Serum-free Culture Research Articles

Sericin Protein: Structure, Properties, and Applications

Silk sericin, the glue protein binding fibroin fibers together, is present in the Bombyx mori silkworms’ cocoons. In recent years, sericin has gained attention for its wide range of properties and possible opportunities for various applications, as evidenced by the meta-analysis conducted in this review. Sericin extraction methods have evolved over the years to become more efficient and environmentally friendly, preserving its structure. Due to its biocompatibility, biodegradability, anti-inflammatory, antibacterial, antioxidant, UV-protective, anti-tyrosinase, anti-aging, and anti-cancer properties, sericin is increasingly used in biomedical fields like drug delivery, tissue engineering, and serum-free cell culture media. Beyond healthcare, sericin shows promise in industries such as textiles, cosmetics, and food packaging. This review aims to highlight recent advancements in sericin extraction, research, and applications, while also summarizing key findings from earlier studies.

Non-pharmacological interventions of intermittent fasting and pulsed radiofrequency energy (PRFE) combination therapy promote diabetic wound healing

ObjectiveThis study aims to conduct an unbiased assessment of the synergistic effects of non-pharmacological Interventions of intermittent fasting and pulsed radiofrequency energy (PRFE) combination therapy on the facilitation of diabetic wound healing, while also exploring the underlying mechanisms. The findings of this research will provide a theoretical framework and innovative strategy for unconventional therapeutic interventions aimed at enhancing the healing process of diabetes-related wounds.MethodsIn vivo experiments involved the induction of diabetic models in C57 mice through streptozotocin injection. To simulate a combined therapeutic approach, diabetic mice underwent fasting on days 2 and 6, accompanied by twice daily PRFE applications for 8 days. In vitro experiments were conducted using a serum-free culture medium to replicate fasting conditions. The investigation encompassed wound healing rate, proliferation, migration, angiogenesis, oxidative stress, fibrogenesis, and sensory nerve growth through histological analysis and functional assessments in vivo. Additionally, this study utilized quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blotting (WB), and immunofluorescence staining techniques to elucidate the potential mechanisms underlying the effects of intermittent Fasting and PRFE combination therapy in diabetic wound healing, both in vitro and in vivo.ResultsThe intermittent fasting and PRFE combination therapy demonstrated superior efficacy in enhancing diabetic wound healing compared to either treatment alone. It harnessed the respective strengths of individual therapies, fostering migration, mitigating oxidative stress, and enhancing fibrogenesis. Furthermore, the combination therapy manifested a synergistic effect in promoting proliferation, tube formation, angiogenesis, and sensory nerve growth.ConclusionThis study demonstrates that intermittent fasting and PRFE combination therapy enhance diabetic wound healing, effectively leveraging the strengths of both therapies and even yielding synergistic benefits. Moreover, it indicates the potential engagement of the P75/HIF1A/VEGFA axis in mediating these effects.

An adaptive Epithelial-Mesenchymal Transition Program Enables Basal Epithelial Cells to Bypass Stress-Induced Stasis and Contributes to Metaplastic Breast Cancer Progenitor State.

