Passage 1 Research Articles

Regenerative Variability of Human Juvenile Chondrocyte Sheets From Different Cell Donors in an Athymic Rat Knee Chondral Defect Model.

This study aimed to establish a combined histological assessment system of neo-cartilage outcomes and to evaluate variations in an established rat defect model treated with human juvenile cartilage-derived chondrocyte (JCC) sheets fabricated from various donors. JCCs were isolated from the polydactylous digits of eight patients. Passage 2 (P2) JCC sheets from all donors were transplanted into nude rat chondral defects for 4 weeks (27 nude rats in total). Defect-only group served as control. Histological samples were stained for safranin O, collagen 1 (COL1), and collagen 2 (COL2). (1) All samples were scored, and correlation coefficients for each score were calculated. (2) Donors were divided into "more effective" and "less effective" groups based on these scores. Then, differences between each group in each category of modified O'Driscoll scoring were evaluated. (1) Modified O'Driscoll scores were negatively correlated with %COL1 area, and positively correlated with %COL2 area and COL2/1 ratio. (2) Four of 8 donors exhibited significantly higher modified O'Driscoll scores and %COL2 areas. JCC donors were divided into two groups by average score values. Significant differences between the two groups were observed in modified O'Driscoll categories of "Nature of predominant tissue," "Reconstruction of subchondral bone," and "Safranin O staining." The combined histological evaluation method is useful for detailed in vivo efficacy assessments of cartilage defect regeneration models. Variations in histological scores among juvenile cartilage-derived chondrocyte donors were correlated to the quality of regenerated cartilage hyaline structure and subchondral bone remodeling observed in the nude rat defect model.

DNA methylation, but not microRNA expression, is affected by in vitro THC exposure in bovine granulosa cells

BackgroundA global increase in cannabis use has led to questions about its effects on fertility. The rise in consumption amongst women of reproductive age is a growing concern, as this group is vulnerable in terms of reproductive health. Ample evidence suggests that the psychoactive component of cannabis, Δ9-Tetrahydrocannabinol (THC), interacts with the endocannabinoid system (ECS), that helps regulate mammalian reproduction. This study aimed to research the epigenetic effects of THC in bovine granulosa cells (GCs) by (1) investigating global DNA methylation via measuring 5-mC and 5-hmC levels; (2) measuring key methylation regulators, including the methylating enzymes DNMT1, DNMT3a, DNMT3b and the demethylases TDG and TET1/2/3; and (3) assessing fertility-associated miRNAs key in developmental competency, including miR-21, -155, -33b, -324 and -346.MethodsBovine GCs were used as a translational model for reproductive toxicity in humans. To determine THC effects, GCs were isolated from Cumulus-Oocyte-Complexes (COCs) from bovine ovaries, cultured in vitro for 7 days, or until confluent, and cryopreserved at passage 1 (P1). For experimentation, cells were thawed, cultured until passage 2 (P2), serum restricted for 24-h and treated for 24-h in one of five groups: control, vehicle (1:1:18 ethanol: tween: saline) and three clinically relevant THC doses (0.032, 0.32 and 3.2 μM). Global methylation was assessed by measuring 5-mC and 5-hmC levels with flow cytometry. To assess mRNA and protein expression of methylation regulators and miRNA profiles, qPCR and Western Blotting were utilized. Shapiro-Wilk test was used to determine normality within datasets. One-way ANOVA was applied to determine statistical significance using GraphPad Prism 6.0.0.ResultsResults indicate a significant decrease (p = 0.0435) in 5-mC levels following low THC exposure, while no changes were observed in 5-hmC levels. A significant increase in DNMT1 following high THC exposure at the RNA level (p < 0.05) and a significant increase following low THC exposure at the protein level (p = 0.0048) were also observed. No significant differences were observed in DNMT3a/3b, TDG, TET1/2/3 mRNAs or in any of the miRNAs analyzed.ConclusionsThis research suggests that THC mainly affects DNA methylation, but not miRNA profiles, ultimately altering gene expression and likely impairing oocyte competence, maturation, and fertilization potential.

