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1.
Engineered Zinc Finger Protein Targeting 2LTR Inhibits HIV Integration in Hematopoietic Stem and Progenitor Cell-Derived Macrophages: In Vitro Study.
Chupradit, K, Khamaikawin, W, Sakkhachornphop, S, Puaninta, C, Torbett, BE, Borwornpinyo, S, Hongeng, S, Wattanapanitch, M, Tayapiwatana, C
International journal of molecular sciences. 2022;(4)
Abstract
Human hematopoietic stem/progenitor cell (HSPC)-based gene therapy is a promising direction for curing HIV-1-infected individuals. The zinc finger protein (2LTRZFP) designed to target the 2-LTR-circle junction of HIV-1 cDNA was previously reported as an intracellular antiviral molecular scaffold that prevents HIV integration. Here, we elucidate the efficacy and safety of using 2LTRZFP in human CD34+ HSPCs. We transduced 2LTRZFP which has the mCherry tag (2LTRZFPmCherry) into human CD34+ HSPCs using a lentiviral vector. The 2LTRZFPmCherry-transduced HSPCs were subsequently differentiated into macrophages. The expression levels of pro-apoptotic proteins of the 2LTRZFPmCherry-transduced HSPCs showed no significant difference from those of the non-transduced control. Furthermore, the 2LTRZFPmCherry-transduced HSPCs were successfully differentiated into mature macrophages, which had normal phagocytic function. The cytokine secretion assay demonstrated that 2LTRZFPmCherry-transduced CD34+ derived macrophages promoted the polarization towards classically activated (M1) subtypes. More importantly, the 2LTRZFPmCherry transduced cells significantly exhibited resistance to HIV-1 integration in vitro. Our findings demonstrate that the 2LTRZFPmCherry-transduced macrophages were found to be functionally and phenotypically normal, with no adverse effects of the anti-HIV-1 scaffold. Our data suggest that the anti-HIV-1 integrase scaffold is a promising antiviral molecule that could be applied to human CD34+ HSPC-based gene therapy for AIDS patients.
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2.
Oxidative stress in retinal pigment epithelium impairs stem cells: a vicious cycle in age-related macular degeneration.
Lazzarini, R, Nicolai, M, Lucarini, G, Pirani, V, Mariotti, C, Bracci, M, Mattioli-Belmonte, M
Molecular and cellular biochemistry. 2022;(1):67-77
Abstract
Aging, chronic oxidative stress, and inflammation are major pathogenic factors in the development and progression of age-related macular degeneration (AMD) with the loss of retinal pigment epithelium (RPE). The human RPE contains a subpopulation of progenitors (i.e., RPE stem cells-RPESCs) whose role in the RPE homeostasis is under investigation. We evaluated the paracrine effects of mature RPE cells exposed to oxidative stress (H2O2) on RPESCs behavior through co-cultural, morphofunctional, and bioinformatic approaches. RPESCs showed a decline in proliferation, an increase of the senescence-associated β-galactosidase activity, the acquisition of a senescent-like secretory phenotype (SASP), and the reduction of their stemness and differentiation competencies. IL-6 and Superoxide Dismutase 2 (SOD2) seem to be key molecules in RPESCs response to oxidative stress. Our results get insight into stress-induced senescent-associated molecular mechanisms implicated in AMD pathogenesis. The presence of chronic oxidative stress in the microenvironment reduces the RPESCs abilities, inducing and/or maintaining a pro-inflammatory retinal milieu that in turn could affect AMD onset and progression.
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3.
Adipose tissue-derived stromal/stem cells + cholecalciferol: a pilot study in recent-onset type 1 diabetes patients.
