Within the immune system, microRNAs (miRNAs) exert key regulatory functions. However, what are the mRNA targets regulated by miRNAs and how miRNAs are transcriptionally regulated themselves remain for the most part unknown. We found that in primary human memory T helper lymphocytes, miR-150 was the most abundantly expressed miRNA, and its expression decreased drastically upon activation, suggesting regulatory roles. Constitutive MIR150 gene expression required the RFX family of transcription factors, and its activation-induced down-regulation was linked to their reduced expression. By performing miRNA pull-down and sequencing experiments, we identified PDGFA-associated protein 1 (PDAP1) as one main target of miR-150 in human T lymphocytes. PDAP1 acted as an RNA-binding protein (RBP), and its CRISPR/Cas-9–mediated deletion revealed that it prominently contributed to the regulation of T-cell proliferation. Overall, using an integrated approach involving quantitative analysis, unbiased genomics, and genome editing, we identified RFX factors, miR-150, and the PDAP1 RBP as the components of a regulatory axis that restrains proliferation of primary human T lymphocytes.

A mechanistic understanding of the regulatory circuits that control the effector responses of memory T helper lymphocytes, and in particular their ability to produce pro‐inflammatory cytokines, may lead to effective therapeutic interventions in all immune‐related diseases. Activation of T lymphocytes induces robust immune responses that in most cases lead to the complete eradication of invading pathogens or tumor cells. At the same time, however, such responses must be both highly controlled in magnitude and limited in time to avoid unnecessary damage. To achieve such sophisticated level of control, T lymphocytes have at their disposal an array of transcriptional and post‐transcriptional regulatory mechanisms that ensure the acquisition of a phenotype that is tailored to the incoming stimulus while restraining unwarranted activation, eventually leading to the resolution of the inflammatory response. Here, we will discuss some of these cell‐intrinsic mechanisms that control T cell responses and involve transcription factors, microRNAs, and RNA‐binding proteins. We will also explore how the same mechanisms can be involved both in anti‐tumor responses and in autoimmunity.

Low back pain related to intervertebral disc (IVD) degeneration has a major socioeconomical impact on our ageing society. Therefore, stem cell therapy to activate self-repair of the IVD remains an exciting treatment strategy. In this respect, tissue-specific progenitors may play a crucial role for IVD regeneration, as these cells are perfectly adapted to this niche. Such a rare progenitor cell population residing in the nucleus pulposus (nucleus pulposus progenitor cells or NPPCs) was found positive for the angiopoietin-1 receptor (Tie2+) and was demonstrated to possess self-renewal capacity and in vitro multipotency. Here, we compared three sorting protocols, i.e., fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS) and a mesh-based label-free cell sorting system (pluriSelect), with respect to cell yield, potential to form colonies (colony forming units = CFUs) and in vitro functional differentiation assays for tripotency. The aim of this study was to demonstrate efficiency of three wide-spread cell sorting methods for picking rare cells (<5%) and how these isolated cells then behave in down-stream functional differentiation in adipogenesis, osteogenesis and chondrogenesis. The cell yield among the isolation methods differed widely, with FACS presenting the highest yield (5.0 ± 4.0%), followed by MACS (1.6 ± 2.9%) and pluriSelect (1.1 ± 1.0%). The number of colonies formed was not significantly different between Tie2+ and Tie2- NPPCs. Only FACS was able to separate into two functionally different populations that showed trilineage multipotency, while MACS and pluriSelect failed to maintain a clear separation between Tie2+ and Tie2- populations in differentiation assays. To conclude, the isolation of NPPC was possible with all three sorting methods, while FACS was the preferred technique for separation of functional Tie2+ cells. .