Advantages of pooling of human bone marrow-derived mesenchymal stromal cells from different donors versus single-donor MSCs

Mesenchymal stromal cells (MSC) from adult bone marrow are the most commonly used cells in clinical trials. MSCs from single donors are the preferred starting material but suffer from a major setback of being heterogeneous that results in unpredictable and inconsistent clinical outcomes. To overcome this, we developed a method of pooling MSCs from different donors and created cell banks to cater clinical needs. Initially, the master cell banks (MCBs) were created at passage 1 (P1) from the bone marrow MSCs isolated from of nine different donors. At this stage, MCBs from three different donors were mixed in equal proportion and expanded till P3 to create working cell banks. Further, the pooled cells and individual donor MSCs were expanded till P5 and cryopreserved and extensively characterised. There was a large heterogeneity among the individual donor MSCs in terms of growth kinetics (90% Coefficient of variation (CV) for cell yield and 44% CV for population doubling time at P5), immunosuppressive ability (30% CV at 1:1 and 300% CV at 1:10 ratio), and the angiogenic factor secretion potential (20% CV for VEGF and71% CV for SDF-1). Comparatively, the pooled cells have more stable profiles (60% CV for cell yield and 7% CV for population doubling time at P5) and exhibit better immunosuppressive ability (15% CV at 1:1 and 32% CV at 1:10 ratio ) and consistent secretion of angiogenic factors (16% CV for VEGF and 51% CV for SDF-1). Further pooling does not compromise the trilineage differentiation capacity or phenotypic marker expression of the MSCs. The senescence and in vitro tumourigenicity characteristics of the pooled cells are also similar to those of individual donor MSCs. We conclude that pooling of MSCs from three different donors reduces heterogeneity among individual donors and produces MSCs with a consistent secretion and higher immunosuppressive profile.

Evolution and virulence of porcine epidemic diarrhea virus following in vitro and in vivo propagation

Practice of inoculating porcine epidemic diarrhea virus (PEDV) in piglets generating feedback material might influence the genetic evolution and attenuation of PEDV. The study was conducted to evaluate evolutionary rate and attenuation following serial in vitro and in vivo propagation. In the study, PED-JPFP0-PJ, Passage 0 (P0), was isolated from infected pigs and serially passaged in Vero cells for 5 consecutive times, P1-P5. P0, P2 and P5 were then subjected to orally inoculate 3-day-old piglets. At 24 h post inoculation, intestines of each passage (F1), were collected, and subsequently sub-passaged in piglets for 2 additional passages (F2-F3). Virus titration, PEDV genomic copies number, VH:CD ratios, and immunohistochemistry were evaluated. S and ORF3 genes were characterized. The results of the study demonstrated that virus titer and virulence were negatively correlated with increased passages, both in vitro and in vivo. Increased substitution rate was observed in higher passages. The evolutionary rate of S gene was higher than that of ORF3. Seven aa changes at positions 223, 291, 317, 607, 694, 1114 and 1199, with reduced N-linked glycan were observed in P5F3. In conclusion, serial passage of PEDV, both in vitro and in vivo, influence the genetic development and the attenuation of PEDV.

Genomic profile of eGFP-tagged senecavirus A subjected to serial plaque-to-plaque transfers

Senecavirus A (SVA) belongs to the genus Senecavirus in the family Picornaviridae. This virus possesses a positive-sense, single-stranded RNA genome, approximately 7200 nt in length, composed of a single 5′ untranslated region, encoding region and 3′ untranslated region. In this study, a recombinant SVA tagged with enhanced green fluorescent protein (eGFP) sequence, rSVA-eGFP, was rescued from its cDNA clone using reverse genetics. The passage-5 (P5) rSVA-eGFP was totally subjected to 55 rounds of consecutive fluorescent plaque-to-fluorescent plaque (FP-FP) transfers, and one extra common passaging in vitro. The P61 viral stock was analyzed by next-generation sequencing. The result showed ten single-nucleotide mutations (SNMs) in the rSVA-eGFP genome, including nine transitions and only one transversion. The P61 progeny still showed a complete eGFP sequence, indicating no occurrence of copy-choice recombination within the eGFP region during serial FP-FP transfers. In other words, this progeny was genetically deficient in the recombination of eGFP sequence (RES), namely, an RES-deficient strain. Out of ten SNMs, three were missense mutations, leading to single-amino acid mutations (SAAMs): F15V in L protein, A74T in VP2, and E53R in 3D protein. The E53R was predicted to be spatially adjacent to the RNA channel of 3D protein, perhaps involved in the emergence of RES-deficient strain. In conclusion, this study uncovered a global landscape of rSVA-eGFP genome after serial FP-FP transfers, and moreover shed light on a putative SAAM possibly related to the RES-deficient mechanism.