Dantas, JR, Araújo, DB, Silva, KR, Souto, DL, de Fátima Carvalho Pereira, M, Luiz, RR, Dos Santos Mantuano, M, Claudio-da-Silva, C, Gabbay, MAL, Dib, SA, et al
Archives of endocrinology and metabolism. 2021;(3):342-351
Abstract
OBJECTIVE Adipose tissue-derived stromal/stem cells (ASCs) and vitamin D have immunomodulatory actions that could be useful for type 1 diabetes (T1D). We aimed in this study to investigate the safety and efficacy of ASCs + daily cholecalciferol (VIT D) for 6 months in patients with recent-onset T1D. METHODS In this prospective, dual-center, open trial, patients with recent onset T1D received one dose of allogenic ASC (1 × 106 cells/kg) and cholecalciferol 2,000 UI/day for 6 months (group 1). They were compared to patients who received chol-ecalciferol (group 2) and standard treatment (group 3). Adverse events were recorded; C-peptide (CP), insulin dose and HbA1c were measured at baseline (T0), after 3 (T3) and 6 months (T6). RESULTS In group 1 (n = 7), adverse events included transient headache (all), mild local reactions (all), tachycardia (n = 4), abdominal cramps (n = 1), thrombophlebitis (n = 4), scotomas (n = 2), and central retinal vein occlusion at T3 (n = 1, resolution at T6). Group 1 had an increase in basal CP (p = 0.018; mean: 40.41+/-40.79 %), without changes in stimulated CP after mixed meal (p = 0.62), from T0 to T6. Basal CP remained stable in groups 2 and 3 (p = 0.58 and p = 0.116, respectively). Group 1 had small insulin requirements (0.31+/- 0.26 UI/kg) without changes at T6 (p = 0.44) and HbA1c decline (p = 0.01). At T6, all patients (100%; n = 7) in group 1 were in honeymoon vs 75% (n = 3/4) and 50% (n = 3/6) in groups 2 and 3, p = 0.01. CONCLUSION Allogenic ASC + VIT D without immunosuppression was safe and might have a role in the preservation of β-cells in patients with recent-onset T1D. ClinicalTrials.gov: NCT03920397.
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4.
Human periodontal ligament stem cells and hormesis: Enhancing cell renewal and cell differentiation.
Calabrese, EJ
Pharmacological research. 2021;:105914
Abstract
This paper provides a detailed assessment of hormetic dose responses by human periodontal ligament stem cells (hPDLSCs). Hormetic dose responses were induced by a broad range of chemicals, including dietary supplements (e.g., curcumin, ginsenoside Rg1), pharmaceutical/commercial substances (e.g., metformin) and endogenous agents (e.g., periostin, TNF-α) for cell proliferation/viability and osteogenic/adipocyte differentiation. This paper clarifies underlying mechanistic foundations of the hPLDSC hormetic dose responses and explores their therapeutic implications. Emerging evidence based on assessments of multiple types of stem cells shows hormetic dose responses to be widespread, reflecting considerable generality and a highly conserved evolutionary trait.
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5.
Natural Compounds and PCL Nanofibers: A Novel Tool to Counteract Stem Cell Senescence.
Bellu, E, Cruciani, S, Garroni, G, Balzano, F, Satta, R, Montesu, MA, Fadda, A, Mulas, M, Sarais, G, Bandiera, P, et al
Cells. 2021;(6)
Abstract
Tissue homeostasis mainly depends on the activity of stem cells to replace damaged elements and restore tissue functions. Within this context, mesenchymal stem cells and fibroblasts are essential for maintaining tissue homeostasis in skin, in particular in the dermis. Modifications in collagen fibers are able to affect stem cell features. Skin properties can be significantly reduced after injuries or with aging, and stem cell niches, mainly comprising extracellular matrix (ECM), may be compromised. To this end, specific molecules can be administrated to prevent the aging process induced by UV exposure in the attempt to maintain a youngness phenotype. NanoPCL-M is a novel nanodevice able to control delivery of Mediterranean plant myrtle (Myrtus communis L.) extracts. In particular, we previously described that myrtle extracts, rich in bioactive molecules and nutraceuticals, were able to counteract senescence in adipose derived stem cells. In this study, we analyzed the effect of NanoPCL-M on skin stem cells (SSCs) and dermal fibroblasts in a dynamic cell culture model in order to prevent the effects of UV-induced senescence on proliferation and collagen depot. The BrdU assay results highlight the significantly positive effect of NanoPCL-M on the proliferation of both fibroblasts and SSCs. Our results demonstrate that-M is able to preserve SSCs features and collagen depot after UV-induced senescence, suggesting their capability to retain a young phenotype.
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6.
Fraxinellone alleviates inflammation and promotes osteogenic differentiation in lipopolysaccharide-stimulated periodontal ligament stem cells by regulating the bone morphogenetic protein 2/Smad pathway.
