Advancements in culture technology of adipose-derived stromal/stem cells: implications for diabetes and its complications.

Stem cell-based therapies exhibit considerable promise in the treatment of diabetes and its complications. Extensive research has been dedicated to elucidate the characteristics and potential applications of adipose-derived stromal/stem cells (ASCs). Three-dimensional (3D) culture, characterized by rapid advancements, holds promise for efficacious treatment of diabetes and its complications. Notably, 3D cultured ASCs manifest enhanced cellular properties and functions compared to traditional monolayer-culture. In this review, the factors influencing the biological functions of ASCs during culture are summarized. Additionally, the effects of 3D cultured techniques on cellular properties compared to two-dimensional culture is described. Furthermore, the therapeutic potential of 3D cultured ASCs in diabetes and its complications are discussed to provide insights for future research.

Establishment of human intestinal organoids derived from commercially available cryopreserved intestinal epithelium and evaluation for pharmacokinetic study.

Human intestinal organoids (HIOs) have been reported to exert their functions in a way that mimics living organs, and HIOs-derived monolayers are expected to be applied to in vitro intestinal pharmacokinetic studies. However, HIOs are established from human tissue, which raises issues of availability and ethics. In the present study, to solve these problems, we have established intestinal organoids using commercially available cryopreserved human intestinal epithelial cells (C-IOs), and compared their functions with biopsy-derived human intestinal organoids (B-IOs) from a pharmacokinetic point of view. Both C-IOs and B-IOs reproduced the morphological features of the intestinal tract and were shown to be composed of epithelial cells. Monolayers generated from C-IOs and B-IOs (C-IO-2D, B-IO-2D, respectively) structurally mimic the small intestine. The C-IOs showed gene expression levels comparable to those of the B-IOs, which were close to those of adult human small intestine. Importantly, the C-IOs-2D showed levels of pharmacokinetics-related protein expression and activity-including cytochrome P450 3A4 (CYP3A4) and carboxylesterase 2 (CES2) enzymatic activities and P-glycoprotein (P-gp) transporter activities -similar to those of B-IOs-2D. This study addresses the difficulties associated with B-IOs and provides fundamental characteristics for the application of C-IOs in pharmacokinetic studies.

Comparison of human biopsy-derived and human iPS cell-derived intestinal organoids established from a single individual.

Rodent-derived intestinal tissues or human colon cancer-derived Caco-2 cells are widely used for in vitro pharmacokinetic tests. However, both entail problems such as species differences from humans and low expression levels of specific pharmacokinetic-related factors, respectively. To solve these problems, many groups, including ours, have been focusing on human biopsy-derived intestinal organoids (b-IOs) and human iPS cell-derived intestinal organoids (i-IOs). However, no reports directly compare the two. Therefore, we established both from a single individual and conducted a comparative study. b-IOs had a shorter doubling time than i-IOs: about 59 h vs 148 h. b-IOs also had higher gene expression levels of major drug transporters and drug-metabolizing enzymes than i-IOs. To evaluate their applicability to pharmacokinetics, both organoids were two-dimensionally cultured. Although the b-IO monolayer had a lower transepithelial electrical resistance than the i-IO monolayer, it had higher gene expression levels of many drug transporters and major drug-metabolizing enzymes than the i-IO monolayer. RNA-seq analysis showed that the i-IOs monolayer had a more complex structure than the b-IOs monolayer because the former contained neuronal and vascular endothelial cells. This study provides basic information for pharmacokinetic applications of human biopsy-derived and human iPS cell-derived intestinal organoids.

TGF-ß1 and -ß3 for Mesenchymal Stem Cells Chondrogenic Differentiation on Poly (Vinyl Alcohol)-Chitosan-Poly (Ethylene Glycol) Scaffold.