Assessing cellular agriculture potential: Population homogeneity and gene expression in cultured bovine mammary epithelial cells

Mammary epithelial cells (MEC) are responsible for milk synthesis in the bovine mammary gland during lactation, and could thereby potentially be used for in-vitro milk components production. Development of such a model, however, requires the MECs to preserve their lactogenic traits following in-vitro growth. Potential impact of growing MEC in the lab following their isolation was studied in relation to their population homogeneity and milk synthesis related gene expressions. Upon isolation, flow cytometry results showed that > 30 % of the obtained cells were positive for cytokeratin 18 (CK18), a marker for the MEC. This proportion increased to 95 % MEC after the first passage. Cell functionality was examined by comparing the expressions of milk synthesis related genes at passage 2 (P2) to passage 6 (P6) following treatment with lactogenic agents. CSN1S1 was found to be expressed significantly higher at P2 compared to P6. In addition, CSN2, CSN3 and PRLR tended to have lower expressions in P6 compared to P2. DGAT1, STAT5A, AKT1, and RPS6KB1 were expressed comparably in both passages. The expressions of LALBA and CSN1S2 were not detectable in any of the samples. Therefore, despite having high levels of CK18 positive cells at later passages, in line with other studies, further passaging affected the expressions of some of the genes related to milk synthesis. A reduced gene expression capacity in the propagated MEC may limit their potential for in-vitro milk components production.

Abstract 6901: Paired colorectal and pancreatic cancer patient-derived tumor and organoids biobank

Abstract Patient-derived xenograft (PDX) models constitute a crucial tool in precision cancer medicine owing to their high accuracy in predicting the response to therapeutic drugs among patients in clinical settings. Our center has established a biobank that collects more than 80 colorectal cancer and 25 pancreatic cancer PDX tissues, along with essential information about patients’ tumors and PDX models. However, cost-intensive and time-consuming limit the usage of PDX model in high throughput screening during drug development. To overcome this hurdle, we are generating a PDX-match paired organoid (PDXO) biobank by using emerging three-dimensional culture techniques. Before large-scaled expansion, the concordance between PDX tissues and PDXOs of partial specimens was evaluated firstly using short-tandem repeat (STR) analysis, immunohistochemistry (IHC) and H&amp;E staining. Data of STR analysis performed by using AmpFLSTR Identifiler PLUS PCR Amplification Kit demonstrated that PDX tissues and PDXOs have identical DNA fingerprinting. IHC and H&amp;E staining revealed PDX tissues and PDXOs have highly similar organ-specific markers expression (e.g., Cytokeratin 20, Caudal Type Homeobox 2, and Pan Cytokeratin) and histological morphology. Even after passing to at least five generations, passed PDXOs maintained high fidelity to their original state, as confirmed by whole-exome sequencing (WES) and STR analysis across passages. The WES analysis for passage 0 (P0) and passage 5 (P5) of PDXOs from different PDX tissues revealed there is more than 95% intersection between P0 and P5 PDXOs with the same missense mutation gene. The results of STR analysis showed that P0 and P5 PDXOs have identical STR profile. To evaluate the capability of PDXOs in clinical response prediction, several effective and ineffective drugs (e.g., oxapliplatin, 5-FU, gemcitabine) were tested. The results of drug testing indicated that PDXOs can be used to accurately predict the response to therapeutic drugs as PDX model but more efficient than PDX model. Therefore, PDXO biobank can serve as a valuable resource for high throughput in vitro drug screening prior in vivo efficacy confirmation using PDX models. Citation Format: Juiling Wang, Wen-Hui Ma, Yun-Chi Lo, Hsiao-Chun Hsieh, Li-Ju Hsiao, Yu-Pin Bau, Nai-Chieh Yang, Shaw-Jenq Tsai, Yan-Shen Shan, Tzu-Yu Chen, Yu-Ling Chen, Pei-Ju Chiang, Tzu-Ling Chen, Hsiu-Ping Chang, Hsian-Jean Chin. Paired colorectal and pancreatic cancer patient-derived tumor and organoids biobank [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6901.

Effect of Passaging on Bovine Chondrocyte Gene Expression and Engineered Cartilage Production.

Tissue engineering strategies show great potential for repairing osteochondral defects in osteoarthritic joints; however, these approaches often rely on passaging cells multiple times to obtain enough cells to produce functional tissue. Unfortunately, monolayer expansion culture causes chondrocyte dedifferentiation, which is accompanied by a phenotypical and morphological shift in chondrocyte properties that leads to a reduction in the quality of de novo cartilage produced. Thus, the objective of this study was to evaluate transcriptional variations during in vitro expansion culture and determine how differences in cell phenotype from monolayer expansion alter development of functional engineered cartilage. We used an unbiased approach to explore genome-wide transcriptional differences in chondrocyte phenotype at passage 1 (P1), P3, and P5, and then seeded cells into hydrogel scaffolds at P3 and P5 to assess cells' abilities to produce cartilaginous extracellular matrix in three dimensional (3D). We identified distinct phenotypic differences, specifically for genes related to extracellular organization and cartilage development. Both P3 and P5 chondrocytes were able to produce chondrogenic tissue in 3D, with P3 cells producing matrix with greater compressive properties and P5 cells secreting matrix with higher glycosaminoglycan/DNA and collagen/DNA ratios. Furthermore, we identified 24 genes that were differentially expressed with passaging and enriched in human osteoarthritis (OA) genome-wide association studies, thereby prioritizing them as functionally relevant targets to improve protocols that recapitulate functional healthy cartilage with cells from adult donors. Specifically, we identified novel genes, such as TMEM190 and RAB11FIP4, which were enriched with human hip OA and may play a role in chondrocyte dedifferentiation. This work lays the foundation for several pathways and genes that could be modulated to enhance the efficacy for chondrocyte culture for tissue regeneration, which could have transformative impacts for cell-based cartilage repair strategies.