Human mammary epithelial cell (HMEC) cultures encounter a stress-associated barrier termed stasis, during which most cells adopt a senescence-like phenotype. From these cultures, rare variants emerge from the basal epithelial population, re-initiating growth. Variants exhibit pre-malignant properties, including an aberrant epigenetic program that enables continued proliferation and acquisition of genetic changes. Following oncogenic transformation, variants produce tumors that recapitulate the histopathological characteristics of metaplastic breast cancer (MBC), a rare subtype characterized by squamous and mesenchymal differentiation. Using the conventional serum-free HMEC culture system, we probed the capacity for phenotypic plasticity inherent to basal epithelial cell populations from human breast tissue as they navigated stasis and emerged as variant populations. We observed robust activation of a TGF-β-dependent epithelial-mesenchymal transition (EMT) program in basal epithelial cells during stasis, followed by subsequent attenuation of this program in emerging variants. Inhibiting the TGF-β pathway or depleting the EMT regulators Snail or Slug allowed basal epithelial cells to collectively bypass stasis, demonstrating that cellular dysfunction and arrest resulting from TGF-β and EMT activation are central to this in vitro barrier. The spontaneous emergence of variants from stasis cultures was associated with a restricted EMT trajectory, which diverted cells away from a complete mesenchymal state characterized by irreversible growth arrest, and instead limited variants to epithelial and intermediate EMT states associated with greater proliferative capacity and stemness. Epigenetic mechanisms, which contributed to the dysregulated growth control characteristic of the variant phenotype, also contributed to the constrained EMT program in variants. By overcoming the cellular dysfunction and growth arrest resulting from TGF-β and EMT activation, variants exhibited increased oncogenic transformation efficiency compared to pre-stasis basal epithelial cells. Inhibiting the TGF-β pathway prior to stasis significantly reduced EMT in the basal epithelial population, alleviated selective pressure driving variant emergence, and enhanced oncogenic transformation efficiency, resulting in tumors with markedly diminished metaplastic differentiation. This study reveals how adaptive EMT reprogramming governs basal epithelial cell fate decisions and contributes to the development of MBC progenitors by restricting access to terminal mesenchymal states that induce growth arrest and, instead, favoring intermediate states with enhanced tumorigenic potential.

Evaluation of the impact of customized serum-free culture medium on the production of clinical-grade human umbilical cord mesenchymal stem cells: insights for future clinical applications

BackgroundThe selection of suitable culture medium is critical for achieving good clinical outcomes in cell therapy. To support the commercial application of stem cell therapy, customized culture media not only need to promote stem cell proliferation, but also need to save costs and meet industrial requirements for inter-batch consistency, efficacy, and biosafety. In this study, we developed a series of serum-free media (SFM) and elucidated the effects between different SFM, as well as between SFM and serum-containing meida (SCM), on human umbilical cord mesenchymal stem cells (hUC-MSCs) phenotype and function. We analyze and emphasize from the perspectives of clinical and commercial application why research on customized culture media is critical for the success of enterprises developing novel cellular therapeutics.MethodsWe cultured hUC-MSCs with identical cell seeding densities in different formulations of SFM and SCM until passage 10 and examined the changes in cell phenotype and function. We analyzed the results with the commercial application requirments of the cellular therapy industry to assess the potential impact of customized culture media on inter-batch consistency, efficacy, stability, biosafety, and cost-effectiveness of industrial-scale cell production.ResultshUC-MSCs cultured in SCM and SFM exhibit consistent cell morphology and surface molecule expression, but hUC-MSCs cultured in SFM demonstrate higher activity, superior proliferative capacity, and greater stability. Furthermore, hUC-MSCs cultured in different SFM exhibit differences in cell activity, proliferative capacity, senescent rate, and S/M ratio of cell cycle, while maintaining a normal karyotype after long-term in vitro cultivation. Moreover, we found that hUC-MSCs cultured in different media exhibit variations in paracrine capacity and in their support of hematopoietic stem cell (HSC) self-renewal.ConclusionConsidering the substantial funding and time required for cell-based drug development, our results underscore the importances of comprehensively optimizing the composition of medium for the specific disease prior to conducting clinical trials of cell-based therapies. The criteria for selecting culture medium should be based on the requirements of the target disease for cellular function. In addition, we provide a way to formulate different customized SFM, which is beneficial for the development of cell therapy industry.

Enhanced production of recombinant coxsackievirus A16 using a serum-free HEK293A suspension culture system for bivalent enterovirus vaccine development

Coxsackievirus A16 (CVA16) is one of the primary pathogens that causes hand, foot, and mouth disease (HFMD) in young children. In previous studies, CVA16 vaccine development has encountered several challenges, such as inefficient replication of the CVA16 virus in present culture systems, the induction of only mild neutralizing antibody titers, and neutralizing antibodies induced by certain vaccine candidates that are unable to protect against CVA16 viral challenge. In this study, we constructed a DNA-launched CVA16 infectious clone (CVA16ic) based on the genomic sequence of the CVA16 N5079 strain to minimize interference from viral quasispecies. The biochemical properties of this CVA16ic strain were similar to those of its parental strain. Serum-free HEK293A suspension cells, which produced higher virus titers than Vero cells, were demonstrated to improve CVA16 production yields. In addition, our study showed that inactivated EV-A71 antigens could enhance the immunogenicity of inactivated CVA16 mature/full particles (F-particles), suggesting that a bivalent CVA16 and EV-A71 vaccine may be an effective strategy for CVA16 vaccine development. These findings are expected to provide novel strategies and accelerate the development of bivalent HFMD vaccines.