Transforming growth factor-beta 1 (TGF-ß1) has been reported to promote chondrogenic differentiation and proliferation in the multipotent stromal cell (MSCs), and the transforming growth factor-beta 3 (TGF-ß3) tends to be exclusively in promoting cell differentiation alone. The objective of this study was to determine the effect of TGF-ß1 and -ß3 on the MSCs chondrogenic differentiation on the poly (vinyl alcohol)-chitosan-poly (ethylene glycol) (PVA-NOCC-PEG) scaffold, compared with that of monolayer and pellet cultures. In this study, P2 rabbit bone marrow-derived MSCs were seeded either on the untreated six-well plate (for monolayer culture) or onto the PVA-NOCC-PEG scaffold or cultured as a pellet culture. The cultures were maintained in a chemically defined serum-free medium supplemented with 10 ng/mL of either TGF-ß1 or TGF-ß3. Cell viability assay, biochemical assay, and real-time polymerase chain reaction were performed to determine the net effect of cell proliferation and chondrogenic differentiation of each of the growth factors. The results showed that the PVA-NOCC-PEG scaffold enhanced MSCs cell proliferation from day 12 to 30 (p < 0.05); however, no significant differences were observed in the cell proliferation between the cultures supplemented with or without TGF-ß1 and TGF-ß3 (p > 0.05). In terms of chondrogenic differentiation, the PVA-NOCC-PEG scaffold augmented the GAGs secretion in MSCs and the mRNA expression levels of Sox9, Col2a1, Acan, and Comp were elevated (p < 0.05). However, there was no significant difference between both the TGF-ß1 and TGF-ß3-treated groups (p > 0.05). In conclusion, TGF-ß1 and TGF-ß3 enhanced the chondrogenic differentiation of MSCs seeded on the PVA-NOCC-PEG scaffold; however, there was no significant difference between the effect of TGF-ß1 and TGF-ß3. Impact statement Transforming growth factor-beta (TGF-ß) superfamily members is a key requirement for the in vitro chondrogenic differentiation of mesenchymal stem cells (MSCs). In this study, the effects of TGF-ß1 and -ß3 on MSC chondrogenic differentiation and proliferation on a novel three-dimensional scaffold, the poly(vinyl alcohol)-chitosan-poly(ethylene glycol) (PVA-NOCC-PEG) scaffold, was evaluated. In this study, the results showed both TGF-ß1 and TGF-ß3 can enhance the chondrogenic differentiation of MSCs seeded on the PVA-NOCC-PEG scaffold.

A One-Step, Monolayer Culture and Chemical-Based Approach to Generate Insulin-Producing Cells From Human Adipose-Derived Stem Cells to Mitigate Hyperglycemia in STZ-Induced Diabetic Rats.

The global population of individuals afflicted with diabetes mellitus has been increasing year by year, and this disease poses a serious threat to human health as well as the economies worldwide. Pancreatic or islet transplantations provide one of the most effective and long-term therapies available to treat diabetes, but the scarcity and quality of pancreatic islets limit their use in treatments. Here, we report the development of a one-step, monolayer culture, and chemical-based protocol that efficiently mediates the differentiation of human adipose-derived stem cells (hADSCs) into insulin-producing cells (IPCs). Our data indicate that hADSCs in monolayer culture that are allowed to differentiate into IPCs are superior to those in suspension cultures with respect to insulin secretion capacity (213-fold increase), cell viability (93.5 ± 3.27% vs. 41.67 ± 13.17%), and response to glucose stimulation. Moreover, the expression of genes associated with pancreatic lineage specification, such as PDX1, ISL1, and INS (encoding insulin), were expressed at significantly higher levels during our differentiation protocol (6-fold for PDX1 and ISL1, 11.5-fold for INS). Importantly, in vivo studies demonstrated that transplantation with IPCs significantly mitigated hyperglycemia in streptozotocin-induced diabetic rats. Our results indicate that this one-step, rapid protocol increases the efficiency of IPC generation and that the chemical-based approach for IPC induction may reduce safety concerns associated with the use of IPCs for clinical applications, thereby providing a safe and effective cell-based treatment for diabetes.

High-efficient serum-free differentiation of endothelial cells from human iPS cells.

