Optimization of cell engineering protocols requires standard, comprehensive quality metrics. We previously developed CellNet, a computational tool to quantitatively assess the transcriptional fidelity of engineered cells compared with their natural counterparts, based on bulk-derived expression profiles. However, this platform and others were limited in their ability to compare data from different sources, and no current tool makes it easy to compare new protocols with existing state-of-the-art protocols in a standardized manner. Here, we utilized our prior application of the top-scoring pair transformation to build a computational platform, platform-agnostic CellNet (PACNet), to address both shortcomings. To demonstrate the utility of PACNet, we applied it to thousands of samples from over 100 studies that describe dozens of protocols designed to produce seven distinct cell types. We performed an in-depth examination of hepatocyte and cardiomyocyte protocols to identify the best-performing methods, characterize the extent of intra-protocol and inter-lab variation, and identify common off-target signatures, including a surprising neural/neuroendocrine signature in primary liver-derived organoids. We have made PACNet available as an easy-to-use web application, allowing users to assess their protocols relative to our database of reference engineered samples, and as open-source, extensible code.

BACKGROUND: Somatic cell fate transition is now gained great importance in tissue regeneration. Currently, research is focused on heart tissue regeneration by reprogramming diverse cells into cardiomyocyte-like cells. Here, we examined the possible effect of miRNAs on the transdifferentiation of fibroblasts into cardiomyocyte-like cells.
METHODS: First heart-specific miRNAs were identified by comparing the gene expression profiles of heart tissue to other body tissues using bioinformatic techniques. After identifying heart-specific miRNAs, their cellular and molecular functions were studied using the miRWalk and miRBase databases. Then the candidate miRNA was cloned into a lentiviral vector. Following, human dermal fibroblasts were cultured and treated with compounds forskolin, valproic acid, and CHIR99021. After 24 h, the lentivector harboring miRNA gene was transfected into the cells to initiate the transdifferentiation process. Finally, after a two-week treatment period, the efficiency of transdifferentiation was examined by inspecting the appearance of the cells and measuring the expression levels of cardiac genes and proteins using RT-qPCR and immunocytochemistry techniques.
RESULTS: Nine miRNAs were identified with higher expression in the heart. The miR-2392 was nominated as the candidate miRNA due to its function and specific expression in the heart. This miRNA has a direct connection with genes involved in cell growth and differentiation; e.g., MAPK and Wnt signaling pathways. According to in vitro results cardiac genes and proteins demonstrated an increase in expression in the fibroblasts that simultaneously received the three chemicals and miR-2392.
CONCLUSIONS: Considering the ability of miR-2392 to induce the expression of cardiac genes and proteins in fibroblast cells, it can induce fibroblasts to differentiate into cardiomyocyte-like cells. Therefore, miR-2392 could be further optimized for cardiomyocyte regeneration, tissue repair, and drug design studies.

Background: Various skeletal disorders display defects in osteoblast development and function. An in vitro model can help to understand underlying disease mechanisms. Currently, access to appropriate starting material for in vitro osteoblastic studies is limited. Native osteoblasts and their progenitors, the bone marrow mesenchymal stem cells, (MSCs) are problematic to isolate from affected patients and challenging to expand in vitro. Human dermal fibroblasts in vitro are a promising substitute source of cells. Method: We developed an in vitro culturing technique to transdifferentiate fibroblasts into osteoblast-like cells. We obtained human fibroblasts from forearm skin biopsy and differentiated them into osteoblast-like cells with ß-glycerophosphate, ascorbic acid, and dexamethasone treatment. Osteoblastic phenotype was confirmed by staining for alkaline phosphatase (ALP), calcium and phosphate deposits (Alizarin Red, Von Kossa) and by a multi-omics approach (transcriptomic, proteomic, and phosphoproteomic analyses). Result: After 14 days of treatment, both fibroblasts and MSCs (reference cells) stained positive for ALP together with a significant increase in bone specific ALP (p = 0.04 and 0.004, respectively) compared to untreated cells. At a later time point, both cell types deposited minerals, indicating mineralization. In addition, fibroblasts and MSCs showed elevated expression of several osteogenic genes (e.g. ALPL, RUNX2, BMPs and SMADs), and decreased expression of SOX9. Ingenuity Pathways Analysis of RNA sequencing data from fibroblasts and MSCs showed that the osteoarthritis pathway was activated in both cell types (p_adj. = 0.003 and 0.004, respectively). Discussion: These data indicate that our in vitro treatment induces osteoblast-like differentiation in fibroblasts and MSCs, producing an in vitro osteoblastic cell system. This culturing system provides an alternative tool for bone biology research and skeletal tissue engineering.