A novel liquid plasma derivative inhibits melanogenesis through upregulation of Nrf2

Non-thermal plasma (NTP) is an emerging technology with extensive applications in biomedicine, including treatment of abnormal pigmentation. However, very few studies have investigated how plasma induces anti-melanogenesis. Here, liquid plasma was prepared by treating an NTP jet with helium and oxygen (as carrier gases) for 15 min in serum-free culture media. In the zebrafish model, pigmentation ratio was observed with or without liquid plasma. The anti-melanogenic effect of liquid plasma was evaluated in human melanocytes by assessing the expression of melanogenesis-related genes using western blotting, RT-PCR, and immunohistochemistry. Liquid plasma reduced pigmentation in the zebrafish model and inhibited melanin synthesis in primary human melanocytes. Intracellular reactive oxygen species levels decreased and Nrf2 expression increased in liquid plasma–treated melanocytes. Liquid plasma affected microphthalmia-associated transcription factor (MITF) and tyrosinase mRNA and protein levels, tyrosinase activity, and melanin content. Considering the role of Wnt/β-catenin and PI3K/Akt pathways in melanogenesis, the effect of liquid plasma on this pathway was determined; liquid plasma decreased active β-catenin, LEF1/TCF4, MITF, and tyrosinase levels in a time-dependent manner and inhibited the nuclear translocation of β-catenin. This inhibition subsequently suppressed melanogenesis by downregulating MITF and tyrosinase. These results suggest that liquid plasma may be used for treating pigmentary disorders.

Urine-derived stem cells serve as a robust platform for generating native or engineered extracellular vesicles

BackgroundMesenchymal stromal cell (MSC) therapy holds great potential yet efficacy and safety concerns with cell therapy persist. The beneficial effects of MSCs are often attributed to their secretome that includes extracellular vesicles (EVs). EVs carry biologically active molecules, protected by a lipid bilayer. However, several barriers hinder large-scale MSC EV production. A serum-free culturing approach is preferred for producing clinical-grade MSC-derived EVs but this can affect both yield and purity. Consequently, new strategies have been explored, including genetically engineering MSCs to alter EV compositions to enhance potency, increase circulation time or mediate targeting. However, efficient transfection of MSCs is challenging. Typical sources of MSC include adipose tissue and bone marrow, which both require invasive extraction procedures. Here, we investigate the use of urine-derived stem cells (USCs) as a non-invasive and inexhaustible source of MSCs for EV production.MethodsWe isolated, expanded, and characterized urine-derived stem cells (USCs) harvested from eight healthy donors at three different time points during the day. We evaluated the number of clones per urination, proliferation capacity and conducted flow cytometry to establish expression of surface markers. EVs were produced in chemically defined media and characterized. PEI/DNA transfection was used to genetically engineer USCs using transposon technology.ResultsThere were no differences between time points for clone number, doubling time or viability. USCs showed immunophenotypic characteristics of MSCs, such as expression of CD73, CD90 and CD105, with no difference at the assessed time points, however, male donors had reduced CD73 + cells. Expanded USCs were incubated without growth factors or serum for 72 h without a loss in viability and EVs were isolated. USCs were transfected with high efficiency and after 10 days of selection, pure engineered cell cultures were established.ConclusionsIsolation and expansion of MSCs from urine is non-invasive, robust, and without apparent sex-related differences. The sampling time point did not affect any measured markers or USC isolation potential. USCs offer an attractive production platform for EVs, both native and engineered.