INTRODUCTION: Endothelial cells (ECs) form the inner lining of all blood vessels of the body play important roles in vascular tone regulation, hormone secretion, anticoagulation, regulation of blood cell adhesion and immune cell extravasation. Limitless ECs sources are required to further in vitro investigations of ECs' physiology and pathophysiology as well as for tissue engineering approaches. Ideally, the differentiation protocol avoids animal-derived components such as fetal serum and yields ECs at efficiencies that make further sorting obsolete for most applications. METHOD: Human induced pluripotent stem cells (hiPSCs) are cultured under serum-free conditions and induced into mesodermal progenitor cells via stimulation of Wnt signaling for 24 h. Mesodermal progenitor cells are further differentiated into ECs by utilizing a combination of human vascular endothelial growth factor A165 (VEGF), basic fibroblast growth factor (bFGF), 8-Bromoadenosine 3',5'-cyclic monophosphate sodium salt monohydrate (8Bro) and melatonin (Mel) for 48 h. RESULT: This combination generates hiPSC derived ECs (hiPSC-ECs) at a fraction of 90.9 ± 1.5% and is easily transferable from the two-dimensional (2D) monolayer into three-dimensional (3D) scalable bioreactor suspension cultures. hiPSC-ECs are positive for CD31, VE-Cadherin, von Willebrand factor and CD34. Furthermore, the majority of hiPSC-ECs express the vascular endothelial marker CD184 (CXCR4). CONCLUSION: The differentiation method presented here generates hiPSC-ECs in only 6 days, without addition of animal sera and at high efficiency, hence providing a scalable source of hiPSC-ECs.

Induction of Human Natural Killer Cells Under Defined Conditions by Seamless Transition from Maintenance Culture of Pluripotent Stem Cells.

The use of pluripotent stem cells (PSCs) as a source of natural killer cells (NK cells) can improve reproducibility in the analysis of the pathogenesis of NK cell-associated diseases and in the production of off-the-shelf cellular medicines. We have developed a method for the differentiation of NK cells from human PSCs under serum-free and two-dimensional condition. Our method enables the seamless transition from maintenance of PSCs to differentiation of NK cells, without the use of any techniques other than medium exchange and whole culture passage.

Is Quercetin Beneficial for Colon Cancer? A Cell Culture Study, Using the Apoptosis Pathways.

BACKGROUND: Quercetin (QCT) is a dietary flavonoid with many beneficial effects (e.g., antioxidant, antiaging, antidiabetic, antifungal effects, and regulation of gastrointestinal motor activity in human); furthermore, it induces apoptosis, cell cycle arrest, and differentiation. OBJECTIVE: The apoptotic effects of OCT were investigated on SW480 human colon cancer cell lines in monolayer and spheroid cultures. METHODS: Quercetin (40-200 µM) was applied, and Inhibitory Concentration (IC50) doses were determined for three time intervals (24, 48, and 72 h). The effective dose was determined and applied for analyses, including staining with BrdU to investigate cell proliferation, terminal deoxynucleotidyl transferase dUTP nick and labeling (TUNEL) to investigate apoptosis, and caspase-3 and Apoptosis Inducing Factor (AIF) to investigate caspase-dependent or independent apoptotic pathways. RESULTS: The effective dose of QCT was determined to be 200 µM and was found to induce apoptosis and inhibit cell proliferation at 24, 48, and 72 h,both in 2D and 3D cultures. Significant increases were observed in both caspase-3 and AIF staining, but cells showed greater caspase-3 staining compared with AIF staining at all time intervals (p<0.05). CONCLUSION: The QCT treatment groups showed more cell death and less cell growth compared with the untreated control groups in both 2D and 3D cultures of SW480 cell lines. The results suggest that quercetin induces apoptosis, inhibits cell proliferation, and has a protective role against colon cancer. However, further studies are needed to clarify its mechanism of action.

Monolayer platform using human biopsy-derived duodenal organoids for pharmaceutical research.

