Full Differentiation Research Articles

Maternal protein restriction affects the differentiation of cells in the epididymal epithelium lining of 44-day-old rats.

Maternal protein restriction delays the differentiation of epididymal mesenchymal cells in newborn rats. However, it's unclear if this delay persists until the full differentiation of the epididymal epithelium at 44 days postnatal. Thus, this study aimed to assess the impact of maternal protein reduction on 44-day-old rats' epididymal epithelium differentiation, following up on the observed delay in newborn animals. Pregnant rats were randomly divided into groups receiving normal-protein (NP - 17% protein) or low-protein (LP - 6% protein) diets during gestation and lactation. On postnatal day (PDN) 44, male offspring were euthanized, and the epididymis (NP n=10, LP n=10) was processed according to immunohistochemical techniques for the detection of aquaporin 9 (AQP9), KI-67, TP63, and ATPase. LP rats showed: a decrease in the intensity of the AQP9 reaction, an increase in cellular proliferation in the initial segment and corpus of the epididymis, an increase in basal cells in the caput and corpus epididymis, and an increase in ATPase-positive clear cells in the cauda region. These findings demonstrate that maternal protein restriction impacts cell differentiation in the epididymal epithelium of 44-day-old rats, persisting even with a normal-protein diet after weaning.

Efficient Generation of CRISPR/Cas9-Mediated Knockout Human Primary Keratinocytes by Electroporation.

Due to their full differentiation capacity in vitro, the culture of human primary keratinocytes (HPKs) represents a physiological model for answering basic biological and dermatological research questions, including those related to skin diseases and the investigation of treatment options. When modified with the CRISPR/Cas9 gene editing approach and cultivated in organotypic 3D epidermal equivalents (EEs), these human cells have the potential to replace established mouse models. However, even when cultivated on feeder cells, HPKs have only a low proliferation capacity in 2D culture, limiting their application potential. This is particularly true for CRISPR/Cas9-modified HPKs, whose generation commonly requires selection of targeted cells, negatively affecting their lifespan. Here, we describe a robust protocol for the rapid, simple, and efficient generation of single- and multi-gene CRISPR/Cas9 knockout HPKs by electroporation of ribonucleoprotein (RNP) complexes, which comprise one or multiple guide RNAs (gRNAs) and Cas9 protein. Unlike DNA transfection or virus-based targeting strategies, electroporation of RNPs represents a targeting approach that minimizes immunological and toxic side effects. Using efficient gRNAs results in the generation of HPKs with a high yield of knockout cells, allowing for their immediate use in experiments without requiring the laborious process of selecting targeted cells or maintaining a feeder cell culture. Furthermore, the use of RNPs and their delivery via electroporation minimizes off-target and other unspecific effects, preventing unintended genomic alterations. Most importantly, CRISPR/Cas9 knockout HPKs generated with this protocol have the ability to form a fully differentiated epidermis in 3D, thus facilitating the understanding of specific protein functions in a highly physiological human skin model. Alternatively, this approach proves valuable for generating models of mono- or polygenic skin diseases via knockouts, providing insights into the underlying molecular mechanisms and facilitating the development of novel therapeutic approaches.

Inactivation of Tumor Suppressor CYLD Inhibits Fibroblast Reprogramming to Pluripotency.