Simple supplementation of serum-free medium produces gametocytes of Plasmodium falciparum that transmit to mosquitoes

BackgroundHuman serum is a major component of Plasmodium falciparum culture medium, and can be replaced with AlbuMAX™ II, a lipid-rich bovine serum albumin, for asexual cultures. However, gametocytes produced without serum are poorly infective to mosquitoes. Serum suffers from high cost, limited availability, and variability in quality.MethodsSeveral commercially-available media supplements were tested for their ability to support parasite growth and production of P. falciparum (3D7) gametocytes in standard RPMI1640 medium containing 0.5% AlbuMAX. The impact on asexual growth and gametocyte production with each supplement was assessed and compared to standard RPMI1640 medium containing 10% human serum, as well as to medium containing 0.5% AlbuMAX alone. The infectivity of gametocytes produced with one supplement to Anopheles gambiae sensu stricto was assessed by standard membrane feeding assay and measuring both prevalence of infection and oocyst intensity.ResultsSupplementation of medium containing 0.5% AlbuMAX with five supplements did not affect asexual growth of P. falciparum, and four of the five supplements supported early gametocyte production. The supplement producing the highest number of gametocytes, ITS-X, was further investigated and was found to support the production of mature gametocytes. Infection prevalence and oocyst intensity did not differ significantly between mosquitoes given a membrane feed containing gametocytes grown in medium with 0.5% AlbuMAX + ITS-X and those grown in medium with 10% human serum. Infection prevalence and oocyst intensity was significantly higher in case of ITS–X supplementation when compared to AlbuMAX alone. Infectious gametocytes were also produced from two field clones using ITS–X supplementation.ConclusionsSerum-free medium supplemented with ITS-X was able to support the growth of gametocytes of P. falciparum that were as infectious to An. gambiae as those grown in medium with 10% serum. This is the first fully serum-free culture system able to produce highly infectious gametocytes, thereby removing the requirement for access to serum for transmission assays.

Distinctive small molecules blend: Promotes lacrimal gland epithelial cell proliferation in vitro and accelerates lacrimal gland injury repair in vivo

PurposeThis study aims to develop a novel serum-free culture strategy containing only two small molecules, Y27632 and SB431542 (2C), for in vitro expansion of mouse lacrimal gland epithelial cells (LGECs) and investigate an innovative therapeutic approach for lacrimal gland (LG) injury. MethodsLGECs proliferative capacity was assessed by cell counting, crystal violet staining, qRT-PCR and immunofluorescence. Cell differentiation was achieved by manipulating culture conditions and assessed by qRT-PCR and AQP5 immunofluorescence. LGECs were seeded in Matrigel for three-dimensional culture and assessed by qRT-PCR and immunofluorescence. Secretory function of the cultures was assayed by ELISA. In vivo, 2C injection verified its reparative capacity in a mouse LG injury model. Corneal fluorescein staining, phenol red cotton thread, H&E, immunofluorescence and Western blot were used to assess LG injury repair. ResultsLGECs cultured with 2C exhibited high expression of stemness/proliferation markers and maintained morphology and proliferative capacity even after the tenth passage. Removal of 2C was efficacious in achieving LGECs differentiation, characterized by the increased AQP5 expression and LTF secretion. 3D spheroids cultured with 2C demonstrated differentiation potential, forming microglandular structures containing multiple LG cell types with secretory functions after 2C removal. In vivo, 2C improved the structural integrity and function of the injured LG. ConclusionsWe present a small molecule combination, 2C, that promotes LGECs expansion and differentiation in vitro and accelerates LG injury repair in vivo. This approach has potential applications for providing a stable source of seed cells for tissue engineering applications, providing new sights for LG-related diseases treatment.

Expression of matrix metalloproteinases and their inhibitors in corneal stromal fibroblasts and keratocytes from healthy and keratoconus corneas.