Expression of HLA-DR by mesenchymal stromal cells in the platelet lysate era: an obsolete release criterion for MSCs?

BackgroundAccording to the definition of the International Society for Cell and Gene Therapy (ISCT), mesenchymal stromal cells (MSCs) do not express HLA-DR. This phenotypic marker as a release criterion for clinical use was established at a time when MSCs were expanded in fetal bovine serum (FBS)-containing media. Replacement of FBS with platelet lysate (PLs) as a medium supplement induced a significantly higher fraction of MSCs to express MHC class II antigens.MethodsAs this raised concerns that such MSCs may play the role of antigen-presenting cells for T cells, in the current study, we studied major factors that may induce HLA-DR on MSCs by means of flow cytometry and real-time polymerase chain reaction. The immunomodulatory potential of MSCs was assessed by a mixed lymphocyte reaction.ResultsOur results demonstrated that a very low percentage of generated and expanded MSCs in FBS express HLA-DR (median: 1.1%, range: 0.3–22%) compared to MSCs generated and expanded in PLs (median: 28.4%, range: 3.3–73.7%). Analysis of the cytokine composition of ten PLs showed a significant positive correlation between the levels of IL-1β, IL-4, IL-10, IL-17, bFGF and expression of HLA-DR, in contrast to no correlation with the age of MSC donors and HLA-DR (r = 0.21). Both MSCs expressing low and high levels of HLA-DR expressed class II transactivator (CIITA), a master gene coding for these molecules. Our results demonstrate for the first time that MSCs with constitutively high levels of HLA-DR also express moderate levels of indoleamine 2,3-dioxygenase (IDO). Treatment of MSCs with multiple doses of TGF-β1 at passage 0 (P0) and passage 1 (P1) completely abrogated HLA-DR and IDO expression. In contrast, treatment of MSCs with a single dose of TGF-β1 after P0 only partially reduced the expression of HLA-DR and CIITA. Remarkably, increased expression of HLA-DR on MSCs that constitutively express high levels of this antigen after overnight incubation with IFN-γ was rather unaffected by incubation with TGF-β1. However, treatment of MSCs with TGF-β1 for 24 h completely abrogated constitutive expression of IDO.ConclusionsIrrespective of HLA-DR expression at the population level, all MSC preparations significantly inhibited the proliferation of stimulated peripheral blood mononuclear cells, indicating that HLA-DR represents an obsolete release marker for the clinical use of MSCs.

Effect of High BMI on Human Bone Marrow-Derived Mesenchymal Stromal Cells.

Bone marrow-derived mesenchymal stromal cells (BMSCs) are attractive candidates in tissue engineering and regenerative medicine. Growing evidence has suggested that a high body mass index (BMI) can affect the properties of BMSCs, resulting in a reduced quality of the cells. However, the results are not consistent. Therefore, this study aimed to investigate the influences of high BMI on human BMSCs (hBMSCs). To avoid gender bias, BMSCs from females and males were studied independently. Finally, hBMSCs from 89 females and 152 males were separately divided into the normal BMI group (18.5 kg/m2 ≤ BMI < 25 kg/m2) and the high BMI group (BMI > 25 kg/m2). The cells were analyzed for the colony-forming potential; proliferation capacity; in vitro adipogenic, osteogenic, and chondrogenic differentiation potentials; and the expression of 32 common surface antigens. The results showed that high BMI did not change the number of colonies at passage 1 in females and males. In contrast, significantly reduced colony numbers at passage 4 (P4) were found in both female and male donors with high BMI. The doubling time of hBMSCs was comparable between the normal and the high BMI groups of females and males. Furthermore, the results of trilineage differentiation did not differ between the different BMI groups of males. In females, the high and the normal BMI groups also showed similar adipogenic and chondrogenic differentiation, while osteogenic differentiation was significantly enhanced in the high-BMI group. Regarding the expression of surface antigens, the expressions of CD200 and SSEA4 on hBMSCs were reduced in the high-BMI group of females and males, respectively. In conclusion, high BMI suppressed the clonogenicity of female and male hBMSCs at P4, improved the in vitro osteogenesis of female hBMSCs, and decreased the expressions of CD200 on hBMSCs in females and SSEA4 in males.

