Spatiotemporal expression of long noncoding RNA Moshe modulates heart cell lineage commitment.

The roles of long non-coding RNA (LncRNA) have been highlighted in various development processes including congenital heart defects (CHD). Here, we characterized the molecular function of LncRNA, Moshe (1010001N08ik-203), one of the Gata6 antisense transcripts located upstream of Gata6, which is involved in both heart development and the most common type of congenital heart defect, atrial septal defect (ASD). During mouse embryonic development, Moshe was first detected during the cardiac mesoderm stage (E8.5 to E9.5) where Gata6 is expressed and continues to increase at the atrioventricular septum (E12.5), which is involved in ASD. Functionally, the knock-down of Moshe during cardiogenesis caused significant repression of Nkx2.5 in cardiac progenitor stages and resulted in the increase in major SHF lineage genes, such as cardiac transcriptional factors (Isl1, Hand2, Tbx2), endothelial-specific genes (Cd31, Flk1, Tie1, vWF), a smooth muscle actin (a-Sma) and sinoatrial node-specific genes (Shox2, Tbx18). Chromatin Isolation by RNA Purification showed Moshe activates Nkx2.5 gene expression via direct binding to its promoter region. Of note, Moshe was conserved across species, including human, pig and mouse. Altogether, this study suggests that Moshe is a heart-enriched lncRNA that controls a sophisticated network of cardiogenesis by repressing genes in SHF via Nkx2.5 during cardiac development and may play an important role in ASD.

CD276 (B7-H3) Maintains Proliferation and Regulates Differentiation in Angiogenic Function in Late Endothelial Progenitor Cells.

Endothelial progenitor cells (EPCs) provide an important source of recovery from blood vessel dysfunction. Late EPCs (LEPCs) are circulating blood cells that are capable of promoting vascular repair. Using transcriptome analysis, we identified distinctive LEPC profiles and found that CD276 (B7-H3) mRNA is strongly expressed in LEPCs. CD276 protein is present abundantly on the cell surface of LEPC when analyzed by fluorescence-activated cell sorter and immunocytochemistry. CD276, a B7 family member, is a type I transmembrane glycoprotein. The role of CD276 in LEPCs remains unknown. CD276 knockdown by lentivirus transduction in LEPCs significantly decreased proliferation and increased apoptosis of LEPCs in vitro. After CD276 silencing, the cell cycle of LEPCs was prone to remain at the G0/G1 phase, and the cell migration rates as well as transwell and wound-healing migration were decreased. CD276 knockdown in LEPCs increased the G1 phase regulators cyclin D2/D3/E1-cyclin-dependent kinases (CDK2/4/6), but decreased the S-G2-M phase regulators cyclin A/B-CDK1. However, LEPCs with CD276 knockdown resulted in increased tube formation in vitro and angiogenesis in a Matrigel plug assay in vivo. FoxC1/C2, an upstream signal of Notch in arterial cell proliferation, and Hey1/2, which is known to promote arterial differentiation in the vasculature, were upregulated in CD276 knockdown LEPCs. In LEPCS, CD276 has a positive effect on proliferation and migration of endothelial cells, but negative effects on angiogenesis, particularly endothelial cell differentiation. Our data indicate, for therapeutic purpose, that CD276 can be used to acquire and maintain cell populations of LEPCs and blocking CD276 will promote angiogenetic differentiation. We found that CD276 (B7-H3) is enriched on the cell membrane of LEPCs. CD276 knockdown reduced proliferation and migration of LEPCs by increasing cell cycle inhibitors such as p21cip1 and pRb and decreasing pErk1/2 and pAkt but promoted angiogenesis and endothelial cell differentiation by elevating vascular endothelial growth factor-vascular endothelial growth factor receptor 1 and p-p38. Stem Cells 2019;37:382-394.

Improved Healing after the Co-Transplantation of HO-1 and BDNF Overexpressed Mesenchymal Stem Cells in the Subacute Spinal Cord Injury of Dogs.

