Early Passage Research Articles

ERASING THE AETHIOPIAN IN CICERO'S POST REDITUM IN SENATU

The Roman attitude toward the Ethiopian as expressed in scattered passages is far less kindly than the Greek. The usage in Terence and the Auctor ad Herennium which imply a vogue for Ethiopians is probably in imitation of Greek usage. How early the Roman attitude crystalized into racial feeling it is hard to say, and as those who express it are chiefly satirists one must be careful in drawing conclusions. Nevertheless in the absence of an expressed good will and in the face of references which have a superior or contemptuous tone it is evident that the Romans had no special affection for Ethiopians at Rome, however romantically they may have spoken of the races of distant India. The earliest passage in which they are spoken of slightingly seems to be in Cicero—cum hoc homine an cum stipite Aethiope, Cicero, De Sen., 6. The word does not occur in all the manuscripts and the Oxford and Teubner texts omit it entirely. In notes it is translated ‘blockhead’ and the statement made that in antiquity the Ethiopians were synonymous with stupidity, a conclusion obviously drawn from the passage and the modern attitude toward them. Even if the word was actually used by Cicero, this passage alone is basis for such a theory.Mrs. Beardsley (op. cit., pp.119–120), in my judgement, is wrong in her conclusion that the Roman attitude toward the Negro crystallized into racial feeling. In support of her view that the Romans referred to the Ethiopians at Rome in a superior and contemptuous tone, Mrs. Beardsley includes the following passages: (1) Cicero, Red. in Sen., 6.14 (cited incorrectly as De Sen., 6); (2) Martial, VI, 39, 6; (3) Juvenal, II, 23. Cicero, Red in Sen., 6.14…cum hoc homine an stipite Aethiope…, as Mrs. Beardsley admits, does not appear in all the manuscripts and is omitted in the best established texts. A consideration of the context leads me to believe that the editors (Oxford, Teubner, Loeb) are right in rejecting Aethiope or stipite Aethiope and in reading stipite. Nevertheless, the appearance of the variant indicates that the author of the reading used Aethiope in a derogatory sense. (It is possible that the pejorative meaning of aethiops was a medieval development.)In these two excerpts, Grace Hadley Beardsley and Frank M. Snowden, Jr., discuss the appearance of the word Aethiops (‘Aethiopian’) in Cicero's Post reditum in senatu 14. Beardsley, whose intellectual project was motivated, as Maghan Keita and, more recently, Najee Olya have discussed, by racial animus and who sought to find evidence of Greco-Roman anti-Blackness that was both consistent with, and therefore a legitimizing exemplum for, contemporary anti-Blackness in 20th-century America, took Cicero's words as ‘the earliest passage in which [Aethiopians] are spoken of slightingly’ at Rome—doing so cautiously, given the fact that most editors had deleted it from the text. Frank M. Snowden, Jr.—whose own work W.E.B. Du Bois explicitly contrasted with Beardsley—responded to Beardsley's assertion that Post reditum in senatu contained evidence of anti-Blackness with scepticism, ultimately doubting the legitimate textual presence of the term and interpreting its presence instead as an artefact of hostile scribal intervention. Indeed, both Beardsley and Snowden discuss the fact that Aethiope does not occur in all of the Cicero manuscripts. While it is true that none of the authoritative textual editions print Aethiope at Post reditum in senatu 14, the textual apparatus nonetheless demonstrates clearly that the term appears in the manuscript tradition more often than it does not:stipe P1: etiope P2stipe uel ethiope Guel aethiope stipe E1esope H

Stabilization of hESCs in two distinct substates along the continuum of pluripotency.

A detailed understanding of the developmental substates of human pluripotent stem cells (hPSCs) is needed to optimize their use in cell therapy and for modeling early development. Genetic instability and risk of tumorigenicity of primed hPSCs are well documented, but a systematic isogenic comparison between substates has not been performed. We derived four hESC lines in naive human stem cell medium (NHSM) and generated isogenic pairs of NHSM and primed cultures. Through phenotypic, transcriptomic, and methylation profiling, we identified changes that arose during the transition to a primed substate. Although early NHSM cultures displayed naive characteristics, including greater proliferation and clonogenic potential compared with primed cultures, they drifted toward a more primed-like substate over time, including accumulation of genetic abnormalities. Overall, we show that transcriptomic and epigenomic profiling can be used to place human pluripotent cultures along a developmental continuum and may inform their utility for clinical and research applications.

