Genetic barcoding systematically comparing genes in del(5q) MDS reveals a central role for CSNK1A1 in clonal expansion

How genetic haploinsufficiency contributes to the clonal dominance of hematopoietic stem cells (HSC) in del(5q) myelodysplastic syndrome (MDS) remains unresolved. Using a genetic barcoding strategy, a systematic comparison was carried out on genes implicated in the pathogenesis of del(5q) MDS in direct competition with each other and wild-type (WT) cells with single clone resolution. Csnk1a1 haploinsufficient HSCs expanded (oligo)clonally and outcompeted all other tested genes and combinations. Csnk1a1-/+ multipotent progenitors showed a pro-proliferative gene signature and HSCs a downregulation of inflammatory signaling/immune response. In validation experiments, Csnk1a1-/+ HSCs outperformed their WT counterparts under a chronic inflammation stimulus, also known to be caused by neighboring genes on chromosome 5. A crucial role for Csnk1a1 haploinsufficiency in the selective advantage of the 5q- HSC is therefore proposed. It is implemented by creation of a unique competitive advantage through increased HSC self-renewal and proliferation capacity, as well as increased fitness under inflammatory stress.

Knockdown of EIF3H inhibits the development and progression of pancreatic cancer by regulating cell proliferation and apoptosis in vitro

Nowadays, pancreatic cancer has been recognized as one of the most fatal malignancies worldwide, the molecular mechanism of which is still not fully understood. In this study, we aimed to uncover the fundamental functions of the eukaryotic translation initiation factor 3H subunit (EIF3H) in the development and progression of pancreatic cancer. Firstly, the results of immunohistochemical (IHC) staining revealed that EIF3H was highly expressed in pancreatic cancer. Moreover, lentiviruses were used to deliver shRNAs into pancreatic cancer cells for silencing EIF3H. Furthermore, the loss-of-function assays demonstrated that knockdown of EIF3H could inhibit the progression of pancreatic cancer cells by reducing proliferation capacity, promoting apoptosis, arresting cell cycle in G2 and suppressing cell migration. In summary, EIF3H may play a critical role in the development and progression of pancreatic cancer, which possesses the potential to act as a therapeutic target for pancreatic cancer treatment.

Mesenchymal Stromal Cells: a Possible Reservoir for HIV-1?

The introduction of antiretroviral therapy (ART) and highly active antiretroviral therapy (HAART) has transformed human immunodeficiency virus (HIV)-1 into a chronic, well-managed disease. However, these therapies do not eliminate all infected cells from the body despite suppressing viral load. Viral rebound is largely due to the presence of cellular reservoirs which support long-term persistence of HIV-1. A thorough understanding of the HIV-1 reservoir will facilitate the development of new strategies leading to its detection, reduction, and elimination, ultimately leading to curative therapies for HIV-1. Although immune cells derived from lymphoid and myeloid progenitors have been thoroughly studied as HIV-1 reservoirs, few studies have examined whether mesenchymal stromal/stem cells (MSCs) can assume this function. In this review, we evaluate published studies which have assessed whether MSCs contribute to the HIV-1 reservoir. MSCs have been found to express the receptors and co-receptors required for HIV-1 entry, albeit at levels of expression and receptor localisation that vary considerably between studies. Exposure to HIV-1 and HIV-1 proteins alters MSC properties in vitro, including their proliferation capacity and differentiation potential. However, in vitro and in vivo experiments investigating whether MSCs can become infected with and harbour latent integrated proviral DNA are lacking. In conclusion, MSCs appear to have the potential to contribute to the HIV-1 reservoir. However, further studies are needed using techniques such as those used to prove that cluster of differentiation (CD)4+ T cells constitute an HIV-1 reservoir before a reservoir function can definitively be ascribed to MSCs. Graphical abstract MSCs may contribute to HIV-1 persistence in vivo in the vasculature, adipose tissue, and bone marrow by being a reservoir for latent HIV-1. To harbour latent HIV-1, MSCs must express HIV-1 entry markers, and show evidence of productive or latent HIV-1 infection. The effect of HIV-1 or HIV-1 proteins on MSC properties may also be indicative of HIV-1 infection.