Abundant expression of proinflammatory cytokines after a spinal cord injury (SCI) creates an inhibitory microenvironment for neuroregeneration. The mesenchymal stem cells help to mitigate the inflammation and improve neural growth and survival. For this purpose, we potentiated the function of adipose-derived mesenchymal stem cells (Ad-MSCs) by transfecting them with brain-derived neurotrophic factor (BDNF) and heme oxygenase-1 (HO-1), through a lentivirus, to produce BDNF overexpressed Ad-MSCs (BDNF-MSCs), and HO-1 overexpressed Ad-MSCs (HO-1-MSCs). Sixteen SCI beagle dogs were randomly assigned into four treatment groups. We injected both HO-1 and BDNF-overexpressed MSCs as a combination group, to selectively control inflammation and induce neuroregeneration in SCI dogs, and compared this with BDNF-MSCs, HO-1-MSCs, and GFP-MSCs injected dogs. The groups were compared in terms of improvement in canine Basso, Beattie, and Bresnahan (cBBB) score during 8 weeks of experimentation. After 8 weeks, spinal cords were harvested and subjected to western blot analysis, immunofluorescent staining, and hematoxylin and eosin (H&E) staining. The combination group showed a significant improvement in hindlimb functions, with a higher BBB score, and a robust increase in neuroregeneration, depicted by a higher expression of Tuj-1, NF-M, and GAP-43 due to a decreased expression of the inflammatory markers interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), and an increased expression of interleukin-10 (IL-10) ( P ≤ 0.05). H&E staining showed more reduced intraparenchymal fibrosis in the combination group than in other groups ( P ≤ 0.05). It was thus suggested that the cotransplantation of HO-1 and BDNF-MSCs is more effective in promoting the healing of SCI. HO-1-MSCs reduce inflammation, which favors BDNF-induced neuroregeneration in SCI of dogs.

DSG2 Is a Functional Cell Surface Marker for Identification and Isolation of Human Pluripotent Stem Cells.

Pluripotent stem cells (PSCs) represent the most promising clinical source for regenerative medicine. However, given the cellular heterogeneity within cultivation and safety concerns, the development of specific and efficient tools to isolate a pure population and eliminate all residual undifferentiated PSCs from differentiated derivatives is a prerequisite for clinical applications. In this study, we raised a monoclonal antibody and identified its target antigen as desmoglein-2 (DSG2). DSG2 co-localized with human PSC (hPSC)-specific cell surface markers, and its expression was rapidly downregulated upon differentiation. The depletion of DSG2 markedly decreased hPSC proliferation and pluripotency marker expression. In addition, DSG2-negative population in hPSCs exhibited a notable suppression in embryonic body and teratoma formation. The actions of DSG2 in regulating the self-renewal and pluripotency of hPSCs were predominantly exerted through the regulation of ß-catenin/Slug-mediated epithelial-to-mesenchymal transition. Our results demonstrate that DSG2 is a valuable PSC surface marker that is essential for the maintenance of PSC self-renewal.

Fibroblast growth factor 2 supports osteoblastic niche cells during hematopoietic homeostasis recovery after bone marrow suppression.