Long-term passages of human clonal mesenchymal stromal cells can alleviate the disease in the rat model of collagen-induced arthritis resembling early passages of different heterogeneous cells.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease of unknown cause. The interaction of immune system cells and the secretion of inflammatory cytokines with synovial cells leads to severe inflammation in the affected joints. Currently, medications, including non-steroidal anti-inflammatory drugs, glucocorticoids, and more recently, disease-modifying anti-rheumatic drugs, are used to reduce inflammation. However, long-term use of these drugs causes adverse effects or resistance in a considerable number of RA patients. Recent findings revealed the safety and efficacy of mesenchymal stromal cells (MSCs)-based therapies both in RA animal models and clinical trials. Here, the beneficial effects of bone marrow-derived heterogeneous MSCs (BM-hMSCs) and Wharton jelly-derived MSCs (WJ-MSCs) at early passages were compared to BM-derived clonal MSCs (BM-cMSCs) at high passage number on a rat model of collagen-induced arthritis. Results showed that systemic delivery of MSCs significantly reversed adverse changes in body weight, paw swelling, and arthritis score in all MSC-treated groups. Radiological images and histological evaluation demonstrated the therapeutic effects of MSCs. There was a decrease in serum level of anti-collagen type II immunoglobulin G and the inflammatory cytokines interleukin (IL)-1β, IL-6, IL-17, and tumor necrosis factor-α in all MSC-treated groups. In contrast, an increase in inhibitory cytokines transforming growth factor-β and IL-10 was seen. Notably, the long-term passages of BM-cMSCs could alleviate RA symptoms similar to the early passages of WJ-MSCs and BM-hMSCs. The importance of BM-cMSCs is the potential to establish cell banks with billions of cells derived from a single donor that could be a competitive cell-based therapy to treat RA.

Derivation of pancreatic acinar cell carcinoma cell line HS-1 as a patient-derived tumor organoid.

Acinar cell carcinoma (ACC) of the pancreas is a malignant tumor of the exocrine cell lineage with a poor prognosis. Due to its rare incidence and technical difficulties, few authentic human cell lines are currently available, hampering detailed investigations of ACC. Therefore, we applied the organoid culture technique to various types of specimens, such as bile, biopsy, and resected tumor, obtained from a single ACC patient. Despite the initial propagation, none of these organoids achieved long-term proliferation or tolerated cryopreservation, confirming the challenging nature of establishing ACC cell lines. Nevertheless, the biopsy-derived early passage organoid developed subcutaneous tumors in immunodeficient mice. The xenograft tumor histologically resembled the original tumor and gave rise to infinitely propagating organoids with solid features and high levels of trypsin secretion. Moreover, the organoid stained positive for carboxylic ester hydrolase, a specific ACC marker, but negative for the duct cell marker CD133 and the endocrine lineage marker synaptophysin. Hence, we concluded the derivation of a novel ACC cell line of the pure exocrine lineage, designated HS-1. Genomic analysis revealed extensive copy number alterations and mutations in EP400 in the original tumor, which were enriched in primary organoids. HS-1 displayed homozygous deletion of CDKN2A, which might underlie xenograft formation from organoids. Although resistant to standard cytotoxic agents, the cell line was highly sensitive to the proteasome inhibitor bortezomib, as revealed by an in vitro drug screen and in vivo validation. In summary, we document a novel ACC cell line, which could be useful for ACC studies in the future.

Nicotinamide Mononucleotide Supplementation Improves Mitochondrial Dysfunction and Rescues Cellular Senescence by NAD+/Sirt3 Pathway in Mesenchymal Stem Cells.

In vitro expansion-mediated replicative senescence has severely limited the clinical applications of mesenchymal stem cells (MSCs). Accumulating studies manifested that nicotinamide adenine dinucleotide (NAD+) depletion is closely related to stem cell senescence and mitochondrial metabolism disorder. Promoting NAD+ level is considered as an effective way to delay aging. Previously, we have confirmed that nicotinamide mononucleotide (NMN), a precursor of NAD+, can alleviate NAD+ deficiency-induced MSC senescence. However, whether NMN can attenuate MSC senescence and its underlying mechanisms are still incompletely clear. The present study herein showed that late passage (LP) MSCs displayed lower NAD+ content, reduced Sirt3 expression and mitochondrial dysfunction. NMN supplementation leads to significant increase in intracellular NAD+ level, NAD+/ NADH ratio, Sirt3 expression, as well as ameliorated mitochondrial function and rescued senescent MSCs. Additionally, Sirt3 over-expression relieved mitochondrial dysfunction, and retrieved senescence-associated phenotypic features in LP MSCs. Conversely, inhibition of Sirt3 activity via a selective Sirt3 inhibitor 3-TYP in early passage (EP) MSCs resulted in aggravated cellular senescence and abnormal mitochondrial function. Furthermore, NMN administration also improves 3-TYP-induced disordered mitochondrial function and cellular senescence in EP MSCs. Collectively, NMN replenishment alleviates mitochondrial dysfunction and rescues MSC senescence through mediating NAD+/Sirt3 pathway, possibly providing a novel mechanism for MSC senescence and a promising strategy for anti-aging pharmaceuticals.