Adaptation of SIVmac to baboon primary cells results in complete absence of in vivo baboon infectivity.

While simian immunodeficiency virus (SIV) infection is non-pathogenic in naturally infected African nonhuman primate hosts, experimental or accidental infection in rhesus macaques often leads to AIDS. Baboons, widely distributed throughout Africa, do not naturally harbor SIV, and experimental infection of baboons with SIVmac results in transient low-level viral replication. Elucidation of mechanisms of natural immunity in baboons could uncover new targets of antiviral intervention. We tested the hypothesis that an SIVmac adapted to replicate in baboon primary cells will gain the capacity to establish chronic infections in vivo. Here, we generated SIVmac variants in baboon cells through serial passage in PBMC from different donors (SIVbn-PBMC s1), in PBMC from the same donors (SIVbn-PBMC s2), or in isolated CD4 cells from the same donors used for series 2 (SIVbn-CD4). While SIVbn-PBMC s1 and SIVbn-CD4 demonstrated increased replication capacity, SIVbn-PBMC s2 did not. Pharmacological blockade of CCR5 revealed SIVbn-PBMC s1 could more efficiently use available CCR5 than SIVmac, a trait we hypothesize arose to circumvent receptor occupation by chemokines. Sequencing analysis showed that all three viruses accumulated different types of mutations, and that more non-synonymous mutations became fixed in SIVbn-PBMC s1 than SIVbn-PBMC s2 and SIVbn-CD4, supporting the notion of stronger fitness pressure in PBMC from different genetic backgrounds. Testing the individual contribution of several newly fixed SIV mutations suggested that is the additive effect of these mutations in SIVbn-PBMC s1 that contributed to its enhanced fitness, as recombinant single mutant viruses showed no difference in replication capacity over the parental SIVmac239 strain. The replicative capacity of SIVbn-PBMC passage 4 (P4) s1 was tested in vivo by infecting baboons intravenously with SIVbn-PBMC P4 s1 or SIVmac251. While animals infected with SIVmac251 showed the known pattern of transient low-level viremia, animals infected with SIVbn-PBMC P4 s1 had undetectable viremia or viral DNA in lymphoid tissue. These studies suggest that adaptation of SIV to grow in baboon primary cells results in mutations that confer increased replicative capacity in the artificial environment of cell culture but make the virus unable to avoid the restrictive factors generated by a complex multicellular organism.

Urokinase-Type Plasminogen Activator Receptor (uPAR) Expression and [64Cu]Cu-DOTA-AE105 uPAR-PET/CT in Patient-Derived Xenograft Models of Oral Squamous Cell Carcinoma

Purpose[64Cu]Cu-DOTA-AE105 urokinase-type plasminogen activator receptor (uPAR)-PET/CT is a novel and promising imaging modality for cancer visualization, although it has not been tested in head and neck cancer patients nor in preclinical models that closely resemble these heterogenous tumors, i.e., patient-derived xenograft (PDX) models. The aim of the present study was to establish and validate oral squamous cell carcinoma (OSCC) PDX models and to evaluate [64Cu]Cu-uPAR-PET/CT for tumor imaging in these models.ProceduresPDX flank tumor models were established by engrafting tumor tissue from three patients with locally advanced OSCC into immunodeficient mice. [64Cu]Cu-DOTA-AE105 was injected in passage 2 (P2) mice, and [64Cu]Cu-uPAR-PET/CT was performed 1 h and 24 h after injection. After the last PET scan, all animals were euthanized, and tumors dissected for autoradiography and immunohistochemical (IHC) staining.ResultsThree PDX models were established, and all of them showed histological stability and unchanged heterogenicity, uPAR expression, and Ki67 expression through passages. A significant correlation between uPAR expression and tumor growth was found. All tumors of all models (n=29) showed tumor uptake of [64Cu]Cu-DOTA-AE105. There was a clear visual concordance between the distribution of uPAR expression (IHC) and [64Cu]Cu-DOTA-AE105 uptake pattern in tumor tissue (autoradiography). No significant correlation was found between IHC (H-score) and PET-signal (SUVmax) (r=0.34; p=0.07).ConclusionsOSCC PDX models in early passages histologically mimic donor tumors and could serve as a valuable platform for the development of uPAR-targeted imaging and therapeutic modalities. Furthermore, [64Cu]Cu-uPAR-PET/CT showed target- and tumor-specific uptake in OSCC PDX models demonstrating the diagnostic potential of this modality for OSCC patients.