To examine the in-vitro expression of matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in corneal stromal cells by distinguishing between fibroblasts and keratocytes of healthy and keratoconus (KC) corneas. Stromal cells were isolated from healthy and KC corneas (n = 8). A normal-glucose, serum-containing cell culture medium (NGSC-medium) was used for cultivation of healthy human corneal fibroblasts (HCFs) and KC human corneal fibroblasts (KC-HCFs). In order to obtain a keratocyte phenotype, the initial cultivation with NGSC-medium was changed to a low-glucose, serum-free cell culture medium for healthy (Keratocytes) and KC cells (KC-Keratocytes). Gene and protein expression of MMP-1, -2, -3, -7, -9 and TIMP-1, -2, -3 were measured by quantitative PCR and Enzyme-Linked Immunosorbent Assay (ELISA) from the cell culture supernatant. KC-HCFs demonstrated a lower mRNA gene expression for MMP-2 compared to HCFs. In contrast to their respective fibroblast groups (either HCFs or KC-HCFs), Keratocytes showed a higher mRNA gene expression of TIMP-3, whereas TIMP-1 mRNA gene expression was lower in Keratocytes and KC-Keratocytes. Protein analysis of the cell culture supernatant revealed lower concentrations of MMP-1 in KC-HCFs compared to HCFs. Compared to Keratocytes, TIMP-1 concentrations was lower in the cell culture supernatant of KC-Keratocytes. In HCFs and KC-HCFs, protein levels of MMP-1 and TIMP-1 were higher and MMP-2 was lower compared to Keratocytes and KC-Keratocytes, respectively. This study indicates an imbalance in MMP and TIMP expression between healthy and diseased cells. Furthermore, differences in the expression of MMPs and TIMPs exist between corneal fibroblasts and keratocytes, which could influence the specific proteolytic metabolism in-vivo and contribute to the progression of KC.

A serum-free culture medium production system by co-culture combining growth factor-secreting cells and l-lactate-assimilating cyanobacteria for sustainable cultured meat production

Large-scale production of cultured meat requires bulk culture medium containing growth-promoting proteins from animal serum. However, animal serum for mammalian cell culture is associated with high costs, ethical concerns, and contamination risks. Owing to its growth factor content, conditioned medium from rat liver epithelial RL34 cells can replace animal serum for myoblast proliferation. More seeded cells and longer culture periods are thought to yield higher growth factor levels, resulting in more effective muscle cell proliferation. However, RL34 cells can deplete nutrients and release harmful metabolites into the culture medium over time, potentially causing growth inhibition and apoptosis. This issue highlights the need for waste clearance during condition medium production. To address this issue, we introduced a lactate permease gene (lldP) and an l-lactate-to-pyruvate conversion enzyme gene (lldD) to generate a recombinant l-lactate-assimilating cyanobacterium Synechococcus sp. KC0110 strain. Transwell co-culture of this strain with RL34 cells exhibited a marked reduction in the levels of harmful metabolites, lactate and ammonium, while maintaining higher concentrations of glucose, pyruvate, and pyruvate-derived amino acids than those seen with RL34 cell monocultures. The co-culture medium supported myoblast proliferation without medium dilution or additional nutrients, which was attributed to the waste clearance and nutrient replenishment effects of the KC0110 strain. This culture system holds potential for the production of low-cost, and animal-free cultured meat.

Multi-omics and pharmacological characterization of patient-derived glioma cell lines

Glioblastoma (GBM) is the most common brain tumor and remains incurable. Primary GBM cultures are widely used tools for drug screening, but there is a lack of genomic and pharmacological characterization for these primary GBM cultures. Here, we collect 50 patient-derived glioma cell (PDGC) lines and characterize them by whole genome sequencing, RNA sequencing, and drug response screening. We identify three molecular subtypes among PDGCs: mesenchymal (MES), proneural (PN), and oxidative phosphorylation (OXPHOS). Drug response profiling reveals that PN subtype PDGCs are sensitive to tyrosine kinase inhibitors, whereas OXPHOS subtype PDGCs are sensitive to histone deacetylase inhibitors, oxidative phosphorylation inhibitors, and HMG-CoA reductase inhibitors. PN and OXPHOS subtype PDGCs stably form tumors in vivo upon intracranial transplantation into immunodeficient mice, whereas most MES subtype PDGCs fail to form tumors in vivo. In addition, PDGCs cultured by serum-free medium, especially long-passage PDGCs, carry MYC/MYCN amplification, which is rare in GBM patients. Our study provides a valuable resource for understanding primary glioma cell cultures and clinical translation and highlights the problems of serum-free PDGC culture systems that cannot be ignored.