The human small intestine is the key organ for absorption, metabolism, and excretion of orally administered drugs. To preclinically predict these reactions in drug discovery research, a cell model that can precisely recapitulate the in vivo human intestinal monolayer is desired. In this study, we developed a monolayer platform using human biopsy-derived duodenal organoids for application to pharmacokinetic studies. The human duodenal organoid-derived monolayer was prepared by a simple method in 3-8 days. It consisted of polarized absorptive cells and had tight junctions. It showed much higher cytochrome P450 (CYP)3A4 and carboxylesterase (CES)2 activities than did the existing models (Caco-2 cells). It also showed efflux activity of P-glycoprotein (P-gp) and inducibility of CYP3A4. Finally, its gene expression profile was closer to the adult human duodenum, compared to the profile of Caco-2 cells. Based on these findings, this monolayer assay system using biopsy-derived human intestinal organoids is likely to be widely adopted.

Effect of dipeptide on intestinal peptide transporter 1 gene expression: An evaluation using primary cultured chicken intestinal epithelial cells.

Peptide transporter 1 (PepT1) is a transporter responsible for absorbing dipeptide and tripeptide in enterocytes and is upregulated by dipeptide in mammals. It has not been certain whether intestinal PepT1 expression is responsive to dipeptides in chickens because of the lack of in vitro study using the cultured enterocytes. This study established a primary culture model of chicken intestinal epithelial cells (IECs) in two-dimensional monolayer culture using collagen gel by which the response of chicken PepT1 gene expression to dipeptide stimuli was evaluated. The cultured chicken IECs showed the epithelial-like morphology attached in a patch-manner and exhibited positive expression of cytokeratin and epithelial cadherin, specific marker proteins of epithelial cells. Moreover, the chicken IECs exhibited the gene expression of intestinal cell type-specific marker, villin1, mucin 2, and chromogranin A, suggesting that the cultured IECs were composed of enterocytes as well as goblet and enteroendocrine cells. PepT1 gene expression was significantly upregulated by synthetic dipeptide, glycyl-l-glutamine, in the cultured IECs. From the results, we herein suggested that dipeptide is a factor upregulating PepT1 gene expression in chicken IECs.

Primary Cultures and Cell Lines for In Vitro Modeling of the Human Adrenal Cortex.

The human adrenal cortex is a complex endocrine organ that produces mineralocorticoids, glucocorticoids and androgens. These steroids are produced in distinct cell types located within the glomerulosa, fasciculata and reticularis of the adrenal cortex. Abnormal adrenal steroidogenesis leads to a variety of diseases that can cause hypertension, metabolic syndrome, infertility and premature adrenarche. The adrenal cortex can also develop steroid-producing adenomas and rarely adrenocortical carcinomas. In vitro cell culture models provide important tools to study molecular and cellular mechanisms controlling both the physiologic and pathologic conditions of the adrenal cortex. In addition, the presence of multiple steroid-metabolizing enzymes within adrenal cells makes it a model for defining possible endocrine disruptors that might block these enzymes. The regulation and dysregulation of human adrenal steroid production and cell division/tumor growth can be studied using freshly isolated cells but this requires access to human adrenal glands, which are not available to most investigators. Immortalized human adrenocortical cell lines have proven to be of considerable value in studying the molecular and biochemical mechanisms controlling adrenal steroidogenesis and tumorigenesis. Current human adrenal cell lines include the original NCI-H295 and its substrains: H295A, H295R, HAC13, HAC15, HAC50 and H295RA as well as the recently established MUC-1, CU-ACC1 and CU-ACC2. The current review will discuss the use of primary cultures of fetal and adult adrenal cells as well as adrenocortical cell lines as in vitro models for the study of human adrenal physiology and pathophysiology.

Secoisolariciresinol diglucoside induces caspase-3-mediated apoptosis in monolayer and spheroid cultures of human colon carcinoma cells.