Poxvirus sensitivity of a novel diploid sheep embryonic heart cell line.

Lumpy skin disease virus (LSDV), camelpox virus (CPV), and orf virus (ORFV) are members of the family Poxviridae. These viruses are usually isolated or produced in embryonated eggs or primary cells because continuous cell lines are less sensitive to infection. Disadvantages of the use of eggs or primary cells include limited availability, potential endogenous contaminants, and a limited ability to perform multiple passages. In this study, we developed a diploid cell culture from sheep embryonic hearts (EHs) and demonstrated its high proliferative and long-term storage capacities. In addition, we demonstrated its sensitivity to representatives of three genera of the family Poxviridae: Capripoxvirus (LSDV), Orthopoxvirus (CPV), and Parapoxvirus (ORFV). The cell culture had a doubling time of 24 h and reached 40 passages with satisfactory yield. This is comparable to that observed in primary lamb testis (LT) cells at passage 5 (P5). After infection, each poxvirus titer was 7.0-7.6 log TCID50/mL for up to five passages and approximately 6.8, 6.4, and 5.6 for the three viruses at P6-P25, P30, and P40, respectively. The sensitivity of sheep EH cells to poxvirus infection did not decrease after long-term storage in liquid nitrogen and was higher than that of primary LT cells, which are used for capripoxvirus and parapoxvirus detection and growth, and Vero cells, which are used for orthopoxvirus detection and growth. Thus, EH diploid cells are useful for poxvirus isolation and production without embryonated eggs or primary cells.

BHLHE40 Maintains the Stemness of PαS Cells In Vitro by Targeting Zbp1 through the Wnt/β-Catenin Signaling Pathway.

Primary bone mesenchymal stem cells (BMSCs) gradually lose stemness during in vitro expansion, which significantly affects the cell therapeutic effects. Here, we chose murine PαS (SCA-1+PDGFRα+CD45-TER119-) cells as representative of BMSCs and aimed to explore the premium culture conditions for PαS cells. Freshly isolated (fresh) PαS cells were obtained from the limbs of C57/6N mice by fluorescence-activated cell sorting (FACS). We investigated the differences in the stemness of PαS cells by proliferation, differentiation, and stemness markers in vitro and by ectopic osteogenesis and chondrogenesis ability in vivo, as well as the changes in the stemness of PαS cells during expansion in vitro. Gain- and loss-of-function experiments were applied to investigate the critical role and underlying mechanism of the basic helix-loop-helix family member E40 (BHLHE40) in maintaining the stemness of PαS cells. The stemness of fresh PαS cells representative in vivo was superior to that of passage 0 (P0) PαS cells in vitro. The stemness of PαS cells in vitro decreased gradually from P0 to passage 4 (P4). Moreover, BHLHE40 plays a critical role in regulating the stemness of PαS cells during in vitro expansion. Mechanically, BHLHE40 regulates the stemness of PαS cells by targeting Zbp1 through the Wnt/β-catenin signaling pathway. This work confirms that BHLHE40 is a critical factor for regulating the stemness of PαS cells during expansion in vitro and may provide significant indications in the exploration of premium culture conditions for PαS cells.

Mechanism of iron on the intestinal epithelium development in suckling piglets.

This study aimed to investigate the mechanism of iron on intestinal epithelium development of suckling piglets. Compared with newborn piglets, 7-day-old and 21-day-old piglets showed changes in the morphology of the jejunum, increased proliferation, differentiated epithelial cells, and expanded enteroids. Intestinal epithelium maturation markers and iron metabolism genes were significantly changed. These results suggest that lactation is a critical stage in intestinal epithelial development, accompanied by changes in iron metabolism. In addition, deferoxamine (DFO) treatment inhibited the activity of intestinal organoids at passage 4 (P4) of 0-day-old piglets, but no significant difference was observed in epithelial maturation markers at passage 1 (P1) and P4, and only argininosuccinate synthetase 1 (Ass1) and β-galactosidase (Gleb) were up-regulated at passage 7 (P7). These results in vitro show that iron deficiency may not directly affect intestinal epithelium development through intestinal stem cells (ISCs). The iron supplementation significantly down-regulated the mRNA expression of interleukin-22 receptor subunit alpha-2 (IL-22RA2) in the jejunum of piglets. Furthermore, the mRNA expression of IL-22 in 7-day-old piglets was significantly higher than that in 0-day-old piglets. Adult epithelial markers were significantly up-regulated in organoids treated with recombinant murine cytokine IL-22. Thus, IL-22 may play a key role in iron-affecting intestinal epithelium development.