Determining epigenetic memory in kidney proximal tubule cell derived induced pluripotent stem cells using a quadruple transgenic reprogrammable mouse

The majority of nucleated somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs). The process of reprogramming involves epigenetic remodelling to turn on pluripotency-associated genes and turn off lineage-specific genes. Some evidence shows that iPSCs retain epigenetic marks of their cell of origin and this “epigenetic memory” influences their differentiation potential, with a preference towards their cell of origin. Here, we reprogrammed proximal tubule cells (PTC) and tail tip fibroblasts (TTF), from a reprogrammable mouse to iPSCs and differentiated the iPSCs to renal progenitors to understand if epigenetic memory plays a role in renal differentiation. This model allowed us to eliminate experimental variability due to donor genetic differences and transfection of the reprogramming factors such as copy number and integration site. In this study we demonstrated that early passage PTC iPSCs and TTF iPSCs expressed low levels of renal progenitor genes and high levels of pluripotency-associated genes, and the transcriptional levels of these genes were not significantly different between PTC iPSCs and TTF iPSCs. We used ChIP-seq of H3K4me3, H3K27me3, H3K36me3 and global DNA methylation profiles of PTC iPSCs and TTF iPSCs to demonstrate that global epigenetic marks were not different between the cells from the two different sets of tissue samples. There were also no epigenetic differences observed when kidney developmental genes and pluripotency-associated genes were closely examined. We did observe that during differentiation to renal progenitor cells the PTC iPSC-derived renal cells expressed higher levels of three renal progenitor genes compared to progenitors derived from TTF iPSCs but the underlying DNA methylation and histone methylation patterns did not suggest an epigenetic memory basis for this.

Abstract 12946: L-Arginine Attenuates Myofibroblast Phenoconversion and Fibrosis in the Human Heart

Introduction. L-Arginine (L-Arg) is a semi-essential amino acid involved in a number of biological processes. L-Arg has been recently shown to have beneficial effects on functional capacity in heart failure. However, the molecular mechanism underlying these actions are mostly unknown and a potential direct effect of L-Arg on cardiac fibroblasts has never been investigated hitherto. Transforming growth factor (TGF)-β is one of the main profibrotic cytokines, known to activate fibroblasts into myofibroblasts stimulating the production of extracellular matrix (ECM). In fibrosis condition, excessive ECM alters myocardial structure, eventually impairing cardiac function. Hypothesis. We hypothesized that L-Arg could mitigate TGF-β-dependent phenoconversion of human cardiac fibroblasts (HCFs). Methods. We isolated primary cardiac fibroblasts from human hearts (protocol approved by the Institutional Review Board for protection of human subjects) and tested a model of fibrosis obtained by treatment with TGF-β. Isolated HCFs were characterized by immunostaining. Early passage HCFs were treated with TGF-β or TGF-β + L-Arg at different doses and time points. The mitochondrial generation of reactive oxygen species (ROS) was assessed by MitoSox at the confocal microscopy. Results. TGF-β induced a significant upregulation of established markers of myofibroblast activation and fibrosis (including Fibronectin, Col1A1, Col1A2, and Periostin ), which was significantly attenuated by L-Arg, as confirmed by RT-qPCR, immunostaining, and immunoblot. Moreover, we observed that TGF-β treatment significantly increased the mitochondrial generation of ROS, which was again prevented by co-treatment with L-Arg. In addition, L-Arg significantly reduced SMAD-2 protein expression. Conclusions. Our findings demonstrate, for the first time to our knowledge, the anti-fibrotic role of L-Arg in HCFs, mediated via a TGF-β/SMAD-2 signaling pathway.

ODP251 Thioredoxin-Interacting Protein (Txnip) And Cellular Senescence: Potential Adverse Effects of Txnip Deficiency.