CYLD is a tumor suppressor gene coding for a deubiquitinating enzyme that has a critical regulatory function in a variety of signaling pathways and biological processes involved in cancer development and progression, many of which are also key modulators of somatic cell reprogramming. Nevertheless, the potential role of CYLD in this process has not been studied. With the dual aim of investigating the involvement of CYLD in reprogramming and developing a better understanding of the intricate regulatory system governing this process, we reprogrammed control (CYLDWT/WT) and CYLD DUB-deficient (CYLDΔ9/Δ9) mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPSCs) through ectopic overexpression of the Yamanaka factors (Oct3/4, Sox2, Klf4, c-myc). CYLD DUB deficiency led to significantly reduced reprogramming efficiency and slower early reprogramming kinetics. The introduction of WT CYLD to CYLDΔ9/Δ9 MEFs rescued the phenotype. Nevertheless, CYLD DUB-deficient cells were capable of establishing induced pluripotent colonies with full spontaneous differentiation potential of the three germ layers. Whole proteome analysis (Data are available via ProteomeXchange with identifier PXD044220) revealed that the mesenchymal-to-epithelial transition (MET) during the early reprogramming stages was disrupted in CYLDΔ9/Δ9 MEFs. Interestingly, differentially enriched pathways revealed that the primary processes affected by CYLD DUB deficiency were associated with the organization of the extracellular matrix and several metabolic pathways. Our findings not only establish for the first time CYLD's significance as a regulatory component of early reprogramming but also highlight its role as an extracellular matrix regulator, which has profound implications in cancer research.

Cutaneous Manifestations of Myeloid Neoplasms Exhibit Broad and Divergent Morphologic and Immunophenotypic Features but Share Ancestral Clonal Mutations With Bone Marrow

In this study, we performed a comprehensive molecular analysis of paired skin and peripheral blood/bone marrow (BM) samples from 17 patients with cutaneous myeloid or cutaneous histiocytic-dendritic neoplasms. The cutaneous manifestations included 10 patients with cutaneous acute myeloid leukemia (c-AML), 2 patients with full or partial Langerhans cell differentiation, 2 patients with blastic plasmacytoid dendritic cell neoplasms (BPDCN), 1 patient with both Langerhans cell differentiation and BPDCN, and 2 patients with full or partial indeterminate dendritic cell differentiation. Seven of the 10 c-AML patients (70%) exhibited concurrent or subsequent marrow involvement by acute myeloid leukemia, with all 7 cases (100%) demonstrating shared clonal mutations in both the skin and BM. However, clonal relatedness was documented in one additional case that never had any BM involvement. Nevertheless, NPM1 mutations were identified in 7 of the 10 (70%) of these c-AML cases while one had KMT2A rearrangement and one showed inv(16). All 3 patients (100%) with Langerhans cell neoplasms, 2 patients with BPDCN (100%), and one of the 2 patients (50%) with other cutaneous dendritic cell neoplasms also demonstrated shared mutations between the skin and concurrent or subsequent myeloid neoplasms. Both BM and c-AML shared identical founding drivers, with a predominance of NPM1, DNMT3A, and translocations associated with monocytic differentiation, with common cutaneous-only mutations involving genes in the signal transduction and epigenetic pathways. Cutaneous histiocytic-dendritic neoplasms shared founding drivers in ASXL1, TET2, and/or SRSF2. However, in the Langerhans cell histiocytosis or histiocytic sarcoma cases, there exist recurrent secondary RAS pathway hits, whereas cutaneous BPDCN cases exhibit copy number or structural variants. These results enrich and broaden our understanding of clonally related cutaneous manifestations of myeloid neoplasms and further illuminate the highly diverse spectrum of morphologic and immunophenotypic features they exhibit.

Development and Characterization of an In Vitro Intestinal Model Including Extracellular Matrix and Macrovascular Endothelium.