CircRNA FAT1 Regulates Osteoblastic Differentiation of Periodontal Ligament Stem Cells via miR-4781-3p/SMAD5 Pathway

The ability of human periodontal ligament stem cells (PDLSCs) to differentiate into osteoblasts is significant in periodontal regeneration tissue engineering. In this study, we explored the role and mechanism of circRNA FAT1 (circFAT1) in the osteogenic differentiation of human PDLSCs. The proliferation capacity of PDLSCs was evaluated by EdU and CCK-8 assay. The abilities of circFAT1 and miR-4781-3p in regulating PDLSC differentiation were analyzed by western blot, reverse transcription-polymerase chain reaction (RT-PCR), alkaline phosphatase (ALP), and Alizarin red staining (ARS). A nucleocytoplasmic separation experiment was utilized for circFAT1 localization. A dual-luciferase reporter assay confirmed the binding relationship between miR-4781-3p and circFAT1. It was showed that circFAT1 does not affect the proliferation of PDLSCs. The osteogenic differentiation of PDLSCs was benefited from circFAT1, which serves as a miRNA sponge for miR-4781-3p targeting SMAD5. Both knockdown of circFAT1 and overexpression of miR-4781-3p suppressed the osteogenic differentiation of PDLSCs. Thus, circFAT1 might be considered as a potential target of PDLSCs mediated periodontal bone regeneration.

Role of Tumor Microenvironment in Cancer Stem Cells Resistance to Radiotherapy

Cancer is a chronic disorder that involves several elements of both the tumor and the host stromal cells. At present, the complex relationship between the various factors presents in the tumor microenvironment (TME) and tumor cells, as well as immune cells located within the TME, is still poorly known. Within the TME, the crosstalk of these factors and immune cells essentially determines how a tumor reacts to the treatment and how the tumor can ultimately be destroyed, remain dormant, or develop and metastasize. Also, in TME, reciprocal crosstalk between cancer-associated fibroblasts (CAFs), extracellular matrix (ECM), hypoxia-inducible factor (HIF) intensifies the proliferation capacity of cancer stem cells (CSCs). CSCs are subpopulation of cells that reside within the tumor bulk and have the capacity to self-renew, differentiate, and repair DNA damage. These characteristics make CSCs develop resistance to a variety of treatments, such as radiotherapy (RT). RT is a frequent and often curative treatment for local cancer which mediates tumor elimination by cytotoxic actions. Also, cytokines and growth factors that are released into TME, have been involved in the activation of tumor radioresistance and the induction of different immune cells, altering local immune responses. In this review, we discuss the pivotal role of TME in resistance of CSCs to RT.

Increasing the efficiency of hyperthermic intraperitoneal chemotherapy (HIPEC) by a combination with a photosensitive drug in pediatric rhabdomyosarcoma