Fu, Z, Wang, X, Li, B, Tang, Y
Archives of oral biology. 2021;:104927
Abstract
OBJECTIVE The aim of the present study was to investigate the role of fraxinellone in periodontitis and identify its potential mechanisms. DESIGN Lipopolysaccharide-induced periodontal ligament stem cells (PDLSCs) was employed to simulate the periodontitis in vitro. The levels of inflammatory factors were evaluated. After treatment with fraxinellone, alkaline phosphatase activity was determined. Additionally, calcium nodules staining was evaluated by alizarin red staining and the expression of osteogenesis differentiation-associated proteins was detected using western blot analysis. Moreover, the levels of proteins in bone morphogenetic protein 2 (BMP2)/Smad pathway were measured. Subsequently, BMP2 was silenced by transfection with small hairpin RNA to explore the underlying mechanisms of fraxinellone in lipopolysaccharide-induced PDLSCs. RESULTS Lipopolysaccharide stimulation significantly upregulated the levels of inflammatory factors, which were inhibited by fraxinellone intervention. Moreover, fraxinellone notably promoted osteogenic differentiation and calcification shown by increasing levels of alkaline phosphatase, calcification and osteogenic marker proteins. Furthermore, the expression of BMP2, phosphorylated Smad1 and phosphorylated Smad5 was remarkably upregulated when fraxinellone exposure in lipopolysaccharide-induced PDLSCs. What's more, BMP2 silencing dramatically restored the effects of fraxinellone on inflammation and osteogenic differentiation of PDLSCs stimulated by lipopolysaccharide. CONCLUSION These data demonstrated that fraxinellone alleviates inflammation and promotes osteogenic differentiation in lipopolysaccharide-stimulated PDLSCs by regulating the BMP2/Smad pathway, providing experimental supports for the clinical application of fraxinellone in the treatment of periodontitis.
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7.
Human Granulosa Cells-Stemness Properties, Molecular Cross-Talk and Follicular Angiogenesis.
Dompe, C, Kulus, M, Stefańska, K, Kranc, W, Chermuła, B, Bryl, R, Pieńkowski, W, Nawrocki, MJ, Petitte, JN, Stelmach, B, et al
Cells. 2021;(6)
Abstract
The ovarian follicle is the basic functional unit of the ovary, comprising theca cells and granulosa cells (GCs). Two different types of GCs, mural GCs and cumulus cells (CCs), serve different functions during folliculogenesis. Mural GCs produce oestrogen during the follicular phase and progesterone after ovulation, while CCs surround the oocyte tightly and form the cumulus oophurus and corona radiata inner cell layer. CCs are also engaged in bi-directional metabolite exchange with the oocyte, as they form gap-junctions, which are crucial for both the oocyte's proper maturation and GC proliferation. However, the function of both GCs and CCs is dependent on proper follicular angiogenesis. Aside from participating in complex molecular interplay with the oocyte, the ovarian follicular cells exhibit stem-like properties, characteristic of mesenchymal stem cells (MSCs). Both GCs and CCs remain under the influence of various miRNAs, and some of them may contribute to polycystic ovary syndrome (PCOS) or premature ovarian insufficiency (POI) occurrence. Considering increasing female fertility problems worldwide, it is of interest to develop new strategies enhancing assisted reproductive techniques. Therefore, it is important to carefully consider GCs as ovarian stem cells in terms of the cellular features and molecular pathways involved in their development and interactions as well as outline their possible application in translational medicine.
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8.
In Vitro Osteoinductivity Assay of Hydroxylapatite Scaffolds, Obtained with Biomorphic Transformation Processes, Assessed Using Human Adipose Stem Cell Cultures.