In vitro intestinal models are used to study biological processes, drug and food absorption, or cytotoxicity, minimizing the use of animals in the laboratory. They usually consist of enterocytes and mucus-producing cells cultured for 3 weeks, e.g., on Transwells, to obtain a fully differentiated cell layer simulating the human epithelium. Other important components are the extracellular matrix (ECM) and strong vascularization. The former serves as structural support for cells and promotes cellular processes such as differentiation, migration, and growth. The latter includes endothelial cells, which coordinate vascularization and immune cell migration and facilitate the transport of ingested substances or drugs to the liver. In most cases, animal-derived hydrogels such as Matrigel or collagen are used as ECM in in vitro intestinal models, and endothelial cells are only partially considered, if at all. However, it is well-known that animal-derived products can lead to altered cell behavior and incorrect results. To circumvent these limitations, synthetic and modifiable hydrogels (Peptigel and Vitrogel) were studied here to mimic xenofree ECM, and the data were compared with Matrigel. Careful rheological characterization was performed, and the effect on cell proliferation was investigated. The results showed that Vitrogel exhibited shear-thinning behavior with an internal structure recovery of 78.9 ± 11.2%, providing the best properties among the gels investigated. Therefore, a coculture of Caco-2 and HT29-MTX cells (ratio 7:3) was grown on Vitrogel, while simultaneously endothelial cells were cultured on the basolateral side by inverse cultivation. The model was characterized in terms of cell proliferation, differentiation, and drug permeability. It was found that the cells cultured on Vitrogel induced a 1.7-fold increase in cell proliferation and facilitated the formation of microvilli and tight junctions after 2 weeks of cultivation. At the same time, the coculture showed full differentiation indicated by high alkaline phosphatase release of Caco-2 cells (95.0 ± 15.9%) and a mucus layer produced by HT29-MTX cells. Drug tests led to ex vivo comparable permeability coefficients (Papp) (i.e., Papp; antipyrine = (33.64 ± 5.13) × 10-6 cm/s, Papp; atenolol = (0.59 ± 0.16) × 10-6 cm/s). These results indicate that the newly developed intestinal model can be used for rapid and efficient assessment of drug permeability, excluding unexpected results due to animal-derived materials.

CSF-1 and Notch signaling cooperate in macrophage instruction and tissue repair during peripheral limb ischemia.

Ischemia causes an inflammatory response featuring monocyte-derived macrophages (MF) involved in angiogenesis and tissue repair. Angiogenesis and ischemic macrophage differentiation are regulated by Notch signaling via Notch ligand Delta-like 1 (Dll1). Colony stimulating factor 1 (CSF-1) is an essential MF lineage factor, but its role in ischemic macrophage development and the interaction with Notch signaling is so far unclear. Using a mouse model of hind limb ischemia with CSF-1 inhibitor studies and Dll1 heterozygous mice we show that CSF-1 is induced in the ischemic niche by a subpopulation of stromal cells expressing podoplanin, which was paralleled by the development of ischemic macrophages. Inhibition of CSF-1 signaling with small molecules or blocking antibodies impaired macrophage differentiation but prolonged the inflammatory response, resulting in impaired perfusion recovery and tissue regeneration. Yet, despite high levels of CSF-1, macrophage maturation and perfusion recovery were impaired in mice with Dll1 haploinsufficiency, while inflammation was exaggerated. In vitro, CSF-1 was not sufficient to induce full MF differentiation from donor monocytes in the absence of recombinant DLL1, while the presence of DLL1 in a dose-dependent manner stimulated MF differentiation in combination with CSF-1. Thus, CSF-1 is an ischemic niche factor that cooperates with Notch signaling in a non-redundant fashion to instruct macrophage cell fate and maturation, which is required for ischemic perfusion recovery and tissue repair.

Application of Microhaplotypes in Sibling Kinship Testing.

To investigate the efficacy of different numbers of microhaplotype (MH) loci and the introduction of different reference samples on the identification of full sibling, half sibling and differentiation between full sibling and half sibling kinships, and to explore the effect of changing mutation rate on sibling testing. First, a family map involving three generations was established, and four full sibling identification models, five half sibling identification models and five models distinguishing full and half siblings were constructed for different reference samples introduced. Based on the results of the previous study, two sets of nonbinary SNP-MH containing 34 and 54 loci were selected. Based on the above MH loci, 100 000 pairs of full sibling vs. unrelated individuals, 100 000 pairs of half sibling vs. unrelated individuals and 100 000 pairs of full sibling vs. half sibling were simulated based on the corresponding sibling kinship testing models, and the efficacy of each sibling kinship testing model was analyzed by the likelihood ratio algorithm under different thresholds. The mutant rate of 54 MH loci was changed to analyze the effect of mutation rate on sibling identification. In the same relationship testing model, the systematic efficacy of sibling testing was positively correlated with the number of MH loci detected. With the same number of MH loci, the efficacy of full sibling testing was better than that of uncle or grandfather when the reference sample introduced was a full sibling of A, but there was no significant difference in the identification efficacy of the four reference samples introduced for full sibling and half sibling differentiation testing. In addition, the mutation rate had a slight effect on the efficacy of sibling kinship testing. Increasing the number of MH loci and introducing reference samples of known relatives can increase the efficacy of full sibling testing, half sibling testing, and differentiation between full and half sibling kinships. The level of mutation rate in sibling testing by likelihood ratio method has a slight but insignificant effect on the efficacy.