Background: Cytoreductive surgery (CRS) in combination with hyperthermic intraperitoneal chemotherapy (HIPEC) is an option in advanced peritoneal sarcomatosis. Nevertheless, CRS and HIPEC are not successful in all patients. An enhancement of HIPEC using photodynamic therapy might be beneficial. Therefore, a combination of the photosensitizer Hypericin (HYP) with HIPEC was evaluated in an animal model. Procedure: An established HIPEC animal model for rhabdomyosarcoma (NOD/LtSz-scid IL2Rγnullmice, n=80) was used. All groups received HYP (100 µg/200 µl) intraperitoneally with and without cisplatin-based (30 or 60 mg/m2) HIPEC (37 or 42 °C, for 60 min) (five groups, each n=16). Tumor dissemination was documented visually and by using HYP-based fluorescence guidance. HYP-based photodynamic therapy (PDT) of the tumor was performed. Finally, tissue samples were evaluated regarding proliferation (Ki-67) and apoptosis (TUNEL). Results: HYP uptake even in smallest tumor nodes (< 1 mm) was found. HYP-based fluorescence guidance allowed a better tumor detection in comparison to visual inspection. Immunohistochemistry revealed HYP penetration across the tumor surface. HYP-based PDT without HIPEC induced marginal apoptotic effects at the tumor surface. Combining HYP with HIPEC revealed cisplatin concentration dependent decrease in proliferation capacity and induction of apoptosis across determined cell layers of the tumor surfaces. Conclusion: HYP as fluorescent photosensitizer offers an intraoperative diagnostic advantage detecting intraperitoneal tumor dissemination. The combination of HYP and cisplatin-based HIPEC was feasible in vivo showing enhanced effects on tumor proliferation and apoptosis induction across the tumor surface. Further studies combining HYP and HIPEC will follow to establish a clinical application.

Obesity affects mitochondrial metabolism and proliferative potential of endometrial progenitor cells

The study aimed to investigate the influence of obesity on cellular features of equine endometrial progenitor cells (Eca EPCs), including viability, proliferation capacity, mitochondrial metabolism, and oxidative homeostasis. Eca EPCs derived from non-obese (Non-OB) and obese (OB) mares were characterised by cellular phenotype and multipotency.Obesity-induced changes in the activity of Eca EPCs include the decline of their proliferative activity, clonogenic potential, mitochondrial metabolism and enhanced oxidative stress. Eca EPCs isolated from obese mares were characterised by an increased occurrence of early apoptosis, loss of mitochondrial dynamics, and senescence-associated phenotype. Attenuated metabolism of Eca EPCs OB was related to increased expression of pro-apoptotic markers (CASP9, BAX, P53, P21), enhanced expression of OPN, PI3K and AKT, simultaneously with decreased signalling stabilising cellular homeostasis (including mitofusin, SIRT1, FOXP3).Obesity alters functional features and self-renewal potential of endometrial progenitor cells. The impaired cytophysiology of progenitor cells from obese endometrium predict lower regenerative capacity if used as autologous transplants.

Hyaluronan and Proteoglycan Link Protein 1 (HAPLN1) Promotes Viability of Rheumatoid Arthritis Fibroblast-Like Synoviocytes

Background To investigate HAPLN1 contribution to the viability of RA-FLSs and identify its potential role in RA pathogenesis. Methods Plasma levels and synovial expression of HAPLN1 were compared between healthy controls, and osteoarthritis (OA) and RA patients. Proliferation and migration of RA-FLSs transfected with siHAPLN1, HAPLN1OE (over-expression vector) and respective controls or treated with rHAPLN1 were measured by MTT and CCK8 assays as well as wound healing and transwell assays. RT-qPCR and automated WB analysis were used to compare the expression of AMPK-ɑ, TNF-ɑ, TGF-β, ACAN, MMPs, Cyclin-D1 and Ki-67 after siHAPLN1 or HAPLN1OE transfection. Proteomics and mRNA-seq analysis was done to study the differentially expressed proteins/genes after siHAPLN1 or rHAPLN1 treatment. Results Expression of HAPLN1 was increased in the plasma samples and synovium tissues of RA patients. HAPLN1OE transfected or rHAPLN1 treated RA-FLSs showed an increased proliferation capacity. However, Si-HAPLN1 has failed to affect the viability of RA-FLSs, though it decreased the migration ability of these cells. On the other hand, HAPLN1OE or rHAPLN1 had inhibitory effect on migration. Both si-HAPLN1 and HAPLN1OE treated RA-FLSs had down-regulated AMPK-ɑ gene expression, while protein level was found to be up-regulated. Furthermore, si-HAPLN1 has down-regulated TNF-ɑ, MMPs, IL-6, TGF-β, fibronectin and ACAN levels, while HAPLN1OE has up-regulated the levels of TNF-ɑ, MMPs, IL-6 and ACAN. Proteomics and mRNA-Seq analysis demonstrated HAPLN1 function in the activation of inflammation, proliferation, increased cell adhesion and strengthening of ECM function. Conclusons: HAPLN1 accelerated the proliferation of RA-FLSs but inhibited its migration ability. HAPLN1 network was found to be mainly involved in the activation of inflammation, cell proliferation and increased cell adhesion.