Isolation and Characterization of Porcine Sapelovirus from the PDCoV-Positive Sample and Its Molecular Epidemiology in Henan Province, China

Porcine sapelovirus (PSV) is an emerging swine enteric virus that can cause various disorders including acute diarrhea, respiratory distress, reproductive failure, and polioencephalomyelitis in pigs. In this study, we isolated a PSV strain HNHB-01 from a clinical porcine deltacoronavirus- (PDCoV-) positive intestinal content of a diarrheic piglet. PSV was first identified using the small RNA deep sequencing and assembly, and further identified by the electron microscopic observation and the immunofluorescence assay. Subsequently, this virus was serially passaged in swine testis (ST) cells, and the complete genomics of PSV HNHB-01 passage 5 (P5), P30, P60, and P100 were sequenced and analyzed. 9 nucleotide mutations and 7 amino acid changes occurred in the PSV HNHB-01 P100 strain when compared with the PSV HNHB-01 P5. Pathogenicity investigation showed that orally inoculation of PSV HNHB-01 P30 could cause obvious clinical symptoms and had broad tissue tropism in 5-day-old piglets. Epidemiological investigation revealed that PSV infections and the coinfections of diarrhea coronaviruses were highly prevalent in swine herds. The complete genomes of 8 representative PSV epidemic strains were sequenced and analyzed. Phylogenetic analysis revealed that the PSV epidemic strains were closely related to other PSV reference strains that located in the Chinese clade. Furthermore, recombination analysis revealed that the recombination events were occurred in downstream of the 2C region in our sequenced PSV HNNY-02/CHN/2018 strain. Our results provided theoretical basis for future research studies of the pathogenic mechanism, evolutionary characteristics, and the development of vaccines against PSV.

LTF induces senescence and degeneration in the meniscus via the NF-κB signaling pathway: A study based on integrated bioinformatics analysis and experimental validation.

Background: The functional integrity of the meniscus continually decreases with age, leading to meniscal degeneration and gradually developing into osteoarthritis (OA). In this study, we identified diagnostic markers and potential mechanisms of action in aging-related meniscal degeneration through bioinformatics and experimental verification. Methods: Based on the GSE98918 dataset, common differentially expressed genes (co-DEGs) were screened using differential expression analysis and the WGCNA algorithm, and enrichment analyses based on Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were further performed. Next, the co-DEGs were imported into the STRING database and Cytoscape to construct a protein‒protein interaction (PPI) network and further validated by three algorithms in cytoHubba, receiver operating characteristic (ROC) curve analysis and the external GSE45233 dataset. Moreover, the diagnostic marker lactotransferrin (LTF) was verified in rat models of senescence and replicative cellular senescence via RT‒qPCR, WB, immunohistochemistry and immunofluorescence, and then the potential molecular mechanism was explored by loss of function and overexpression of LTF. Results: According to the analysis of the GSE98918 dataset, we identified 52 co-DEGs (42 upregulated genes and 10 downregulated genes) in the OA meniscus. LTF, screened out by Cytoscape, ROC curve analysis in the GSE98918 dataset and another external GSE45233 dataset, might have good predictive power in meniscal degeneration. Our experimental results showed that LTF expression was statistically increased in the meniscal tissue of aged rats (24 months) and senescent passage 5th (P5) meniscal cells. In P5 meniscal cells, LTF knockdown inhibited the NF-κB signaling pathway and alleviated senescence. LTF overexpression in passage 0 (P0) meniscal cells increased the expression of senescence-associated secretory phenotype (SASP) and induced senescence by activating the NF-κB signaling pathway. However, the senescence phenomenon caused by LTF overexpression could be reversed by the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). Conclusion: For the first time, we found that increased expression of LTF was observed in the aging meniscus and could induce meniscal senescence and degeneration by activating the NF-κB signaling pathway. These results revealed that LTF could be a potential diagnostic marker and therapeutic target for age-related meniscal degeneration.

Stability and biosafety of human epidermal stem cell for wound repair: preclinical evaluation