Abstract PR007: ERG-driven luminal prostate cancers emerge from Ck5+/Nkx3.1+ basal cells

Abstract TMPRSS2-ERG translocations are initiating lesions in nearly 50% of human prostate adenocarcinomas, often in conjunction with PTEN loss. In mice, prostate-specific expression of ERG using a Probasin-Cre driver (Pb-Cre4) is sufficient to induce invasive Ck8+ luminal adenocarcinoma with high penetrance in the context of Pten loss. Because TMPRSS2 expression in the human prostate is androgen-regulated and thereby largely restricted to luminal epithelial cells, it has been assumed that luminal cells are the likely cell of origin for TMPRSS2-ERG positive prostate cancers. To gain further support for this hypothesis, we examined the early stages of luminal adenocarcinoma initiation in the Pb-Cre ERG/Pten model at a single cell level. To our surprise, we found a highly proliferative population of ERG+ Ck5+ (a putative basal marker) cells that are enriched at the invasive front in a background of more canonical ERG+/Ck8+/Ck5- luminal tumor cells. We next performed lineage tracing experiments using Ck5-, Ck8- as well as Nkx3.1-Cre drivers, introduced via viral injection or tamoxifen induction, which collectively support the hypothesis that ERG-driven tumors originate in a subset of basal cells defined by Ck5+/Nkx3.1+. This conclusion is further supported by orthotopic transplantation of freshly isolated primary mouse prostate basal (Cd49f high) or luminal (Cd24 high) cells showing that ERG+/Ck8+ tumors arise preferentially from basal cells following ex vivo ERG/Pten activation. Time course experiments in organoids and in vivo show that ERG promotes Ck8+ luminal differentiation from highly proliferative Ck5+ cells, progressing through a Ck5+/Ck8+ intermediate cell state. Collectively, these data implicate a tumor initiating cell population defined by a mix of basal (Ck5) and luminal (Nkx3.1) markers that, under the influence of ERG expression, can expand and invade while retaining full luminal differentiation potential. Our results also suggest that widely held assumptions regarding luminal-specific expression of canonical markers such as Nkx3.1 and Probasin may need revision. Citation Format: Weiran Feng, Erik Ladewig, Nazifa Salsabeel, Huiyong Zhao, Young Sun Lee, Anuradha Gopalan, Hanzhi Luo, Wenfei Kang, Ning Fan, Eric Rosiek, Ignas Masilionis, Katia Manova-Todorova, Ronan Chaligné, Elisa de Stanchina, Brett S. Carver, Yu Chen, Dong Gao, Christina S. Leslie, Charles L. Sawyers. ERG-driven luminal prostate cancers emerge from Ck5+/Nkx3.1+ basal cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr PR007.