Abstract Thioredoxin-interacting protein (TXNIP) is a ubiquitously expressed protein that belongs to the α-arrestin family of proteins but is unique in that it interacts with and inhibits the function of the endogenous antioxidant, Thioredoxin (Trx). TXNIP is strongly upregulated by hyperglycemia and is associated with increased oxidative stress and promotes pancreatic beta cell apoptosis accelerating the progression of diabetes. Age-dependent dysregulation of the Trx/TXNIP redox system has been implicated in cellular senescence and aging. However, the mechanisms are not fully understood. While Trx overexpression has been reported to protect against aging and age-related diseases, and we found that TXNIP-/- (KO) mice were protected from Diabetic kidney disease, paradoxically, others reported TXNIP KO mice had reduced survival in response to paraquat mediated oxidative stress. To investigate the potential effects of TXNIP in cellular senescence we used primary mouse renal glomerular mesangial cells (MC) from WT and TXNIP KO mice. Passaging of cells leads to replicative senescence. In WT MC this was associated with a significant reduction in TXNIP expression in late passage (LP-passage 25) compared to early passage (EP-passage 5-7) (p<0. 05), and this was accompanied by appearance of the senescence phenotype determined by significant increases in expression of the cellular senescence markers, p53, p16, p21, phosphorylation of histone H2AX (p<0. 05) and senescence associated beta-galactosidase staining (13-fold increase) (p<0. 01). To determine whether TXNIP downregulation played a causal role, primary MC from TXNIP KO were compared to WT at EP 5. TXNIP KO showed significantly higher expression of senescence markers P53 and P16 (p<0. 05), and beta-galactosidase staining (15-fold). AKT activation has been implicated in replicative senescence. Here, increased phosphorylation of AKT and its substrate FOXO were found in LP versus EP WT, and EP KO versus WT MC (p<0. 05). These studies indicate that TXNIP downregulation is a mediator of replicative senescence associated with increased AKT signaling. Thus, while beneficial for diabetes, targeting TXNIP may have long term untoward effects on aging. Presentation: No date and time listed

Culturing and Neuronal Differentiation of Human Dental Pulp Stem Cells.

A major issue in studying human neurogenetic disorders, especially rare syndromes affecting the nervous system, is the ability to grow neuronal cultures that accurately represent these disorders for analysis. Although there has been some success in generating induced pluripotent stem cells (iPSC) from both skin and blood, there are still limitations to the collection, production and use of iPSC derived neurons. We have had significant success in collecting and growing human dental pulp stem cells (DPSC) from exfoliated teeth sent directly to our laboratory by the parents of children with a variety of rare neurogenetic syndromes. This protocol outlines our current methods for the growth and expansion of DPSC from exfoliated (baby) teeth. These DPSC can be differentiated into a variety of cell types including osteoblasts, chondrocytes, and mixed neuron and glial cultures. Here we provide our protocol for the differentiation of early passage DPSC cultures into neurons for molecular and cellular studies. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Collection and transportation of exfoliated teeth Basic Protocol 2: Dental pulp extraction Basic Protocol 3: Passage, freezing, and thawing of DPSC cultures Basic Protocol 4: Differentiation of DPSC into mixed neuronal cultures.

Low Oxygen Tension Inhibits Senescence in Bone Marrow Mesenchymal Stem Cells (BMSCs)

Background: Stem cells have been used in regenerative medicine but are so few in the body that they require cell culture. Stem cell culture was performed under normal oxygen tension and passage was carried out until the number of cells was sufficient for therapy. Stem cell cultures under normal oxygen tension do not match the stem cell microenvironment, which can lead to premature senescence. This study aims to determine the association of low oxygen tension with premature senescence of mesenchymal stem cells (MSCs) through inhibition of p21 expression by HIF-1a. Methode: The research method used rabbit bone marrow in New Zealand as a source of MSCs. The results of isolation of MSCs were divided into two groups for cultured on normal and low oxygen tension until 10 passages. Cells were identified using flowcytometry for cd105 and cd34. At early and late passage, the expression of p21 and HIF-1a was examined using immunofluorescence while senescence was examined using β-galactosidase assay. Results: The results showed that in low oxygen cultures HIF-1a expression increased significantly (p <0.05) while p21 expression decreased significantly (p <0.05) as did the β-galactosidase assay. Conclusions: The conclusion of this research is low oxygen tension culture able to decrease premature senescence culture of invitro stem cells mesenchymal through obstacles p21 by HIF-1a.
 Keywords: p21 expression, HIF-1a expression, late passage, premature senescence.

Semicarbazide-Sensitive Amine Oxidase (SSAO) and Lysyl Oxidase (LOX) Association in Rat Aortic Vascular Smooth Muscle Cells.