Apoptotic effects of secoisolariciresinol diglucoside (SDG) in 2D and 3D cultures of SW480 cells were investigated. 40-200 µM SDG was used and IC50 values were determined for three different time intervals as 24, 48, or 72 hr for further experiments. BrdU, TUNEL, AIF, and caspase-3 stainings were used. SDG inhibited cell proliferation almost half and half for all time intervals in 2D and 3D cultures and also, induced apoptosis. Apoptotic cell percentages in the control group for 24, 48, and 72 hr were 27.00%, 29.00%, and 28.00%, respectively, while in the SDG treatment group were 59.00%, 61.00%, and 62.00%, respectively. In the spheroid cell culture, apoptotic cell percentages in the control group for 24, 48, and 72 hr were 6.90%, 7.20%, and 7.10%, respectively, while in the SDG treatment group were 19.50%, 19.50%, and 20.70%, respectively. Caspase-3 and AIF antibodies were used to indicate caspase-dependent and -independent apoptotic pathways. Significant increases were seen in both AIF and caspase-3 stainings when compared to the control group but caspase-3 staining results were significantly greater when compared to the AIF staining at all time intervals (p < .05). To prove this, CASP3 gene expression was evaluated by RT-qPCR. Unlike staining results, there was no statistically significant change at 24 hr in 2D and 3D cultures. But, significant upregulation at 48 (2.32-fold in 2D and 2.46-fold in 3D) and 72 hr (5.04-fold in 2D and 6.45-fold in 3D) were seen. PRACTICAL APPLICATIONS: Colon cancer is one of the most prevalent cancer in the developed countries and its etiology is complex. Although the underlying mechanisms are mostly unknown, the link between diet and colon cancer is known and dietary habits can promote cancer or protect against it. In recent years, flaxseed is accepted as a significant functional food ingredient and feeding with it could help in to prevent cancer. Secoisolariciresinol diglucoside is a flaxseed lignan and is metabolized to mammalian lignans by the gut. In the present study, SDG was evaluated for its apoptotic effects in colon carcinoma cell line via monolayer and spheroid cultures using immunohistochemical and gene expression techniques. Findings of this study suggest that SDG may protect against cancers and in particularly against colon cancer and further investigations has to be carried out for detailed underlying mechanisms.

Establishment and characterization of a novel cell line, NCC-DDLPS2-C1, derived from a patient with dedifferentiated liposarcoma.

Dedifferentiated liposarcoma (DDLPS) is a highly aggressive subtype of liposarcoma that is histologically a transition form between an atypical lipomatous tumor/well-differentiated liposarcoma and a non-lipogenic sarcoma. DDLPS is genetically characterized by a complex karyotype with copy number variations and genomic complexity. DDLPS has a poor prognosis, a high local recurrence rate, and refractory behaviors for chemotherapy and radiation, which indicate a requirement for a novel therapeutic strategy for better clinical outcomes. We report here, a novel DDLPS cell line (NCC-DDLPS2-C1) developed from a tumor tissue. NCC-DDLPS2-C1 cells showed an amplified 12q13-15 region and exhibited constant growth, spheroid formation, and invasion. High-throughput drug screening revealed distinct sensitivity between monolayer- and three-dimensional cells. Romidepsin and trabectedin especially showed high anti-proliferative effects in both culture methods of NCC-DDLPS2-C1. Thus, the NCC-DDLPS2-C1 cell line may serve as a useful resource for DDLPS studies.

Transient loss of membrane integrity following intracellular ice formation in dimethyl sulfoxide-treated hepatocyte and endothelial cell monolayers.

