A micro-fragmented collagen gel as a cell-assembling platform for critical limb ischemia repair.

Critical limb ischemia (CLI) is a devastating disease characterized by the progressive blockage of blood vessels. Although the paracrine effect of growth factors in stem cell therapy made it a promising angiogenic therapy for CLI, poor cell survival in the harsh ischemic microenvironment limited its efficacy. Thus, an imperative need exists for a stem-cell delivery method that enhances cell survival. Here, a collagen microgel (CMG) cell-delivery scaffold (40 × 20 µm) was fabricated via micro-fragmentation from collagen-hyaluronic acid polyionic complex to improve transplantation efficiency. Culturing human adipose-derived stem cells (hASCs) with CMG enabled integrin receptors to interact with CMG to form injectable 3-dimensional constructs (CMG-hASCs) with a microporous microarchitecture and enhanced mass transfer. CMG-hASCs exhibited higher cell survival (p < 0.0001) and angiogenic potential in tube formation and aortic ring angiogenesis assays than cell aggregates. Injection of CMG-hASCs intramuscularly into CLI mice increased blood perfusion and limb salvage ratios by 40 % and 60 %, respectively, compared to cell aggregate-treated mice. Further immunofluorescent analysis revealed that transplanted CMG-hASCs have greater muscle regenerative and angiogenic potential, with enhanced cell survival than cell aggregates (p < 0.05). Collectively, we propose CMG as a cell-assembling platform and CMG-hASCs as promising therapeutics to treat CLI.

Functionally enhanced cell spheroids for stem cell therapy: Role of TIMP1 in the survival and therapeutic effectiveness of stem cell spheroids.

Stem cell therapy has emerged as a promising regenerative medicine strategy but is limited by poor cell survival, leading to low therapeutic outcomes. We developed cell spheroid therapeutics to overcome this limitation. We utilized solid-phase FGF2 to form functionally enhanced cell spheroid-adipose derived (FECS-Ad), a type of cell spheroid that preconditions cells with intrinsic hypoxia to increase the survival of transplanted cells. We demonstrated an increase in hypoxia-inducible factor 1-alpha (HIF-1α) levels in FECS-Ad, which led to the upregulation of tissue inhibitor of metalloproteinase 1 (TIMP1). TIMP1 enhanced the survival of FECS-Ad, presumably through the CD63/FAK/Akt/Bcl2 anti-apoptotic signaling pathway. Cell viability of transplanted FECS-Ad was reduced by TIMP1 knockdown in an in vitro collagen gel block and a mouse model of critical limb ischemia (CLI). TIMP1 knockdown in FECS-Ad inhibited angiogenesis and muscle regeneration induced by FECS-Ad transplanted into ischemic mouse tissue. Genetic overexpression of TIMP1 in FECS-Ad further promoted the survival and therapeutic efficacy of transplanted FECS-Ad. Collectively, we suggest that TIMP1 acts as a key survival factor to improve the survival of transplanted stem cell spheroids, which provides scientific evidence for enhanced therapeutic efficacy of stem cell spheroids, and FECS-Ad as a potential therapeutic agent to treat CLI. STATEMENT OF SIGNIFICANCE: We used FGF2-tethered substrate platform to form adipose-derived stem cell spheroids, as we named as functionally enhanced cell spheroid-adipose derived (FECS-Ad). In this paper, we showed that intrinsic hypoxia of spheroids upregulated expression of HIF-1α, which in turn upregulated expression of TIMP1. Our paper highlights TIMP1 as a key survival factor to improve survival of transplanted stem cell spheroids. We believe that our study has a very strong scientific impact as extending transplantation efficiency is essential for successful stem cell therapy.

Effects of the Extracts from Fruit and Stem of Camellia japonica on Induced Pluripotency and Wound Healing.