Vascular smooth muscle cells (VSMCs) are the main stromal cells in the medial layer of the vascular wall. These cells produce the extracellular matrix (ECM) and are involved in many pathological changes in the vascular wall. Semicarbazide-sensitive amine oxidase (SSAO) and lysyl oxidase (LOX) are vascular enzymes associated with the development of atherosclerosis. In the vascular smooth muscle cells, increased SSAO activity elevates reactive oxygen species (ROS) and induces VSMCs death; increased LOX induces chemotaxis through hydrogen peroxide dependent mechanisms; and decreased LOX contributes to endothelial dysfunction. This study investigates the relationship between SSAO and LOX in VSMCs by studying their activity, protein, and mRNA levels during VSMCs passaging and after silencing the LOX gene, while using their respective substrates and inhibitors. At the basal level, LOX activity decreased with passage and its protein expression was maintained between passages. βAPN abolished LOX activity (** p < 0.01 for 8 vs. 3 and * p < 0.05 for 5 vs. 8) and had no effect on LOX protein and mRNA levels. MDL72527 reduced LOX activity at passage 3 and 5 (##&nbsp;p < 0.01) and had no effect on LOX protein, and mRNA expression. At the basal level, SSAO activity also decreased with passage, and its protein expression was maintained between passages. MDL72527 abolished SSAO activity (****&nbsp;p < 0.0001 for 8 vs. 3 and * p < 0.05 for 5 vs. 8), VAP-1 expression at passage 5 (** p < 0.01) and 8 (**** p < 0.0001), and Aoc3 mRNA levels at passage 8 (* p < 0.05). βAPN inhibited SSAO activity (**** p < 0.0001 for 5 vs. 3 and 8 vs. 3 and * p < 0.05 for 5 vs. 8), VAP-1 expression at passage 3 (* p < 0.05), and Aoc3 mRNA levels at passage 3 (* p < 0.05). Knockdown of the LOX gene (**** p < 0.0001 for Si6 vs. Sictrl and *** p < 0.001 for Si8 vs. Sictrl) and LOX protein (** p < 0.01 for Si6 and Si8 vs. Sictrl) in VSMCs at passage 3 resulted in a reduction in Aoc3 mRNA (####&nbsp;p < 0.0001 for Si6 vs. Sictrl and ###&nbsp;p < 0.001 for Si8 vs. Sictrl) and VAP-1 protein (#&nbsp;p < 0.05 for Si8 vs. Sictrl). These novel findings demonstrate a passage dependent decrease in LOX activity and increase in SSAO activity in rat aortic VSMCs and show an association between both enzymes in early passage rat aortic VSMCs, where LOX was identified as a regulator of SSAO activity, protein, and mRNA expression.

Paracrine Senescence of Mesenchymal Stromal Cells Involves Inflammatory Cytokines and the NF-κB Pathway.

It has been known that senescence-associated secretory phenotype (SASP) triggers senescence of the surrounding normal cells. However, SASP signaling regarding mesenchymal stromal cell aging remains to be fully elucidated. Therefore, the present study aimed to clarify the molecular mechanism of late (passage) MSC-induced paracrine SASP-mediated senescence of early (passage) MSCs during ex vivo expansion. Here, we conducted an extensive characterization of senescence features in bone-marrow (BM)-derived MSCs from healthy human donors. Late MSCs displayed an enlarged senescent-like morphology, induced SASP-related proinflammatory cytokines (IL-1α and IL-8), and reduced clonogenic capacity and osteogenic differentiation when compared to early MSCs. Of note, paracrine effects of SASP-related IL-1α and IL-8 from late MSCs induced cellular senescence of early MSCs via an NF-κB-dependent manner. Moreover, cellular senescence of early MSCs was promoted by the synergistic action of IL-1α and IL-8. However, inhibition of NF-κB by shRNA transfection or using inhibitors in early MSCs blocked early MSCs cellular senescence caused by paracrine SASP of late MSCs. In conclusion, these findings reveal that late MSCs display features of senescence and that, during ex vivo expansion, SASP-related proinflammatory cytokines contribute to activate a cellular senescence program in early MSCs that may ultimately impair their functionality.

Improved Sendai viral system for reprogramming to naive pluripotency

Naive human induced pluripotent stem cells (iPSCs) can be generated by reprogramming somatic cells with Sendai virus (SeV) vectors. However, only dermal fibroblasts have been successfully reprogrammed this way, and the process requires culture on feeder cells. Moreover, SeV vectors are highly persistent and inhibit subsequent differentiation of iPSCs. Here, we report a modified SeV vector system to generate transgene-free naive human iPSCs with superior differentiation potential. The modified method can be applied not only to fibroblasts but also to other somatic cell types. SeV vectors disappear quickly at early passages, and this approach enables the generation of naive iPSCs in a feeder-free culture. The naive iPSCs generated by this method show better differentiation to trilineage and extra-embryonic trophectoderm than those derived by conventional methods. This method can expand the application of iPSCs to research on early human development and regenerative medicine.