Generating Neural Progenitors from Embryonic Stem Cells in Serum-Free Monolayer Cultures

Generating Neural Progenitors from Embryonic Stem Cells in Serum-Free Monolayer Cultures

Spheroid trilineage differentiation model of primary mesenchymal stem/stromal cells under hypoxia and serum-free culture conditions.

Due to their unique properties, human mesenchymal stem/stromal cells (MSCs) possess tremendous potential in regenerative medicine, particularly in cell-based therapies where the multipotency and immunomodulatory characteristics of MSCs can be leveraged to address a variety of disease states. Although MSC-based cell therapeutics have emerged as one of the most promising medical treatments, the clinical translation is hampered by the variability of MSC-based cellular products caused by tissue source-specific differences and the lack of physiological cell culture approaches that closely mimic the human cellular microenvironment. In this study, a model for trilineage differentiation of primary adipose-, bone marrow-, and umbilical cord-derived MSCs into adipocytes, chondrocytes and osteoblasts was established and characterized. Differentiation was performed in spheroid culture, using hypoxic conditions and serum-free and antibiotics-free medium. This platform was characterized for spheroid diameter and trilineage differentiation capacity reflecting functionality of differentiated cells, as indicated by lineage-specific extracellular matrix (ECM) accumulation and expression of distinct secreted markers. The presented model shows spheroid growth during the course of differentiation and successfully supports trilineage differentiation for MSCs from almost all tissue sources except for osteogenesis of umbilical cord-derived MSCs. These findings indicate that this platform provides a suitable and favorable environment for trilineage differentiation of MSCs from various tissue sources. Therefore, it poses a promising model to generate highly relevant biological data urgently required for clinical translation and therefore might be used in the future to generate in vitro microtissues, building blocks for tissue engineering or as disease models.

Life cycle assessment of Beefy-9 and Beefy-R serum-free culture media for cell-cultivated beef production

The sustainable production of cell-cultivated meat requires the use of environmentally friendly nutrients. Culture media, which provides the necessary nutrients for cell proliferation and differentiation is a major contributor to the environmental impacts of cultivated meat production. Fetal bovine serum (FBS) is problematic due to animal sourcing, ethical concerns, and high cost. Beefy-R and Beefy-9 are experimentally validated, serum-free cell culture media designed for bovine myoblast culture. While both formulations include basal media, ascorbic acid, sodium selenite, recombinant proteins, and growth factors, Beefy-9 and Beefy-R contain recombinant albumin and rapeseed protein isolates (RPI) to replace FBS, respectively. While these culture media do not include animal-sourced components, their environmental impacts have not yet been investigated. In this study, life cycle assessment (LCA) was performed to estimate the environmental burdens of Beefy-9 and Beefy-R culture media production and identify hotspots for optimization. Uncertainty was assessed through sensitivity and scenario analyses, as well as Monte Carlo simulation. Our results demonstrate that Beefy-R has significantly lower environmental impacts compared to Beefy-9 in 13 out of 18 evaluated impact categories. For instance, the global warming potential (GWP) of Beefy-R was almost five times lower when compared against that of Beefy-9 production. In Beefy-R, despite the absence of recombinant albumin, recombinant proteins and growth factors were hotspots, accounting for 41 %, 5.9 %, and 12 % of GWP, land use, and water consumption impacts, respectively. This study confirms that RPI can be a more sustainable substitute for recombinant albumin in bovine myoblast serum-free media formulations. Additional research is required to identify low-impact alternatives for other recombinant proteins and growth factors in culture media.