INTRODUCTION: Nucleus Pulposus Progenitor Cells (NPPCs), positive for the angiopoietin-1 receptor (Tie2), were demonstrated in human, mouse, canine and bovine NP tissue [1,2,3]. Tie2+ NPPCs possess a multi-lineage differentiation potential, and regeneration potential is attributed to them. However, the isolation of Tie2+ NPPCs can be cumbersome. Hence, three isolation methods were compared. METHODS: Bovine NP cells were isolated from 10-14-month-old animals. Cell sorting was performed with an antibody against Tie2 (bs-1300R, Bioss) using FACS, magnetic-activated cell sorting (MACS) and pluriSelect, a size-based sorting method. Outcomes were evaluated by cell yield of Tie2+ cells, the ability of sorted cells to form colonies and tri-lineage differentiation assays. RESULTS & DISCUSSION: FACS resulted in the highest Tie2+ cell yield (5.0 ± 4.0%) followed by MACS (1.6 ± 2.9%) and pluriSelect (1.1 ± 1.4%). Colony forming ability did not differ between Tie2+ and Tie2- cells for any isolation method. However, Tie2+ cells obtained by MACS tended to have more colonies than FACS and pluriSelect. Osteogenic and adipogenic differentiation of Tie2+ and Tie2- cells did not result in a clear distinction for MACS and pluriSelect; Tie2+ FACS-sorted cells demonstrated superior osteogenic and adipogenic differentiation over Tie2- cells. Also for chondrogenesis, the Tie2+ FACS-sorted Tie2+ NPPCs tended to produce more proteoglycan versus Tie2- NPPCs, whereas for MACS and pluriSelect no difference was found. CONCLUSIONS: Based on the parameters tested, isolation of NPPC is possible with all three methods. However, cell yields differed widely. FACS although most invasive, appears to be the most specific sorting method for these Tie2 + cells among the tested methods as Tie2 + cells do not demonstrate osteogenic and adipogenic differentiation. As for cell yield MACS seems to reveal the most, possibly this is due to inclusion of cells expressing Tie2 less strongly.

Low back pain related to intervertebral disk (IVD) degeneration has a major socioeconomic impact on our aging society. Therefore, stem cell therapy to activate self-repair of the IVD remains an exciting treatment strategy. In this respect, tissue-specific progenitors may play a crucial role in IVD regeneration, as these cells are perfectly adapted to this niche. Such a rare progenitor cell population residing in the nucleus pulposus (NP) (NP progenitor cells [NPPCs]) was found positive for the angiopoietin-1 receptor (Tie2 + ), and was demonstrated to possess self-renewal capacity and in vitro multipotency. Here, we compared three sorting protocols; that is, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and a mesh-based label-free cell sorting system (pluriSelect), with respect to cell yield, potential to form colonies (colony-forming units), and in vitro functional differentiation assays for tripotency. The aim of this study was to demonstrate the efficiency of three widespread cell sorting methods for picking rare cells ( < 5%) and how these isolated cells then behave in downstream functional differentiation in adipogenesis, osteogenesis, and chondrogenesis. The cell yields among the isolation methods differed widely, with FACS presenting the highest yield (5.0% – 4.0%), followed by MACS (1.6% – 2.9%) and pluriSelect (1.1% – 1.0%). The number of colonies formed was not significantly different between Tie2 + and Tie2 - NPPCs. Only FACS was able to separate into two functionally different populations that showed trilineage multipotency, while MACS and pluriSelect failed to maintain a clear separation between Tie2 + and Tie2 - populations in differentiation assays. To conclude, the isolation of NPPCs was possible with all three sorting methods, while FACS was the preferred technique for separation of functional Tie2 + cells.

Introduction: Nucleus pulposus progenitor cells (NPPC), Tie2+ cells (positive for angiopoietin receptor), which possess multi-lineage differential potential is a potential cell population for cell therapy. However, the number of Tie2+ cells in NP is extremely limited. Referring to the recent research of Tie2+ hematopoietic stem cells we attempted to increase the Tie2+ cell sub-population in nucleus pulposus cells (NPC) by PPARδ agonist treatment and increasing mitophagy. Methods: Cells were isolated from fresh human IVD tissue from spinal surgery with written consent. The passage 1 human NP cells were cultured in low glucose Dulbecco’s Modified Eagle’s Medium media containing PPARδ agonist (GW501516, Sigma), i.e., 25 µM, or vehicle control (N = 2 donors). After 10 days NP, the Tie2 marker expression was then detected by flow cytometry cells and relative gene expression was determined by real-time qPCR, i.e. at ACAN, col1, col2, and PTEN-induced kinase 1 (PINK1). Results: PPARδ-agonist-treated NP population had ~3 times more Tie2+ cells and PINK1 gene expression tended to be higher than in the vehicle control group. Conclusion: PPARδ agonist possibly increases the Tie2+ cell population in NPC by increasing mitophagy similar to hematopoietic stem cells.