GD2-targeting CAR-T cells enhanced by transgenic IL-15 expression are an effective and clinically feasible therapy for glioblastoma

BackgroundAggressive primary brain tumors such as glioblastoma are uniquely challenging to treat. The intracranial location poses barriers to therapy, and the potential for severe toxicity. Effective treatments for primary brain...

Biological activity reduction and mitochondrial and lysosomal dysfunction of mesenchymal stem cells aging in vitro

BackgroundMesenchymal stem cells (MSCs) have been extensively used for the treatment of various diseases in preclinical and clinical trials. In vitro propagation is needed to attain enough cells for clinical use. However, cell aging and viability reduction caused by long-time culture have not been thoroughly investigated, especially for the function of mitochondria and lysosomes. Therefore, this study was designed to detect mitochondrial and lysosomal activity, morphological and functional changes in human umbilical cord MSCs (UMSCs) after long-time culture.MethodsFirst, we examined cell activities, including proliferation and immigration ability, differentiation potential, and immunosuppressive capacity of UMSCs at an early and late passages as P4 (named UMSC-P4) and P9 (named UMSC-P9), respectively. Then, we compared the mitochondrial morphology of UMSC-P4 and UMSC-P9 using the electronic microscope and MitoTracker Red dyes. Furthermore, we investigated mitochondrial function, including mitochondrial membrane potential, antioxidative ability, apoptosis, and ferroptosis detected by respective probe. Cell energy metabolism was tested by mass spectrometry. In addition, we compared the lysosomal morphology of UMSC-P4 and UMSC-P9 by electronic microscope and lysoTracker Red dyes. Finally, the transcriptome sequence was performed to analyze the total gene expression of these cells.ResultsIt was found that UMSC-P9 exhibited a reduced biological activity and showed an impaired mitochondrial morphology with disordered structure, reduced mitochondrial crista, and mitochondrial fragments. They also displayed decreased mitochondrial membrane potential, antioxidative ability, tricarboxylic acid cycle activity and energy production. At the same time, apoptosis and ferroptosis were increased. In addition, UMSC-P9, relative to UMSC-P4, showed undegraded materials in their lysosomes, the enhancement in lysosomal membrane permeability, the reduction in autophagy and phagocytosis. Moreover, transcriptome sequence analysis also revealed a reduction of cell function, metabolism, mitochondrial biogenesis, DNA replication and repair, and an increase of gene expression related to cell senescence, cancer, diseases, and infection in UMSC-P9.ConclusionThis study indicates that in vitro long-time culturing of MSCs can cause mitochondrial and lysosomal dysfunction, probably contributing to the decline of cell activity and cell aging. Therefore, the morphology and function of mitochondria and lysosomes can be regarded as two important parameters to monitor cell viability, and they can also serve as two important indicators for optimizing in vitro culture conditions.

Improved biological performance of human cartilage-derived progenitors in platelet lysate xenofree media in comparison to fetal bovine serum media

Primary articular cartilage-derived cells are among the preferred contenders for cell-based therapy approaches for cartilage repair. Limited access to primary human cartilage tissue necessitates the process of in vitro cell expansion to obtain sufficient cells for therapeutic purposes. Therapeutic outcomes of such cell-based approaches become highly dependent on the quality of the in vitro culture-expanded cells. The objective of this study was to determine the differential biological effects of human platelet lysate (hPL) xeno-free defined media vs FBS containing traditional media on primary human cartilage-derived cells. Our goal in pursuing this work was to identify a preferred xenofree media alternative, that can be used as a platform for expansion of cells intended for clinical applications. Primary cartilage-derived cells obtained from five patients were simultaneously cultured in two expansion media's: (1) traditional (DMEM+10%FBS+1%P/S) and (2) defined xenofree (Nutristem® complete media+0.5%hPL). Connective tissue progenitors (CTPs) were assayed by standard colony forming unit assay, morphology, proliferation in early and late passages, expression of MSC associated cell-surface markers (CD73, CD90 and CD105) and trilineage differentiation (adipogenesis, osteogenesis and chondrogenesis) were considered for comparison of biological performance. Early biological performance of primary cartilage-derived cells was significantly improved in Nutristem® expansion media in comparison to traditional expansion media with respect to (1) Colony forming efficiency tended to be higher (p = 0.058) and (2) CTPs formed larger colonies with respect to total cells per colony and colony area (p < 0.01). In the culture expanded cell population, Nutristem® expansion media was superior to traditional expansion media with respect to: (1) overall proliferation rate through passages 1–4 (p = 0.027), (2) total cells harvested at end of passage 4 (p = 0.028) and (3) total positive stain area of CD73 (p = 0.006), CD90 (p = 0.001) and CD105 (p = 0.049). Nutristem®-hPL expanded cells when differentiated in respective xenofree serum-free defined MSCgo™ differentiated media's, also showed significant improvement in adipogenic, osteogenic and chondrogenic marker expression. Overall, we convincingly demonstrated that a low concentration of hPL in combination with defined xenofree media is an effective and economic growth supplement to culture expand primary cartilage-derived cells. It can be manufactured under cGMP conditions to improve clinical-grade cell products’ quality for therapeutic applications.