Immediate post-thaw evaluation of membrane integrity has proven to yield overestimates of cell survival under conditions that preclude intracellular ice formation (IIF). However, prominent theories on the mechanisms of intracellular nucleation suggest a damaged membrane can reseal, prompting us to evaluate whether immediate post-thaw assessments of membrane integrity can in fact underestimate cell survival under conditions that promote IIF. HUVEC and HepG2 monolayers were treated with 1.4 M DMSO and frozen to -25 °C under conditions that formed either 0% or 100% IIF. Membrane integrity was evaluated both immediately and 24 h post-thaw, with metabolic activity assessments performed 24 h post-thaw as a secondary measure of survival. Treatment with 1.4 M DMSO and nucleation of 100% IIF resulted in a drastic increase in the relative percent of membrane intact cells following a 24 h culture period (HUVEC: 90.2% ± 0.7%; HepG2: 70.4% ± 4.0%), which correlated with 24 h post-thaw metabolic activity. These differences between the immediate and 24 h post-thaw membrane integrity assessments were significantly more than those seen in the absence of either IIF or DMSO treatment. Therefore, a high incidence of IIF in DMSO-treated monolayers may lead to erroneous underestimates of cell survival when conducting immediate post-thaw assessments of membrane integrity.

Establishment of a Simple Method for Inducing Neuronal Differentiation of P19 EC Cells without Embryoid Body Formation and Analysis of the Role of Histone Deacetylase 8 Activity in This Differentiation.

P19 pluripotent embryonic carcinoma (EC) stem cells are derived from pluripotent germ cell tumours and can differentiate into three germ layers. Treatment of these cells in suspension culture with retinoic acid induces their differentiation into neurons and glial cells. Hence, these cells are an excellent in vitro model to study the transition from the upper blastoderm to the neuroectoderm. However, because of the complex nature of the techniques involved, the results are highly dependent on the skills of the experimenter. Herein, we developed a simple method to induce neuronal differentiation of adherent P19 EC cells in TaKaRa NDiff® 227 serum-free medium (originally N2B27 medium). This medium markedly induced neuronal differentiation of P19 EC cells. The addition of retinoic acid to the NDiff® 227 medium further enhanced differentiation. Furthermore, cells differentiated by the conventional method, as well as the new method, showed identical expression of the mature neuronal marker, neuronal nuclei. To determine whether our approach could be applied for neuronal studies, we measured histone deacetylase 8 (HDAC8) activity using an HDAC8 inhibitor and HDAC8-knockout P19 EC cells. Inhibition of HDAC8 activity suppressed neuronal maturation. Additionally, HDAC8-knockout cell lines showed immature differentiation compared to the wild-type cell line. These results indicate that HDAC8 directly regulates the neuronal differentiation of P19 EC cells. Thus, our method involving P19 EC cells can be used as an experimental system to study the nervous system. Moreover, this method is suitable for screening drugs that affect the nervous system and cell differentiation.

Cell culture dimensionality influences mesenchymal stem cell fate through cadherin-2 and cadherin-11.

The acquisition of a specific cell fate is one of the core aims of tissue engineering and regenerative medicine. Significant evidence shows that aggregate cultures have a positive influence on cell fate decisions, presumably through cell-cell interactions, but little is known about the specific mechanisms. To investigate the difference between cells cultured as a monolayer and as aggregates, we started by looking at cadherin expression, an important protein involved in cell adhesion, during the differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs) in aggregate and monolayer cultures. We observed that proliferating hMSCs in monolayer culture expressed lower levels of cadherin-2 and increased cadherin-11 expression at cell-cell contact sites over time, which was not evident in the aggregate cultures. By knocking down cadherin-2 and cadherin-11, we found that both cadherins were required for adipogenic differentiation in a monolayer as well as aggregate culture. However, during osteogenic differentiation, low levels of cadherin-2 were found to be favorable for cells cultured as a monolayer and as aggregates, whereas cadherin- 11 was dispensable for cells cultured as aggregates. Together, these results provide compelling evidence for the important role that cadherins play in regulating the differentiation of hMSCs and how this is affected by the dimensionality of cell culture.

Alterations in characteristics of canine articular chondrocytes in non-passaged long-term monolayer culture: Matter of differentiation, dedifferentiation and redifferentiation.