Transition of signal requirement in hematopoietic stem cell development from hemogenic endothelial cells

Hematopoietic stem cells (HSCs) develop from hemogenic endothelial cells (HECs) in vivo during mouse embryogenesis. When cultured in vitro, cells from the embryo phenotypically defined as pre-HSC-I and pre-HSC-II have the potential to differentiate into HSCs. However, minimal factors required for HSC induction from HECs have not yet been determined. In this study, we demonstrated that stem cell factor (SCF) and thrombopoietin (TPO) induced engrafting HSCs from embryonic day (E) 11.5 pre-HSC-I in a serum-free and feeder-free culture condition. In contrast, E10.5 pre-HSC-I and HECs required an endothelial cell layer in addition to SCF and TPO to differentiate into HSCs. A single-cell RNA sequencing analysis of E10.5 to 11.5 dorsal aortae with surrounding tissues and fetal livers detected TPO expression confined in hepatoblasts, while SCF was expressed in various tissues, including endothelial cells and hepatoblasts. Our results suggest a transition of signal requirement during HSC development from HECs. The differentiation of E10.5 HECs to E11.5 pre-HSC-I in the aorta-gonad-mesonephros region depends on SCF and endothelial cell-derived factors. Subsequently, SCF and TPO drive the differentiation of E11.5 pre-HSC-I to pre-HSC-II/HSCs in the fetal liver. The culture system established in this study provides a beneficial tool for exploring the molecular mechanisms underlying the development of HSCs from HECs.

Leucine rich α2 glycoprotein 1 derived from malignant pleural mesothelioma cells facilitates macrophage M2 phenotypes

Background: Macrophages constitute the main part of infiltrating immune cells in Malignant pleural mesothelioma (MPM) and abnormally high ratios of M2 macrophages are present in both pleural effusion and tissue samples of MPM patients. Whether MPM cells affect formation of M2 macrophages is poorly understood. In this study, we focused on identification of MPM-cells-derived soluble factors with M2-promoting effects. Methods: Media of malignant pleural mesothelioma cells were collected and soluble factors affecting macrophages were analyzed by mass spectrometry. TGF-β receptor inhibitor SB431542 was used as the entry point to explore the downstream mechanism of action by qRT-PCR, WB and immunofluorescence. Results: The serum-free culture media collected from the human MPM cells Meso1 and Meso2 significantly enhanced expression of the M2 signature molecules including IL-10, TGF-β and CD206 in the human macrophages THP-1, while the culture medium of the human MPM cells H2452 did not show such M2-promoting effects. Analysis of proteins by mass spectrometry and ELISA suggested that Leucine rich α2 glycoprotein 1(LRG1) was a potential candidate. LRG1 time- and dose-dependently increased expression of the M2 signature molecules, confirming its M2-promoting effects. Furthermore, LRG1’s M2-promoting effects were reduced by the TGF-β receptor inhibitor SB431542, and LRG1 increased phosphorylation of Smad2, indicating that M2-promoting effects of LRG1 were via the TGF-β receptor/Smad2 signaling pathway. Conclusions: Our results provide a potential M2-promoting new member, LRG1, which contributes to the immune escape of MPM via the TGF-β receptor/Smad2 signaling pathway.

A retrospective analysis of sheep generated by somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) is one of the primary methods for production of genetically engineered sheep, which allows for gene editing or transgene introduction in somatic cells. The use of SCNT eliminates the risk of genetic mosaicism in embryos and animals that is commonly observed after zygote micromanipulations. This retrospective analysis of SCNT in sheep performed at Utah State University, spanning from 2016 to 2021, examined parameters that may impact pregnancy and full-term development, including donor oocytes (donor age), donor cell lines, SCNT parameters (time of oocyte activation following SCNT, number of transferred embryos, in vitro maturation and culture conditions), and recipients (surgical number and ovulatory status), as well as factors that may correlate with large offspring syndrome or abnormal offspring syndrome (LOS/AOS) in the fetuses and lambs. Our findings indicated that compared to prepubertal oocytes, the SCNT embryos produced from adult sheep oocytes had comparable in vitro maturation rates, pregnancy and full-term development rates, as well as SCNT efficiency. In addition, earlier activation time of SCNT embryos (e.g. 24–26 h post maturation) was correlated to the early pregnancy loss rate, full-term rate, and SCNT efficiency. Compared to our standard serum-containing medium, commercial serum-free culture medium showed a positive correlation with the full-term development of sheep SCNT embryos. Transferring 15–30 embryos per recipient resulted in consistently good pregnancy rates. Surgical numbers and ovulatory status (having at least one follicle between 6 and 12 mm in size or a corpus hemorrhagicum (CH)) of recipients did not affect pregnancy and full-term development rates. In summary, this retrospective analysis identified parameters for improving pregnancy and full-term development of SCNT embryos in sheep.

