation factors of schools in Indonesia. The research method used is descriptive analytic. Based on the research results, it was found ten differences in schools in Indonesia, namely provision, language used, cultural background, curriculum, study time at school, ethical values, pathways to tertiary institutions, facilities, and prices. Keywords Differentiation of schools, private schools, public schools

Biofabrication of a Tubular Model of Human Urothelial Mucosa Using Human Wharton Jelly Mesenchymal Stromal Cells

Several models of bioartificial human urothelial mucosa (UM) have been described recently. In this study, we generated novel tubularized UM substitutes using alternative sources of cells. Nanostructured fibrin–agarose biomaterials containing fibroblasts isolated from the human ureter were used as stroma substitutes. Then, human Wharton jelly mesenchymal stromal cells (HWJSC) were used to generate an epithelial-like layer on top. Three differentiation media were used for 7 and 14 days. Results showed that the biofabrication methods used here succeeded in generating a tubular structure consisting of a stromal substitute with a stratified epithelial-like layer on top, especially using a medium containing epithelial growth and differentiation factors (EM), although differentiation was not complete. At the functional level, UM substitutes were able to synthesize collagen fibers, proteoglycans and glycosaminoglycans, although the levels of control UM were not reached ex vivo. Epithelial differentiation was partially achieved, especially with EM after 14 days of development, with expression of keratins 7, 8, and 13 and pancytokeratin, desmoplakin, tight-junction protein-1, and uroplakin 2, although at lower levels than controls. These results confirm the partial urothelial differentiative potential of HWJSC and suggest that the biofabrication methods explored here were able to generate a potential substitute of the human UM for future clinical use.