This study investigated the effects of culture time on phenotype stability of canine articular chondrocytes (CACs) in non-passaged long-term monolayer culture. Third passage (P3) CACs isolated from four cartilage samples were seeded at three different initial seeding densities (0.2 × 104, 1.0 × 104 and 5.0 × 104 cells/cm2) and maintained in monolayer condition up to 8 weeks without undergoing subculture after confluence. The characteristic changes of chondrocytes during the culture period were evaluated based on the cell morphology, cell proliferation, glycosaminoglycans (GAGs) content, DNA quantification, mRNA expression and ultrastructure of chondrocytes. Chondrocytes maintained under post-confluence condition exhibited a capability to grow and proliferate up to 4 weeks. Alcian blue staining and Dimethylmethylene blue (DMMB) assay revealed that the extracellular matrix (ECM) synthesis was increased in a time-dependent manner from 2 to 8 weeks. The chondrocyte mRNA expression profile was dramatically affected by prolonged culture time, with a significant downregulation of collagen type I, whereas the expression of collagen type II, aggrecan, Sox9 and matrix metalloproteinase 13 (MMP-13) were significantly upregulated. In addition, transmission electron microscopy (TEM) result indicated dilation of rough endoplasmic reticulum (RER) in these long-term monolayer cultured chondrocytes. These findings demonstrate that the chondrocytes phenotype could be partially redifferentiated through the spontaneous redifferentiation process in long-term cultures using standard culture medium without the addition of chondrogenic supplements or tissue-culture scaffolds.

Comparative Molecular Analysis of Cancer Behavior Cultured In Vitro, In Vivo, and Ex Vivo.

Current pre-clinical models of cancer fail to recapitulate the cancer cell behavior in primary tumors primarily because of the lack of a deeper understanding of the effects that the microenvironment has on cancer cell phenotype. Transcriptomic profiling of 4T1 murine mammary carcinoma cells from 2D and 3D cultures, subcutaneous or orthotopic allografts (from immunocompetent or immunodeficient mice), as well as ex vivo tumoroids, revealed differences in molecular signatures including altered expression of genes involved in cell cycle progression, cell signaling and extracellular matrix remodeling. The 3D culture platforms had more in vivo-like transcriptional profiles than 2D cultures. In vivo tumors had more cells undergoing epithelial-to-mesenchymal transition (EMT) while in vitro cultures had cells residing primarily in an epithelial or mesenchymal state. Ex vivo tumoroids incorporated aspects of in vivo and in vitro culturing, retaining higher abundance of cells undergoing EMT while shifting cancer cell fate towards a more mesenchymal state. Cellular heterogeneity surveyed by scRNA-seq revealed that ex vivo tumoroids, while rapidly expanding cancer and fibroblast populations, lose a significant proportion of immune components. This study emphasizes the need to improve in vitro culture systems and preserve syngeneic-like tumor composition by maintaining similar EMT heterogeneity as well as inclusion of stromal subpopulations.

Differentiation of Human Mesenchymal Stem Cells from Wharton's Jelly Towards Neural Stem Cells Using A Feasible and Repeatable Protocol.

The transplantation of neural stem cells (NSCs) capable of regenerating to the cells of the central nervous system (CNS) is a promising strategy in the treatment of CNS diseases and injury. As previous studies have highlighted mesenchymal stem cells (MSCs) as a source of NSCs, this study aimed to develop a feasible, efficient, and reproducible method for the neural induction of MSCs isolated from Wharton's jelly (hWJ-MSCs). We induced neural differentiation in a monolayer culture using epidermal growth factor, basic fibroblast growth factor, N2, and B27 supplements. This resulted in a homogenous population of proliferating cells that expressed certain neural markers at both the protein and mRNA levels. Flow cytometry and immunocytochemistry confirmed the expression of neural markers: nestin, sex-determining region Y (SRY) box 1 and 2 (SOX1 and SOX2), microtubule-associated protein 2 (MAP2), and glial fibrillary acidic protein (GFAP). The qRT-PCR analysis revealed significantly enhanced expression of nestin and MAP2 in differentiated cells. This study confirms that it is possible to generate NSCs-like cells from hWJ-MSCs in a 2D culture using a practical method. However, the therapeutic effectiveness of such differentiated cells should be extended to confirm the terminal differentiation ability and electrophysiological properties of neurons derived from them.