BackgroundCell therapy is a key technology to prevent sacrificing normal skin. Although some studies have shown the promise of human epidermal stem cells (EpiSCs), the efficacy, biosafety and quality control of EpiSC therapy have not been systematically reported.MethodsThe biosafety, stemness maintenance and wound repair of EpiSC were systematically verified by in vitro and in vivo experiments. EpiSC were prepared from the foreskin using a collagen type IV rapid adherence method. The EpiSCs were identified by flow cytometry, immunofluorescence staining and cell morphology. The well-growing passage 1 (P1) EpiSCs were used to determine the proliferation curve (counting method). EpiSC clone formation assay was performed by Giemsa staining. Nude mice were used to prepare a full-thickness skin defect wound model to detect the repair effect of EpiSCs. The biosafety of EpiSCs was double tested in vitro and in vivo.ResultsThe results showed that the expression of specific markers and clone formation efficiency was stable when passage 1 (P1) to P8 cells were cultured, and the stemness rate of P8 cells was close to 85.1%. EpiSCs were expanded in vitro for 25 days, the number of cells reached 2.5 × 108, and the transplantable area was approximately 75% of the total body surface area (TBSA). At 45 days, the total number of cells was approximately 30 billion, and the transplantable area was approximately the size of a volleyball court. A nude mouse wound model indicated that EpiSCs could rapidly close a wound. On postinjury day 7, the wound epithelialization rate in the cell transplantation group was significantly higher than that in the NaCl group (P < 0.05). In vitro, cell senescence increased, and telomerase activity decreased in P1 to P8 EpiSCs. In vivo, there were no solid tumors or metastatic tumors after EpiSC (P8) transplantation. In addition, the quality control of cultured cells met the clinical application criteria for cell therapy.ConclusionThis preclinical study showed the stability and biosafety of human EpiSC therapy for wound repair.Graphical

Simple Procedure for the Isolation of Mesenchymal Stem Cells from Different Parts of the Human Umbilical Cord

The umbilical cord and placenta are both sources of mesenchymal stem cells (MSCs) that are promising for cell-based therapy. Furthermore, compared to other MSCs sources, they are easy to obtain with no invasive procedures. This study presents an adapted method for stem cell isolation from three different parts of the human umbilical cord, including Wharton’s jelly (WJ), cord lining (CL), and cord-placenta junction (CPJ). The isolation consists of sample preparation, tissue dissection into distinct anatomical regions, mincing and enzyme digestion, and explant culturing. In addition, we monitored when the cells migrated from the explant to the bottom of the cell culture dish and passed the cells after they became confluent. This study found that WJ cells were the first to reach confluence at Passage 0 (P0). In contrast, CL cells needed the longest time to get confluence at P0 but displayed faster cell growth after subsequent passages (P1-P2). In addition, CPJ cells showed growth retardation after P1 and P2. Altogether, we could extract the MSCs from umbilical cord tissue explants by using DMEM supplemented with 10% FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin as general cell culture medium and omitting the use of gentamicin. However, the MSCs may need a more complex specified medium for optimum cell regeneration for further cell expansion.Keywords: mesenchymal stem cells, umbilical cord, Wharton’s jelly, cord lining, cord-placenta junction.

Morphology-based noninvasive early prediction of serial-passage potency enhances the selection of clone-derived high-potency cell bank from mesenchymal stem cells

BackgroundRapidly expanding clones (RECs) are one of the single-cell-derived mesenchymal stem cell clones sorted from human bone marrow mononuclear cells (BMMCs), which possess advantageous features. The RECs exhibit long-lasting proliferation potency that allows more than 10 repeated serial passages in vitro, considerably benefiting the manufacturing process of allogenic MSC-based therapeutic products. Although RECs aid the preparation of large-variation clone libraries for a greedy selection of better-quality clones, such a selection is only possible by establishing multiple-candidate cell banks for quality comparisons. Thus, there is a high demand for a novel method that can predict “low-risk and high-potency clones” early and in a feasible manner given the excessive cost and effort required to maintain such an establishment.MethodsLNGFR and Thy-1 co-positive cells from BMMCs were single-cell-sorted into 96-well plates, and only fast-growing clones that reached confluency in 2 weeks were picked up and passaged as RECs. Fifteen RECs were prepared as passage 3 (P3) cryostock as the primary cell bank. From this cryostock, RECs were passaged until their proliferation limitation; their serial-passage limitation numbers were labeled as serial-passage potencies. At the P1 stage, phase-contrast microscopic images were obtained over 6–90 h to identify time-course changes of 24 morphological descriptors describing cell population information. Machine learning models were constructed using the morphological descriptors for predicting serial-passage potencies. The time window and field-of-view-number effects were evaluated to identify the most efficient image data usage condition for realizing high-performance serial-passage potency models.ResultsSerial-passage test results indicated variations of 7–13-repeated serial-passage potencies within RECs. Such potency values were predicted quantitatively with high performance (RMSE < 1.0) from P1 morphological profiles using a LASSO model. The earliest and minimum effort predictions require 6–30 h with 40 FOVs and 6–90 h with 15 FOVs, respectively.ConclusionWe successfully developed a noninvasive morphology-based machine learning model to enhance the efficiency of establishing cell banks with single-cell-derived RECs for quantitatively predicting the future serial-passage potencies of clones. Conventional methods that can make noninvasive and quantitative predictions without wasting precious cells in the early stage are lacking; the proposed method will provide a more efficient and robust cell bank establishment process for allogenic therapeutic product manufacturing.