TRAF3 regulates IL-6 receptor signaling in T cells through the phosphatase PTPN22

Abstract IL-6 is an important signal three cytokine for T helper 17 (Th17) and T follicular helper (Tfh) differentiation. Previous work has suggested a possible role for TRAF3 in the function and/or differentiation of these two subsets, but no direct studies have yet been performed. In this study, we examine the role of TRAF3 in IL-6R signaling in T cells. TRAF3 regulates IL-2R and type I interferon receptor signaling in T cells by altering recruitment of the phosphatases PTPN2 and PTPN22, respectively. B cell TRAF3 curtails IL-6R signaling by recruiting PTPN22, thus we hypothesized that TRAF3 regulates T cell IL-6R signaling through a PTPN22-dependent mechanism. We investigated the role of T cell TRAF3 using a TRAF3-deficient human T cell lymphoma line and a T cells from a T cell-specific TRAF3 knockout mouse (T-Traf3−/−). We found reduced IL-6-induced STAT1 and STAT3 activation in human and mouse T cells. STAT1 and STAT3 activation are both required for full differentiation of mouse Tfh cells, whereas STAT3 is required for and STAT1 antagonizes Th17 differentiation. Interestingly, in vitrodifferentiation of naïve CD4 T cells to Th17 cells was not affected by TRAF3 deficiency. To determine the mechanism by which TRAF3 promotes STAT1 and STAT3 activation in response to IL-6R signaling, we treated WT and Traf3−/−T cells with IL-6 in the presence of PTPN22 inhibitor or vehicle control. We found that PTPN22 inhibition restored activation of STAT3, but did not restore activation of STAT1, suggesting different mechanisms of regulation. Together these data implicate TRAF3 in regulation of IL-6R signaling in a partly PTPN22-dependent manner, lending an additional level of understanding to TRAF3 regulation of Th17 and Tfh differentiation. Supported by NIH R01 AI123107, R01 AI162656, T32 AI007260

Plant growth and early in vitro floral differentiation of vanilla (Vanilla planifolia Jacks. ex Andrews)

Objective: To induce in vitro flowering of vanilla (Vanilla planifolia) with different plant growth regulators (PGRs) using the double-layer technique. Design/methodology/approach: A layer of semi-solid Knudson C (KC) medium, added with 40 g L-1 sucrose, 15% coconut water (CW; v/v) and 7 g L-1 agar, was placed in 100 mL flasks. A liquid layer of the same composition without agar was placed on top. It was supplemented with different doses (mg L-1) of PGRs: 6-benzyladenine (BA) (7), thidiazuron (TDZ) (6), paclobutrazol (PBZ) (0.5) and gibberellic acid (AG3) (2). Plus two controls, C1: no PGRs + no CW; C2: no PGRs + CW. Vanilla shoots of 2 cm in length and with at least one axillary shoot were placed. They were incubated at a temperature of 26±2 °C day and 18 °C darkness, with light intensity of 55 µmol m-2 s-1 during 13 weeks. The number of shoots, leaves and roots was evaluated, as well as the length of shoots and fresh weight. Floral differentiation was evaluated at the tenth week by conventional microtechnique. Results: The number shoots and leaves and shoot length were significantly higher in C2. The number of roots increased with PBZ 0.5 mgL-1. C1 and C2 promoted higher fresh weight. Floral differentiation was observed with AG3 2 and PBZ 0.5 mg L-1 treatments. Limitations on study/implications: Further evaluation of other PGR doses and environmental conditions is required to achieve full floral differentiation of vanilla. Findings/conclusions: CW increased vegetative growth. AG3 and PBZ showed early floral differentiation in Vanilla planifolia, which is the first report of this phenomenon for the species.

Effect of farmland cost on the scale efficiency of agricultural production based on farmland price deviation