WNT Activation and TGFβ-Smad Inhibition Potentiate Stemness of Mammalian Auditory Neuroprogenitors for High-Throughput Generation of Functional Auditory Neurons In Vitro.

Hearing loss affects over 460 million people worldwide and is a major socioeconomic burden. Both genetic and environmental factors (i.e., noise overexposure, ototoxic drug treatment and ageing), promote the irreversible degeneration of cochlear hair cells and associated auditory neurons, leading to sensorineural hearing loss. In contrast to birds, fish and amphibians, the mammalian inner ear is virtually unable to regenerate due to the limited stemness of auditory progenitors, and no causal treatment is able to prevent or reverse hearing loss. As of today, a main limitation for the development of otoprotective or otoregenerative therapies is the lack of efficient preclinical models compatible with high-throughput screening of drug candidates. Currently, the research field mainly relies on primary organotypic inner ear cultures, resulting in high variability, low throughput, high associated costs and ethical concerns. We previously identified and characterized the phoenix auditory neuroprogenitors (ANPGs) as highly proliferative progenitor cells isolated from the A/J mouse cochlea. In the present study, we aim at identifying the signaling pathways responsible for the intrinsic high stemness of phoenix ANPGs. A transcriptomic comparison of traditionally low-stemness ANPGs, isolated from C57Bl/6 and A/J mice at early passages, and high-stemness phoenix ANPGs was performed, allowing the identification of several differentially expressed pathways. Based on differentially regulated pathways, we developed a reprogramming protocol to induce high stemness in presenescent ANPGs (i.e., from C57Bl6 mouse). The pharmacological combination of the WNT agonist (CHIR99021) and TGFβ/Smad inhibitors (LDN193189 and SB431542) resulted in a dramatic increase in presenescent neurosphere growth, and the possibility to expand ANPGs is virtually limitless. As with the phoenix ANPGs, stemness-induced ANPGs could be frozen and thawed, enabling distribution to other laboratories. Importantly, even after 20 passages, stemness-induced ANPGs retained their ability to differentiate into electrophysiologically mature type I auditory neurons. Both stemness-induced and phoenix ANPGs resolve a main bottleneck in the field, allowing efficient, high-throughput, low-cost and 3R-compatible in vitro screening of otoprotective and otoregenerative drug candidates. This study may also add new perspectives to the field of inner ear regeneration.

Ezh2 Inhibits Replicative Senescence of Atrial Fibroblasts Through Promotion of H3K27me3 in the Promoter Regions of CDKN2a and Timp4 Genes.

BackgroundIn most cell types, replicative senescence (RS) is supposed to be a principle causative factor for aging. Atrial fibrosis, pathologically characterized by proliferation of atrial fibroblasts (AFs) and excessive accumulation of extracellular matrix proteins, is the most common substrate of atrial fibrillation (Afib) in the elderly. However, whether AFs’ RS develops in the aged and fibrotic left atrium (LA) and, if yes, what is the key regulator for the pathogenesis of AFs’ RS remain largely unknown.MethodsWe obtained the left atrial tissues from young (6–8 weeks old) and aged (24 months old) C57BL/6 male mice. Screening and validation of differential genes were performed using comparative analysis of RNA-seq results. Replicative senescence was examined in primary AFs after cell passage. Further gain-of-function and loss-of-function experiments were performed to explore the regulation of the AFs’ RS progression.ResultsIn the present study, we demonstrated that there was a considerable extent of AFs’ RS in the aged and fibrotic LA. Transcriptome screening showed that Ezh2 (Enhancer of zeste homolog 2) was significantly downregulated in the LA tissue of aged mice. Ezh2 is a histone methyltransferase that catalyzes H3K27me3 and mediates transcriptional silencing. We confirmed that Ezh2 was downregulated in the isolated pure senescent AFs. Knockdown of Ezh2 by siRNA or inhibition of Ezh2ʹs methyltransferase activities by GSK-126 and GSK-343 accelerated RS in the early passage of AFs, while its overexpression deaccelerated RS in the late passage of AFs. Mechanistically, Ezh2 suppressed CDKN2a (p16, p19) and Timp4 gene transcription by forming canonical H3K27me3 modifications in their promoter regions. Furthermore, the functional balance between Timp4 and MMP8 in AFs could be collapsed by changes in Ezh2 expression.ConclusionThese results thus indicate that Ezh2 is a key regulator of AFs’ RS and this work may provide a basis for future treatments for atrial fibrosis in the elderly.