Introduction: Mesenchymal stromal cells (MSC) have been identified as the most prominent cell-based therapy candidates for cartilage, bone and intervertebral disc diseases due to their intrinsic ability to differentiate into chondrocytes and osteocytes.1–4 One of the major problems involved in MSC-based cell therapy is the necessity to expand the cells in vitro to obtain sufficient cells for administration.5 During prolonged expansion, MSC become senescent which impairs their therapeutic potential.5,6 Here, we aimed to investigate whether extracellular nicotinamide riboside (NR), a precursor of nicotinamide adenine dinucleotide (NAD), is beneficial for MSC expansion in respective of growth kinetics, mitochondrial activity and senescence. Methods: MSC were isolated from human bone marrow aspirates by gradient centrifugation and subsequent expansion in α-MEM + 10% FBS + 2.5 ng/ml bFGF-2. Immunophenotyping was performed by flow cytometry. The cytotoxicity of NR was measured at day 4 for 29 concentrations in a range from 0.005 μM to 4’000 μM. The long-term effect of NR was tested at concentrations of 10, 100 and 1’000 μM by measuring the population doubling level (PDL), relative confluency (IncuCyte S3®), mitochondrial activity by resazurin reduction, senescence-associated β-galactosidase assay (SA-β-gal) and NAD/NADH ratio. Results: The isolated cells displayed a typical MSC immunophenotype (CD73+, CD90+, CD105+, CD34-, CD45-, CD14-). NR exhibited no acute cytotoxicity at any tested concentration. MSC treated with 3000 μM and 4’000 μM NR had a significantly higher mitochondrial activity than the negative control (p=0.0027 and p<0.0001 respectively, N=3). However, in the weeks 3 to 8, cells treated with ≥100 μM NR died reaching a maximum PDL of 13.43 (N=4). In two donors, the experimental group with 10 μM NR reached a 2-fold higher PDL than the negative control, on the other hand, two donors exhibited no difference in PDL between the two groups. The relative confluency at passage (P) 2 after 6 days in culture was higher with 10 μM NR compared to the negative control (35.00 ± 9.29% and 26.19 ± 5.41% respectively, mean ± SD, N=2). The absolute mitochondrial activity was significantly higher with 10 μM NR at P4, P8 and P10 (p<0.01, N=4), albeit, when normalized to the cell count, no difference in mitochondrial activity was observed suggesting that the higher absolute mitochondrial activity was caused by the higher cell count, rather than higher cellular activity. At all passages, the percentage of SA-β-gal positive cells was under 5%, except in the negative control medium at P11 (18.17% ± 18.18%, mean ± SD, N=1). All experimental groups treated with NR had a higher NAD/NADH ratio which exhibited a dose-dependent trend (N=1). Conclusion: Extracellular NR elevated the intracellular NAD/NADH ratio presumably by serving as a precursor of NAD. NR is not cytotoxic within 4 days of culture at concentrations up to 4’000 μM, though in long-term culture already 100 μM proved to be cytotoxic. Long-term culture with 10 μM NR improved the growth kinetics and mitochondrial activity markedly in two donors. The mechanism of this has yet to be determined by measuring the telomere lengths and by analyzing the gene expression of sirtuins (which are triggered by the NAD/NADH ratio) and senescence-associated genes. 1. Squillaro, T., Peluso, G. & Galderisi, U. Clinical Trials with Mesenchymal Stem Cells: An Update. Cell Transplant. 25, 1–53 (2015). 2. Trounson, A. & McDonald, C. Stem Cell Therapies in Clinical Trials: Progress and Challenges. Cell Stem Cell 17, 11–22 (2015). 3. Sakai, D. & Schol, J. Cell therapy for intervertebral disc repair: Clinical perspective. Journal of Orthopaedic Translation (2017). doi:10.1016/j.jot.2017.02.002 4. Vedicherla, S. & Buckley, C. T. Cell-based therapies for intervertebral disc and cartilage regeneration— Current concepts, parallels, and perspectives. Journal of Orthopaedic Research (2017). doi:10.1002/jor.23268 5. Turinetto, V., Vitale, E. & Giachino, C. Senescence in Human Mesenchymal Stem Cells: Functional Changes and Implications in Stem Cell-Based Therapy. Int. J. Mol. Sci. 17, 1164 (2016). 6. Sepulveda, J. C. et al. Cell senescence abrogates the therapeutic potential of human mesenchymal stem cells in the lethal endotoxemia model. Stem Cells 32, 1865–77 (2014).