The relationship between agricultural production factor prices and production performance has long been a topic of academic discussion. However, current research has paid relatively little attention to the problem of scale efficiency losses in agricultural production caused by deviations in farmland prices. In order to explore the development direction needed to improve China's agricultural competitiveness, we analyzed the deviation mechanism of farmland prices in China and the loss of scale efficiency of agricultural production due to high farmland prices, starting from the perspective of farmland price deviation. Accordingly, based on 1188 micro-farm household survey samples, a threshold regression model was used to provide empirical support for our theoretical analysis in two dimensions: full samples and differentiation of farmland use types. The empirical results show that the problem of high farmland prices prevails throughout China, with the mean farmland price deviation (abbreviated as DEV) in Chinese provinces reaching 1.42, and it is especially high in Shandong (1.88), Henan (1.67), and Heilongjiang (1.59). The deviation is mainly caused by China's special resource endowment and farming culture. At the same time, the current scale efficiency of agricultural production in Chinese provinces is inadequate, with a mean value of only 0.70, and approximately 65% of farmers' actual input size is significantly lower than the optimal input size. The threshold effect test shows that the effect of farmland cost increase on agricultural production scale efficiency exhibits a structural shift from promoting to inhibiting as the degree of farmland price changes from low to high. In particular, in the scenario of overpriced farmland (DEV ≥ 1.16), farmland cost increase inhibits the growth of China's agricultural production scale efficiency. In addition, there is heterogeneity across farmland uses, with grain crops being more likely to be scale-efficient (0.74) than nongrain crops (0.62). The threshold regression results between the grain and nongrain groups further reveal that in the high farmland price scenario, grain crops are more severely inhibited by the rising cost of farmland than nongrain crops. Ultimately, we propose an optimal solution to improve the performance of agricultural production in China in two dimensions: reducing farmland cost and promoting service scale operation.

Volcano–Plutonic Complex of the Tumrok Range (Eastern Kamchatka): An Example of the Ural-Alaskan Type Intrusion and Related Volcanic Series

Zoned plutons, composed of dunites, pyroxenites, and gabbroic rocks, have been referred to as the Ural-Alaskan type complexes (UA-complexes) and occur in numerous paleo-arc settings worldwide. Many of these complexes are source rocks for economic placers of platinum-group metals. Thus, it is important to understand how UA-complexes form and the origin and behavior of platinum-group elements (PGEs). It is widely assumed that the UA-complexes result from differentiation of supra-subduction high-Ca high-Mg sub-alkaline magmas. However, there is a lack of direct evidence for the existence and differentiation of such magmas, mainly because cases of UA-complexes being spatially and temporally linked to co-genetic volcanics are unknown. We studied an UA-complex from the Tumrok range (Eastern Kamchatka) where a dunite-clinopyroxenite-gabbro assemblage is spatially and temporary related to high-Ca volcanics (i.e., picrites and basalts). Based on the mineral and chemical composition of the rocks, mineral chemistry, and composition of melt inclusions hosted within rock-forming minerals, we conclude that the intrusive assemblage and the volcanics are co-genetic and share the same parental magma of ankaramitic composition. Furthermore, the compositions of the plutonic rocks are typical of UA-complexes worldwide. Finally, the rocks studied exhibit a full differentiation sequence from olivine-only liquidus in picrites and dunites to eutectic crystallization of diopside or hornblende, plagioclase, and K-Na feldspar in plagio-wehrlites and gabbroic rocks. All these results make the considered volcano–plutonic complex a promising case for petrological studies and modelling of UA-complex formation.

Translational control in cortical development.

Differentiation of specific neuronal types in the nervous system is worked out through a complex series of gene regulation events. Within the mammalian neocortex, the appropriate expression of key transcription factors allocates neurons to different cortical layers according to an inside-out model and endows them with specific properties. Precise timing is required to ensure the proper sequential appearance of key transcription factors that dictate the identity of neurons within the different cortical layers. Recent evidence suggests that aspects of this time-controlled regulation of gene products rely on post-transcriptional control, and point at micro-RNAs (miRs) and RNA-binding proteins as important players in cortical development. Being able to simultaneously target many different mRNAs, these players may be involved in controlling the global expression of gene products in progenitors and post-mitotic cells, in a gene expression framework where parallel to transcriptional gene regulation, a further level of control is provided to refine and coordinate the appearance of the final protein products. miRs and RNA-binding proteins (RBPs), by delaying protein appearance, may play heterochronic effects that have recently been shown to be relevant for the full differentiation of cortical neurons and for their projection abilities. Such heterochronies may be the base for evolutionary novelties that have enriched the spectrum of cortical cell types within the mammalian clade.