Factors and optimizations of healthcare workers' perception in alternative care facilities.

BackgroundDiverse measures have been carried out worldwide to establish Alternative Care Facilities (ACFs) for different ends, such as receiving, curing or isolating patients, aiming to cope with tremendous shock in the urban medical system during the early passage of the COVID-19 epidemic. Healthcare workers always felt anxious and stressed during multiple major public health emergencies in medical facilities. Some active measures to improve healthcare workers' perceptions, such as temporary training, workflow improvement, and supplementary facilities, were proved insufficient in several past public health emergencies. Therefore, this study aims to analyze the contributing factors of the healthcare workers' perceptions of the ACFs in this pandemic, which can help find an innovative path to ensure their health, well-being and work efficiency.MethodThis paper conducted semi-structured in-depth interviews with the world's first batch of healthcare workers who have worked in ACFs through a qualitative study based on Grounded Theory. The healthcare workers interviewed from Heilongjiang, Shandong, Fujian, and Hubei provinces, have worked in one of the four different ACFs built in Wuhan. The results are obtained through the three-level codes and analyses of the interview recordings.ResultsThe factors affecting the perception of healthcare workers in ACFs during the epidemic situation can be summarized into five major categories: individual characteristics, organization management, facilities and equipment, space design, and internal environment. The five major categories affecting the composition of perception can be further divided into endogenous and exogenous factors, which jointly affect the perception of healthcare workers in ACFs. Among them, individual characteristics belong to endogenous factors, which are the primary conditions, while other categories belong to exogenous factors, which are the decisive conditions.ConclusionThis paper clarifies factors affecting the perception of healthcare workers in ACFs and analyzes the mechanism of each factor. It is posited that the passive strategies are a promising solution to protect healthcare workers' health, improve their work efficiency, and help reduce the operation stress of ACFs. We should train multidisciplinary professionals for future healthcare and enhance collaborations between healthcare workers and engineers. To sum up, this paper broadens new horizons for future research on the optimization of ACFs and finds new paths for alleviating healthcare workers' adverse perceptions of ACFs.

CD26 is a senescence marker associated with reduced immunopotency of human adipose tissue-derived multipotent mesenchymal stromal cells

IntroductionHuman mesenchymal stromal cells (MSCs) have immunomodulatory, anti-inflammatory, and tolerogenic effects. Long-term in vitro expansion of MSCs to generate clinical grade products results in the accumulation of senescent-functionally impaired MSCs. Markers to assess the ‘senescent load’ of MSC products are needed.MethodsEarly and late passage human adipose tissue (AT) MSCs from pediatric and adult donors were characterized using established senescent markers [i.e., MSC size, granularity, and autofluorescence by flow cytometry; β-galactosidase staining (SA-β-gal); CDKN2A and CDKN1A by qRT-PCR]. In gene set enrichment analysis, DPP4 (also known as adenosine deaminase complexing protein 2 or CD26) was found as a prominent dysregulated transcript that was increased in late passage MSC(AT). This was confirmed in a larger number of MSC samples by PCR, flow cytometry, Western blotting, and immunofluorescence. In vitro immunopotency assays compared the function of CD26high and CD26low MSC(AT). The effect of senolytics on the CD26high subpopulation was evaluated in senescent MSC(AT).ResultsLate passage MSC(AT) had a senescence transcriptome signature. DPP4 was the most differentially enriched gene in senescent MSCs. Late passage senescent MSC(AT) had higher CD26 surface levels and total protein abundance. Moreover, CD26 surface levels were higher in early passage MSC(AT) from adults compared to pediatric donors. CD26 abundance correlated with established senescence markers. CD26high MSC(AT) had reduced immunopotency compared to CD26low MSC(AT). Senolytic treatment induced MSC apoptosis, which decreased the frequencies of CD26high MSC(AT).ConclusionsDPP4 gene expression and DPP4/CD26 protein abundance are markers of replicative senescence in MSC(AT). Samples enriched in CD26high MSC(AT) have reduced immunopotency and CD26high MSCs are reduced with senolytics.