Expansion of human allogeneic liver-derived progenitor cells for liver regenerative therapy in serum-free culture conditions

Human allogeneic liver-derived progenitor cells (HALPCs) display advanced ability to differentiate into hepatocyte-like cells and exhibit potent immunomodulatory, anti-inflammatory, and anti-fibrotic properties. HALPCs have been successfully manufactured under good manufacturing practice (GMP) and are currently in clinical development. A previous phase 2a trial demonstrated the safety of peripheral intravenous infusions of HALPCs and preliminary evidence of the cells’ properties to restore liver function in patients with acute-on-chronic liver failure (ACLF), thus potentially improving their survival. A phase 2b trial is currently ongoing across multiple centers (NCT04229901) to obtain proof-of-concept on efficacy and additional safety. HALPCs are currently manufactured using fetal bovine serum (FBS), which can reveal qualitative and quantitative variations between batches. The use of serum-free medium (SFM) represents an alternative means to overcome this variability while also complying fully with regulations. The aim of this study was to compare current FBS-containing culture conditions with two industry-available GMP-compliant SFMs: StemMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) and PRIME-XV (FUJIFILM Irvine Scientific, Santa Ana, California, USA).The proliferation of HALPCs was significantly stimulated by both SFMs, which shortened both their emergence period and population doubling time. This effect was correlated with a significant improvement in their genetic stability as analyzed by conventional karyotyping. The expression profile (identity and purity) and functionality of HALPCs cultured in SFM were maintained, as demonstrated by flow cytometry and enzyme-linked immunoassay (ELISA), respectively. Their potency, evaluated via prostaglandin E2 (PGE2) secretion, showed a similar effect on CD4+ T-cell proliferation in FBS and SFM conditions. Furthermore, a greater proportion of HALPCs cultured in SFM showed enhanced expression of tissue factor (CD142) compared with the FBS condition.Altogether, SFM conditions enabled consistent HALPC quality to be achieved without altering their expression and functional profiles.

A convenient model of serum-induced reactivity of human astrocytes to investigate astrocyte-derived extracellular vesicles.

Extracellular vesicles (EVs) are secreted by all cells in the CNS, including neurons and astrocytes. EVs are lipid membrane enclosed particles loaded with various bioactive cargoes reflecting the dynamic activities of cells of origin. In contrast to neurons, the specific role of EVs released by astrocytes is less well understood, partly due to the difficulty in maintaining primary astrocyte cultures in a quiescent state. The aim of this study was to establish a human serum-free astrocyte culture system that maintains primary astrocytes in a quiescent state to study the morphology, function, and protein cargoes of astrocyte-derived EVs. Serum-free medium with G5 supplement and serum-supplemented medium with 2% FBS were compared for the culture of commercially available human primary fetal astrocytes. Serum-free astrocytes displayed morphologies similar to in vivo astrocytes, and surprisingly, higher levels of astrocyte markers compared to astrocytes chronically cultured in FBS. In contrast, astrocyte and inflammatory markers in serum-free astrocytes were upregulated 24 h after either acute 2% FBS or cytokine exposure, confirming their capacity to become reactive. Importantly, this suggests that distinct signaling pathways are involved in acute and chronic astrocyte reactivity. Despite having a similar morphology, chronically serum-cultured astrocyte-derived EVs (ADEVs) were smaller in size compared to serum-free ADEVs and could reactivate serum-free astrocytes. Proteomic analysis identified distinct protein datasets for both types of ADEVs with enrichment of complement and coagulation cascades for chronically serum-cultured astrocyte-derived EVs, offering insights into their roles in the CNS. Collectively, these results suggest that human primary astrocytes cultured in serum-free medium bear similarities with in vivo quiescent astrocytes and the addition of serum induces multiple morphological and transcriptional changes that are specific to human reactive astrocytes and their ADEVs. Thus, more emphasis should be made on using multiple structural, molecular, and functional parameters when evaluating ADEVs as biomarkers of astrocyte health.