MEK1 activation enhances the ex vivo proliferation of haematopoietic stem/progenitor cell.

Haematopoietic stem/progenitor cell (HSPC) integrates intracellular signal network from growth factors (GFs) and utilizes its proliferation feature to generate high yields of transplantable cells upon ex vivo culture. However, the molecular basis for HSPC activation and proliferation is not completely understood. The goal of this study was to investigate proliferation regulator in the downstream of GFs and develop HSPC expansion strategy. Microarray and Ingenuity Pathway Analysis were performed to evaluate differentially expressed genes in cytokine-induced CD34+ cells after ex vivo culture. We identified that MEK1 was a potential HSPC proliferation regulator, which represented indispensable roles and MEK1 silence attenuated the proliferation of HSPC. Notably, 500 nM MEK1 agonist, PAF C-16, increased the numbers of phenotypic HSPC and induced cell cycling of HSPC. The PAF C-16 expanded HSPC demonstrated comparative clonal formation ability and secondary expansion capacity compared to the vehicle control. Our results provide insights into regulating the balance between proliferation and commitment of HSPC by targeting the HSPC proliferation-controlling network. This study demonstrates that MEK1 critically regulates HSPC proliferation and cell production in the ex vivo condition for transplantation.

Trehalose glycopolymers for cryopreservation of tissue-engineered constructs.

The development of an effective cryopreservation method to achieve off-the-shelf and bioactive tissue-engineered constructs (TECs) is important to meet the requirements for clinical applications. The trehalose, a non-permeable cryoprotectant (CPA), has difficulty in penetrating the plasma membranes of mammalian cells and has only been used in combination with other cell penetrating CPA (such as DMSO) to cryopreserve mammalian cells. However, the inherent cytotoxicity of DMSO results in increasing risks with respect to cryopreserved cells. Therefore, in this study, permeable trehalose glycopolymers were synthesised for cryopreservation of TECs. The trehalose glycopolymers exhibited good ice inhibiting activities and biocompatibilities. Furthermore, the viability and function of TECs after cryopreservation with 5.0 wt% S2 were similar to those of the non-cryopreserved TECs. We developed an effective preservation strategy for the off-the-shelf availability of TECs.

Three-Dimension Co-culture of Hematopoietic Stem Cells and Differentiated Osteoblasts on Gallic Acid Grafted-Chitosan Scaffold as a Model of Hematopoietic Stem Cells Niche.

The existing approaches of hematopoietic stem cells (HSCs) expansion in vitro were difficult to meet the needs of clinical application. While in vivo, HSCs efficiently self-renew in niche where they interact with three dimension extracellular matrix and stromal cells. Osteoblasts (OBs) are one of most significant stromal cells of HSCs niche. Here, we proposed a three-dimensional environment based on gallic acid grafted-chitosan (2c) scaffold and OBs differentiated from human umbilical cord mesenchymal stem cells (HUMSCs) to recapitulate the main components of HSCs niche. The results of alkaline phosphatase staining and alizarin red staining demonstrated that HUMSCs were successfully induced into OBs. The results showed that the expansions of CD34+cells, CD34+CD38- cells and CD34+CD38-CD45RA-CD49f+CD90+ cells (primitive hematopoietic stem cells, pHSCs) harvested from the biomimetic HSCs niche based on 2c scaffold and OBs (IV) group were larger than those harvested from other three culture groups. Importantly, it was found that the CD34+ cells harvested from IV group had better secondary expansion capability and colony forming potential, indicating better self-renewal ability. In addition, the biomimetic HSCs niche based on 2c scaffold and OBs protected HSCs apoptosis and promoted HSCs division. Taken together, the biomimetic HSCs niche based on 2c scaffold and OBs was an effective strategy for ex vivo expansion of HSCs in clinical scale.

The change of plasma metabolic profile and gut microbiome dysbiosis in patients with rheumatoid arthritis.

Objective: Rheumatoid arthritis (RA) is a chronic inflammatory joint disease, which is associated with progressive disability, systemic complications, and early death. But its etiology and pathogenesis are not fully understood. We aimed to investigate the alterations in plasma metabolite profiles, gut bacteria, and fungi and their role of them in the pathogenesis of RA. Methods: Metabolomics profiling of plasma from 363 participants including RA (n = 244), systemic lupus erythematosus (SLE, n = 50), and healthy control (HC, n = 69) were performed using the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry. The differentially expressed metabolites were selected among groups and used to explore important metabolic pathways. Gut microbial diversity analysis was performed by 16S rRNA sequencing and ITS sequencing (RA = 195, HC = 269), and the specific microbial floras were identified afterward. The diagnosis models were established based on significant differential metabolites and microbial floras, respectively. Results: There were 63 differential metabolites discovered between RA and HC groups, mainly significantly enriched in the arginine and proline metabolism, glycine, serine, and threonine metabolism, and glycerophospholipid metabolism between RA and HC groups. The core differential metabolites included L-arginine, creatine, D-proline, ornithine, choline, betaine, L-threonine, LysoPC (18:0), phosphorylcholine, and glycerophosphocholine. The L-arginine and phosphorylcholine were increased in the RA group. The AUC of the predictive model was 0.992, based on the combination of the 10 differential metabolites. Compared with the SLE group, 23 metabolites increased and 61 metabolites decreased in the RA group. However, no significant metabolic pathways were enriched between RA and SLE groups. On the genus level, a total of 117 differential bacteria genera and 531 differential fungal genera were identified between RA and HC groups. The results indicated that three bacteria genera (Eubacterium_hallii_group, Escherichia-Shigella, Streptococcus) and two fungal genera (Candida and Debaryomyces) significantly increased in RA patients. The AUC was 0.80 based on a combination of six differential bacterial genera and the AUC was 0.812 based on a combination of seven differential fungal genera. Functional predictive analysis displayed that differential bacterial and differential fungus both were associated with KEGG pathways involving superpathway of L-serine and glycine biosynthesis I, arginine, ornithine, and proline interconversion. Conclusion: The plasma metabolism profile and gut microbe profile changed markedly in RA. The glycine, serine, and threonine metabolism and arginine and proline metabolism played an important role in RA.

Magnetic-driven dynamic culture promotes osteogenesis of mesenchymal stem cell.

Effective nutrient transport and appropriate mechanical stimulation play important roles in production of tissue-engineered bone grafts. In this study, an experimental set-up for magnetic-driven dynamic culture of cells was designed to mimic the microenvironment of the bone tissue. Here, its ability to contribute to osteogenic differentiation was investigated by inoculating human umbilical cord mesenchymal stem cells (HUMSCs) on magnetic scaffolds. The cytocompatibility of the developed magnetic scaffolds was verified for HUMSCs. Magnetic scaffolds seeded with HUMSCs were exposed to magnetic fields. The results showed that magnetic fields did not affect cell activity and promoted HUMSCs osteogenic differentiation. The magnetic scaffolds were magnetically driven for dynamic culture in the experimental set-up to evaluate the influence of HUMSCs osteoblast differentiation. The results indicated that magnetic-driven dynamic culture increased cell alkaline phosphatase (ALP) activity (p < 0.05) and calcium release (p < 0.05) compared with static culture. The effect was demonstrated in the expression of bone-associated genes. Overall, this study showed that magnetic-driven dynamic culture is a promising tool for regenerative bone engineering.