BACKGROUND: Hematopoietic stem cell (HSC) maintenance requires a specific microenvironment. HSC niches can be activated by tissue damaging chemotherapeutic drugs and various cell signaling molecules such as SDF-1 and FGF, which might also result in bone marrow stress. Recent research has insufficiently shown that endosteal osteolineage cells and other niche constituents recover after marrow injury. METHODS: We investigated the role of FGF2 in the osteoblastic niche cells during hematopoietic homeostasis recovery after bone marrow injury. Mice were treated with 5-fluorouracil (5FU) to eliminate actively cycling cells in the bone marrow. Primary osteoblasts were isolated and subjected to cell culture. Real-time PCR, western blot and immunohistochemical staining were performed to study niche-related genes, osteoblast markers, and FGF2 signaling. Proliferation rate were analyzed by marker gene Ki67 and colony formation assay. Also, osterix-positive osteoprogenitor cells were isolated by FACS from Osx-GFP-Cre mice after 5FU treatment, and subjected to RNA-sequencing and analyzed for Fgf receptors and niche markers. RESULTS: The endosteal osteolineage cells isolated from 5FU-treated mice showed increased expression of the niche-related genes Sdf-1, Jagged-1, Scf, N-cad, Angpt1 and Vcam-1 and the osteoblast marker genes Osx, Opn, Runx2, and Alp, indicating that BM stress upon 5FU treatment activated the osteoblastic niche. Endosteal osteoblast expanded from a single layer to several layers 3 and 6 days after 5FU treatment. During the early recovery phase in 5FU-activated osteoblastic niches increased FGF2 expression and activated its downstream pERK. FGF2 treatment resulted in increased proliferation rate and the expression of niche marker genes in 5FU-activated osteoblastic niche cells. RNA-seq analysis in Osterix-positive osteoprogenitor cells isolated from 5FU-treated Osx-GFP mice showed significantly increased expression of Fgf receptors Fgfr1, 2 and 3. Although osteoblastic niche cells were damaged by 5FU treatment in the beginning, the increased number of OB layers in the recovery phase may be derived from resident osteoprogenitor cells by FGF2 activation under stress. CONCLUSIONS: Taken together, FGF2 signaling can regulate osteoblastic niche cells to support HSC homeostasis in response to bone marrow damage.

Nectin-2 (CD112) Is Expressed on Outgrowth Endothelial Cells and Regulates Cell Proliferation and Angiogenic Function.

Outgrowth endothelial cells (OECs) are a subpopulation of endothelial progenitor cells (EPCs) that have the capacity for proliferation and the ability to promote angiogenesis. In this study, we identified Nectin-2 as a surface protein of OECs through unbiased quantitative proteomics analysis. Using immunocytochemistry and flow cytometry, we confirmed that Nectin-2 is highly expressed on OECs. Nectin-2 (CD112) expression was limited or lower on mononuclear cells (MNCs) and mature tube-forming endothelial cells (ECs). Blocking Nectin-2 with a neutralizing monoclonal antibody significantly increased the trans-well migration and tube forming capacity of OECs. Similarly, Nectin-2 knockdown resulted in enhanced tube formation, cell migration and proliferation with p-Erk activation. Moreover, Nectin-2 deficiency resulted in compensatory increase of other Nectin family genes including Nectin-3 and Necl-4 which promote VEGFR signaling. These results indicate that Nectin-2 is a surface marker and an important regulator of OECs, with significant implications for the isolation of OECs and blocking Nectin-2 on OECs by an antibody for angiogenic applications.

Inducible HGF-secreting Human Umbilical Cord Blood-derived MSCs Produced via TALEN-mediated Genome Editing Promoted Angiogenesis.

Mesenchymal stem cells (MSCs) promote therapeutic angiogenesis to cure serious vascular disorders. However, their survival period and cytokine-secretory capacity are limited. Although hepatocyte growth factor (HGF) can accelerate the rate of angiogenesis, recombinant HGF is limited because of its very short half-life (<3-5 minutes). Thus, continuous treatment with HGF is required to obtain an effective therapeutic response. To overcome these limitations, we produced genome-edited MSCs that secreted HGF upon drug-specific induction. The inducible HGF expression cassette was integrated into a safe harbor site in an MSC chromosome using the TALEN system, resulting in the production of TetOn-HGF/human umbilical cord blood-derived (hUCB)-MSCs. Functional assessment of the TetOn-HGF/hUCB-MSCs showed that they had enhanced mobility upon the induction of HGF expression. Moreover, long-term exposure by doxycycline (Dox)-treated TetOn-HGF/hUCB-MSCs enhanced the anti-apoptotic responses of genome-edited MSCs subjected to oxidative stress and improved the tube-formation ability. Furthermore, TetOn-HGF/hUCB-MSCs encapsulated by arginine-glycine-aspartic acid (RGD)-alginate microgel induced to express HGF improved in vivo angiogenesis in a mouse hindlimb ischemia model. This study showed that the inducible HGF-expressing hUCB-MSCs are competent to continuously express and secrete HGF in a controlled manner. Thus, the MSCs that express HGF in an inducible manner are a useful therapeutic modality for the treatment of vascular diseases requiring angiogenesis.