Small molecules that improve reprogramming, stem cell properties, and regeneration can be widely applied in regenerative medicine. Natural plant extracts represent an abundant and valuable source of bioactive small molecules for drug discovery. Natural products themselves or direct derivatives of them have continued to provide small molecules that have entered clinical trials, such as anticancer and antimicrobial drugs. Here, we tested 3695 extracts from native plants to examine whether they can improve induced pluripotent stem cell (iPSC) generation using genetically homogeneous secondary mouse embryonic fibroblasts (MEFs) harboring doxycycline (dox)-inducible reprograming transgenes. Among the tested extracts, extracts from the fruit and stem of Camellia japonica (CJ) enhanced mouse and human iPSC generation and promoted efficient wound healing in an in vivo mouse wound model. CJ is one of the best-known species of the genus Camellia that belongs to the Theaceae family. Our findings identified the natural plant extracts from the fruit and stem of CJ as novel regulators capable of enhancing cellular reprogramming and wound healing, providing a useful supplement in the development of a more efficient and safer method to produce clinical-grade iPSCs and therapeutics.

ESRP1-Induced CD44 v3 Is Important for Controlling Pluripotency in Human Pluripotent Stem Cells.

The importance of alternative splicing (AS) events in pluripotency regulation has been highlighted by the determination of different roles and contributions of different splice isoforms of pluripotency-related genes and by the identification of distinct pluripotency-related splicing factors. In particular, epithelial splicing regulatory protein 1 (ESRP1) has been characterized as an essential splicing factor required for the regulation of human pluripotency and differentiation. Nevertheless, a detailed molecular characterization of ESRP1 (mRNA splice variants 1-6) in human pluripotency is lacking. In this study, we determined that ESRP1 splice variants are differentially expressed in undifferentiated and differentiated human pluripotent stem cells (PSCs). Undifferentiated human PSCs predominantly expressed the ESRP1 v1, v4, and v5, and their expression was downregulated upon differentiation. Ectopic expression of ESRP1 v1, v4, or v5 enhanced the pluripotent reprogramming of human fibroblasts and restored the ESRP1 knockdown-mediated reduction of reprogramming efficiency. Notably, undifferentiated human PSCs expressed the cell surface protein CD44 variant 3 (CD44 v3), and isoform switching from CD44 v3 to CD44 variant 6 (CD44 v6) occurred upon differentiation. Importantly, the human PSC-specific ESRP1 variants influenced CD44 v3 expression. CD44 knockdown or inhibition of binding of CD44 with its major ligand, hyaluronan, significantly induced the loss of human PSC pluripotency and the reduction of reprogramming efficiency. Our results demonstrate that the effect of ESRP1 and CD44 on human PSC pluripotency is isoform-dependent and that ESRP1-induced CD44 v3 is functionally associated with human PSC pluripotency control. Stem Cells 2018;36:1525-1534.

The unique spliceosome signature of human pluripotent stem cells is mediated by SNRPA1, SNRPD1, and PNN.

Spliceosomes are the core host of pre-mRNA splicing, allowing multiple protein isoforms to be produced from a single gene. Herein, we reveal that spliceosomes are more abundant in human pluripotent stem cells (hPSs), including human embryonic stem cells (hESs) and human induced pluripotent stem cells (hiPSs), than non-hPSs, and their presence is associated with high transcriptional activity. Supportively, spliceosomal components involved in the catalytically active pre-mRNA splicing step were mainly co-localized with hPS spliceosomes. By profiling the gene expression of 342 selected splicing factors, we found that 71 genes were significantly altered during the reprogramming of human somatic cells into hiPSs. Among them, SNRPA1, SNRPD1, and PNN were significantly up-regulated during the early stage of reprogramming, identified as hub genes by interaction network and cluster analysis. SNRPA1, SNRPD1, or PNN depletion led to a pronounced loss of pluripotency and significantly blocked hiPS generation. SNRPA1, SNRPD1, and PNN co-localized with the hPS spliceosomes, physically interacted with each other, and positively influenced the appearance of hPS spliceosomes. Our data suggest that SNRPA1, SNRPD1, and PNN are key players in the regulation of pluripotency-specific spliceosome assembly and the acquisition and maintenance of pluripotency.