Intratumoral heterogeneity (ITH) is an intrinsic feature of malignant tumors that eventually allows a subfraction of resistant cancer cells to clonally evolve and cause therapy failure or relapse. ITH, cellular plasticity and tumor progression are driven by epithelial-mesenchymal transition (EMT) and the reverse process, MET. During these developmental programs, epithelial (E) cells are successively converted to invasive mesenchymal (M) cells, or back to E cells, by passing through a series of intermediate E/M states, a phenomenon termed E-M plasticity (EMP). The induction of MET has clinical potential as it can block the initial EMT stages that favor tumor cell dissemination, while its inhibition can curb metastatic outgrowth at distant sites. In pancreatic ductal adenocarcinoma (PDAC), cellular models with which to study EMP or MET induction are scarce. Here, we have generated single cell-derived clonal cultures of the quasimesenchymal PDAC-derived cell line, PANC-1, and found that these differ strongly with respect to cell morphology and EMT marker expression, allowing for their tentative classification as E, E/M or M. Interestingly, the different EMT phenotypes were found to segregate with differences in tumorigenic potential in vitro, as measured by colony forming and invasive activities, and in circadian clock function. Moreover, the individual clones the phenotypes of which remained stable upon prolonged culture also responded differently to treatment with transforming growth factor (TGF)β1 in regard to regulation of growth and individual TGFβ target genes, and to culture conditions that favour ductal-to-endocrine transdifferentiation as a more direct measure for cellular plasticity. Of note, stimulation with TGFβ1 induced a shift in parental PANC-1 cultures towards a more extreme M and invasive phenotype, while exposing the cells to a combination of the proinflammatory cytokines IFNγ, IL1β and TNFα (IIT) elicited a shift towards a more E and less invasive phenotype resembling a MET-like process. Finally, we show that the actions of TGFβ1 and IIT both converge on regulating the ratio of the small GTPase RAC1 and its splice isoform, RAC1b. Our data provide strong evidence for dynamic EMT-MET transitions and qualify this cell line as a useful model with which to study EMP.

OBJECTIVE: Endochondral ossification, which involves transdifferentiation of chondrocytes into osteoblasts, is an important process involved in the development and postnatal growth of most vertebrate bones as well as in bone fracture healing. To study the basic molecular mechanisms of this process, a robust and easy-to-use in vitro model is desirable. Therefore, we aimed to develop a standardized in vitro assay for the transdifferentiation of chondrogenic cells towards the osteogenic lineage.
METHODS: Murine chondrogenic ATDC5 cells were differentiated into the chondrogenic lineage for seven days and subsequently differentiated towards the osteogenic direction. Gene expression analysis of pluripotency, as well as chondrogenic and osteogenic markers, cell-matrix staining, and immunofluorescent staining, were performed to assess the differentiation. In addition, the effects of Wnt3a and lipopolysaccharides (LPS) on the transdifferentiation were tested by their addition to the osteogenic differentiation medium.
RESULTS: Following osteogenic differentiation, chondrogenically pe-differentiated cells displayed the expression of pluripotency and osteogenic marker genes as well as alkaline phosphatase activity and a mineralized matrix. Co-expression of Col2a1 and Col1a1 after one day of osteogenic differentiation indicated that osteogenic cells had differentiated from chondrogenic cells. Wnt3a increased and LPS decreased transdifferentiation towards the osteogenic lineage.
CONCLUSIONS: We successfully established a rapid, standardized in vitro assay for the transdifferentiation of chondrogenic cells into osteogenic cells, which is suitable for testing the effects of different compounds on this cellular process.

Osteoblasts play a key role in bone remodeling. Recent studies have reported that some hypertrophic chondrocytes co-expressing collagen I(Col I) and collagen X (ColX) could directly transdifferentiate into osteoblasts during endochondral ossification. However, whether nutrition intervention is beneficial to this transformation to improve osteoporosis (OP) remains unknown. In this study, ovariectomy (OVX)-induced OP mice were orally administered with docosahexaenoic acid (DHA) in different molecular forms for 13 weeks. The results showed that both DHA-triglyceride (DHA-TG) and DHA-phosphatidylcholine (DHA-PC) increased the bone mineral density and bone mineral apposition rate in ovariectomized mice, while DHA-ethyl esters (DHA-EE) had little effect. Interestingly, we found that both DHA-PC and DHA-TG increased the height of the growth plate, mainly increasing the number of hypertrophic chondrocytes. Further investigation by simultaneously labeling ColX and ColI indicated that DHA-PC and DHA-TG promoted the number of chondrocyte-transdifferentiated osteoblasts in the growth plate close to the diaphysis, in which DHA-PC performed better than DHA-TG. Apoptosis was not the only fate of hypertrophic chondrocytes. Western blot results showed that both DHA-TG and DHA-PC downregulated the Bax and cleaved-caspase3 expression and upregulated Bcl-2 expression in the growth plate, suggesting that chondrocyte apoptosis is inhibited. Runx2, the key regulator of chondrocyte-to-osteoblast transdifferentiation, was significantly increased by DHA-TG and DHA-PC, while DHA-EE had no effect on the above indicators. To our best knowledge, this is the first report that both DHA-PC and DHA-TG enhanced bone formation via promoting the chondrocyte-to-osteoblast transdifferentiation in the growth plate, contributing to the amelioration of OP. These activities depend on the molecular forms of DHA and their bioavailabilities. Our results provide guidance for the application of fish oil for bone health.