Iaquinta, MR, Torreggiani, E, Mazziotta, C, Ruffini, A, Sprio, S, Tampieri, A, Tognon, M, Martini, F, Mazzoni, E
International journal of molecular sciences. 2021;(13)
Abstract
In this study, the in vitro biocompatibility and osteoinductive ability of a recently developed biomorphic hydroxylapatite ceramic scaffold (B-HA) derived from transformation of wood structures were analyzed using human adipose stem cells (hASCs). Cell viability and metabolic activity were evaluated in hASCs, parental cells and in recombinant genetically engineered hASC-eGFP cells expressing the green fluorescence protein. B-HA osteoinductivity properties, such as differentially expressed genes (DEG) involved in the skeletal development pathway, osteocalcin (OCN) protein expression and mineral matrix deposition in hASCs, were evaluated. In vitro induction of osteoblastic genes, such as Alkaline phosphatase (ALPL), Bone gamma-carboxyglutamate (gla) protein (BGLAP), SMAD family member 3 (SMAD3), Sp7 transcription factor (SP7) and Transforming growth factor, beta 3 (TGFB3) and Tumor necrosis factor (ligand) superfamily, member 11 (TNFSF11)/Receptor activator of NF-κB (RANK) ligand (RANKL), involved in osteoclast differentiation, was undertaken in cells grown on B-HA. Chondrogenic transcription factor SRY (sex determining region Y)-box 9 (SOX9), tested up-regulated in hASCs grown on the B-HA scaffold. Gene expression enhancement in the skeletal development pathway was detected in hASCs using B-HA compared to sintered hydroxylapatite (S-HA). OCN protein expression and calcium deposition were increased in hASCs grown on B-HA in comparison with the control. This study demonstrates the biocompatibility of the novel biomorphic B-HA scaffold and its potential use in osteogenic differentiation for hASCs. Our data highlight the relevance of B-HA for bone regeneration purposes.
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9.
Epigenetic memory: gene writer, eraser and homocysteine.
Tyagi, SC, Stanisic, D, Singh, M
Molecular and cellular biochemistry. 2021;(2):507-512
Abstract
Naturally chromatin remodeling is highly organized, consisting of histone acetylation (opening/relaxation of the compact chromatin structure), DNA methylation (inhibition of the gene expression activity) and sequence rearrangement by shifting. All this is essentially required for proper "in-printing and off-printing" of genes thus ensuring the epigenetic memory process. Any imbalance in ratios of DNA methyltransferase (DNMT, gene writer), fat-mass obesity-associated protein (FTO, gene eraser) and product (function) homocysteine (Hcy) could lead to numerous diseases. Interestingly, a similar process also happens in stem cells during embryogenesis and development. Despite gigantic unsuccessful efforts undertaken thus far toward the conversion of a stem cell into a functional cardiomyocyte, there has been hardly any study that shows successful conversion of a stem cell into a multinucleated cardiomyocyte. We have shown nuclear hypertrophy during heart failure, however; the mechanism(s) of epigenetic memory, regulation of genes during fertilization, embryogenesis, development and during adulthood remain far from understanding. In addition, there may be a connection of aging, loosing of the memory leading to death, and presumably to reincarnation. This review highlights some of these pertinent issues facing the discipline of biology as a whole today.
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10.
Protocol and Baseline Data on Renal Autologous Cell Therapy Injection in Adults with Chronic Kidney Disease Secondary to Congenital Anomalies of the Kidney and Urinary Tract.
Stavas, J, Diaz-Gonzalez de Ferris, M, Johns, A, Jain, D, Bertram, T
Blood purification. 2021;(4-5):678-683
Abstract
BACKGROUND Advanced cell therapies with autologous, homologous cells show promise to affect reparative and restorative changes in the chronic kidney disease (CKD) nephron. We present our protocol and preliminary analysis of an IRB-approved, phase I single-group, open-label trial that tests the safety and efficacy of Renal Autologous Cell Therapy (REACT; NCT04115345) in adults with congenital anomalies of the kidney and urinary tract (CAKUT). METHODS Adults with surgically corrected CAKUT and CKD stages 3 and 4 signed an informed consent and served as their "own" baseline control. REACT is an active biological ingredient acquired from a percutaneous tissue acquisition from the patient's kidney cortex. The specimen undergoes a GMP-compliant manufacturing process that harvests the selected renal cells composed of progenitors for renal repair, followed by image-guided locoregional reinjection into the patient's renal cortex. Participants receive 2 doses at 6-month intervals. Primary outcomes are stable renal function and stable/improved quality of life. Additional exploratory endpoints include the impact of REACT on blood pressure, vitamin D levels, hemoglobin, hematocrit and kidney volume by MRI analysis. RESULTS Four men and 1 woman were enrolled and underwent 5 cell injections. Their characteristics were as follows: mean 52.8 years (SD 17.7 years), 1 Hispanic, 4 non-Hispanic, and 5 white. There were no renal tissue acquisition, cell injection, or cell product-related complications at baseline. CONCLUSION REACT is demonstrating feasibility and patient safety in preliminary analysis. Autologous cell therapy treatment has the potential to stabilize or improve renal function in CAKUT-associated CKD to delay or avert dialysis. Patient enrollment and follow-up are underway.