Cytological Effects of Serum Isolated from Polytraumatized Patients on Human Bone Marrow-Derived Mesenchymal Stem Cells

Due to their immunomodulatory and regenerative capacity, human bone marrow-derived mesenchymal stem cells (hBMSCs) are promising in the treatment of patients suffering from polytrauma. However, few studies look at the effects of sera from polytraumatized patients on hBMSCs. The aim of this study was to explore changes in hBMSC properties in response to serum from polytrauma patients taken at different time points after the trauma incident. For this, sera from 84 patients with polytrauma (collected between 2010 and 2020 in our department) were used. In order to test the differential influence on hBMSC, sera from the 1st (D1), 5th (D5), and 10th day (D10) after polytrauma were pooled, respectively. As a control, sera from three healthy donors (HS), matched with respect to age and gender to the polytrauma group, were collected. Furthermore, hBMSCs from four healthy donors were used in the experiments. The pooled sera of HS, D1, D5, and D10 were analyzed by multicytokine array for pro-/anti-inflammatory cytokines. Furthermore, the influence of the different sera on hBMSCs with respect to cell proliferation, colony forming unit-fibroblast (CFU-F) assay, cell viability, cytotoxicity, cell migration, and osteogenic and chondrogenic differentiation was analyzed. The results showed that D5 serum significantly reduced hBMSC cell proliferation capacity compared with HS and increased the proportion of dead cells compared with D1. However, the frequency of CFU-F was not reduced in polytrauma groups compared with HS, as well as the other parameters. The serological effect of polytrauma on hBMSCs was related to the time after trauma. It is disadvantageous to use BMSCs in polytraumatized patients at least until the fifth day after polytrauma as obvious cytological changes could be found at that time point. However, it is promising to use hBMSCs to treat polytrauma after five days, combined with the concept of “Damage Control Orthopedics” (DCO).

Developing a pro-angiogenic placenta derived amniochorionic scaffold with two exposed basement membranes as substrates for cultivating endothelial cells

Decellularized and de-epithelialized placenta membranes have widely been used as scaffolds and grafts in tissue engineering and regenerative medicine. Exceptional pro-angiogenic and biomechanical properties and low immunogenicity have made the amniochorionic membrane a unique substrate which provides an enriched niche for cellular growth. Herein, an optimized combination of enzymatic solutions (based on streptokinase) with mechanical scrapping is used to remove the amniotic epithelium and chorion trophoblastic layer, which resulted in exposing the basement membranes of both sides without their separation and subsequent damages to the in-between spongy layer. Biomechanical and biodegradability properties, endothelial proliferation capacity, and in vivo pro-angiogenic capabilities of the substrate were also evaluated. Histological staining, immunohistochemistry (IHC) staining for collagen IV, and scanning electron microscope demonstrated that the underlying amniotic and chorionic basement membranes remained intact while the epithelial and trophoblastic layers were entirely removed without considerable damage to basement membranes. The biomechanical evaluation showed that the scaffold is suturable. Proliferation assay, real-time polymerase chain reaction for endothelial adhesion molecules, and IHC demonstrated that both side basement membranes could support the growth of endothelial cells without altering endothelial characteristics. The dorsal skinfold chamber animal model indicated that both side basement membranes could promote angiogenesis. This bi-sided substrate with two exposed surfaces for cultivating various cells would have potential applications in the skin, cardiac, vascularized composite allografts, and microvascular tissue engineering.