In vitro culturing of cells in two-dimensional (2D) environments is a widespread used methodology in biomedical research. Most commonly, cells are cultured on artificial plastic dish surfaces, which lead to abnormal functional behaviors, as plastic does not reflect the native microenvironment found in vivo or in situ. Therefore, a multitude of three-dimensional (3D) cell culture systems were developed in the past years, which aim to bridge the gap between 2D cell culture dishes and the in vivo situation. One of the more recent development in the field, the generation of viable precision-cut tissue slices from various organs emerged as an exciting approach to study complex interactions and biological processes ex vivo in 3D. Decellularization of such tissue slices leads to the removal of all functional cells, and leaves behind a scaffold of extracellular matrix (ECM), which closely recapitulates the molecular composition, mechanical properties, topology, and microarchitecture of native ECM. Subsequently, decellularized precision-cut lung slices (PCLS), also called 3D lung tissue culture (3D-LTCs), can be successfully reseeded with a variety of cell types, including fibroblasts, which attach to and engraft into the matrix. Here, we describe the generation of PCLS from resected human lung tissue and their decellularization and recellularization with primary human fibroblasts. This novel 3D tissue culture model allows for various functional studies of fibroblast behavior on native ECM composition and topology.

Mutations in genes encoding proteins of the smooth muscle cell (SMC) contractile apparatus contribute to familial aortic aneurysms. To investigate the pathogenicity of these mutations, SMC are required. We demonstrate a novel method to generate SMC-like cells from human dermal fibroblasts by transdifferentiation to study the effect of variants in genes encoding proteins of the SMC contractile apparatus (ACTA2 and MYH11) in patients with aortic aneurysms. Dermal fibroblasts from seven healthy donors and cells from seven patients with MYH11 or ACTA2 variants were transdifferentiated into SMC-like cells within a 2-week duration using 5 ng/ml TGFβ1 on a scaffold containing collagen and elastin. The induced SMC were comparable to primary human aortic SMC in mRNA expression of SMC markers which was confirmed on the protein level by immunofluorescence quantification analysis and Western blotting. In patients with MYH11 or ACTA2 variants, the effect of intronic variants on splicing was demonstrated on the mRNA level in the induced SMC, allowing classification into pathogenic or nonpathogenic variants. In conclusion, direct conversion of human dermal fibroblasts into SMC-like cells is a highly efficient method to investigate the pathogenicity of variants in proteins of the SMC contractile apparatus.

BACKGROUND: The purpose of this study is to investigate whether ordinary hepatocellular carcinomas (HCCs) show positivity of stem/progenitor cell markers and cholangiocyte markers during the process of tumor progression.
METHODS: Ninety-four HCC lesions no larger than 8 cm from 94 patients were immuno-histochemically studied using two hepatocyte markers (Hep par 1 and α-fetoprotein), five cholangiocyte markers (cytokeratin CK7, CK19, Muc1, epithelial membrane antigen and carcinoembryonic antigen) and three hepatic stem/progenitor cell markers (CD56, c-Kit and EpCAM). The tumors were classified into three groups by tumor size: S1, < 2.0 cm; S2, 2.0-5.0 cm; S3, 5.0-8.0 cm. The tumors were also classified according to tumor differentiation: well, moderately and poorly differentiated. The relationship between the positive ratios of these markers, tumor size and tumor differentiation was examined.
RESULTS: The positive ratios of cholangiocyte markers tended to be higher in larger sized and more poorly differentiated tumors (except for CK7). The positive ratios of stem/progenitor cell markers tended to be higher in larger sized and more poorly differentiated tumors (except for c-Kit).
CONCLUSIONS: Ordinary HCC can acquire the characteristic of positivity of cholangiocyte and stem/progenitor cell markers during the process of tumor progression.

The appearance of proliferating bile ductular structures, which is called the \"atypical ductular reaction\" is frequently observed in various chronic liver diseases associated. However, the origin of these increased bile ductules has been a matter of controversy. In this study, we investigated the origin of ductular cells as an aspect of relation between epithelial to mesenchymal transition (EMT) and epithelial members of liver parenchyme, such as hepatocyte and cholangiocyte by immunohistochemical staining of human liver. Thirteen specimens of surgically resected liver with biliary cirrhosis were selected. Three sets of double immunohistochemical stains were done; Hep-Par 1 - cytokeratin 19 (CK19), Hep-Par 1 - α-sm ooth mus cle actin (α-SMA) and CK19 - α-SMA. As a result, we investigated the dual expression of the markers of hepatocyte and cholangiocyte in the same cell; in ductular cell and surrounding hepatocyte. However, there seems to be no dual expression of markers for EMT with epithelial markers. This study suggests a possibility of phenotypic change of mature hepatocyte into cholangiocyte. Future studies will be necessary to determine the role that proliferating cholangiocytes play in the pathogenesis of biliary fibrosis and how cholangiocytes interact with other cell types of the liver such as hepatic stellate cells or Kupffer cells.