Visualization of Differentiated Cells in 3D and 2D Intestinal Organoid Cultures.

The intestinal epithelium maintains self-renewal and differentiation capacities via coordination of key signaling pathways, including the Wnt, bone morphogenetic protein (BMP), epidermal growth factor (EGF), and Notch signaling pathways. Based on this understanding, a combination of stem cell niche factors, EGF, Noggin, and the Wnt agonist R-spondin was shown to enable the growth of mouse intestinal stem cells and the formation of organoids with indefinite self-renewal and full differentiation capacity. Two small-molecule inhibitors, including a p38 inhibitor and a TGF-beta inhibitor, were added to propagate cultured human intestinal epithelium but at the cost of differentiation capacity. There have been improvements in culture conditions to overcome these issues. Substitution of the EGF and a p38 inhibitor with insulin-like growth factor-1 (IGF-1) and fibroblast growth factor-2 (FGF-2) enabled multilineage differentiation. Monolayer culture with mechanical flow to the apical epithelium promoted the formation of villus-like structures with mature enterocyte gene expression. Here, we summarize our recent technological improvements in human intestinal organoid culture that will deepen the understanding of intestinal homeostasis and diseases.

Liposome-Mediated Gene Transfer in Differentiated HepaRG™ Cells: Expression of Liver Specific Functions and Application to the Cytochrome P450 2D6 Expression

The goal of this study was to establish a procedure for gene delivery mediated by cationic liposomes in quiescent differentiated HepaRG™ human hepatoma cells. We first identified several cationic lipids promoting efficient gene transfer with low toxicity in actively dividing HepG2, HuH7, BC2 and progenitor HepaRG™ human hepatoma cells. The lipophosphoramidate Syn1-based nanovector, which allowed the highest transfection efficiencies of progenitor HepaRG™ cells, was next used to transfect differentiated HepaRG™ cells. Lipofection of these cells using Syn1-based liposome was poorly efficient most likely because the differentiated HepaRG™ cells are highly quiescent. Thus, we engineered the differentiated HepaRG™ Mitogenic medium supplement (ADD1001) that triggered robust proliferation of differentiated cells. Importantly, we characterized the phenotypical changes occurring during proliferation of differentiated HepaRG™ cells and demonstrated that mitogenic stimulation induced a partial and transient decrease in the expression levels of some liver specific functions followed by a fast recovery of the full differentiation status upon removal of the mitogens. Taking advantage of the proliferation of HepaRG™ cells, we defined lipofection conditions using Syn1-based liposomes allowing transient expression of the cytochrome P450 2D6, a phase I enzyme poorly expressed in HepaRG cells, which opens new means for drug metabolism studies in HepaRG™ cells.

IL6 supports long-term expansion of hepatocytes in vitro

Hepatocytes are very difficult to expand in vitro. A few studies have demonstrated that chemical cocktails with growth factors or Wnt ligands can support long-term expansion of hepatocytes via dedifferentiation. However, the culture conditions are complex, and clonal expansion of hepatic progenitors with full differentiation capacity are rarely reported. Here, we discover IL6, combined with EGF and HGF, promotes long-term expansion (>30 passages in ~150 days with theoretical expansion of ~1035 times) of primary mouse hepatocytes in vitro in simple 2D culture, by converting hepatocytes into induced hepatic progenitor cells (iHPCs), which maintain the capacity of differentiation into hepatocytes. IL6 also supports the establishment of single hepatocyte-derived iHPC clones. The summation of the downstream STAT3, ERK and AKT pathways induces a number of transcription factors which support rapid growth. This physiological and simple way may provide ideas for culturing previously difficult-to-culture cell types and support their future applications.

Generation of patient-derived pluripotent stem cell-lines and CRISPR modified isogenic controls with mutations in the Parkinson’s associated GBA gene

The GBA gene encodes the lysosomal enzyme glucocerebrosidase (GCase), responsible for the hydrolysis of glucocerebroside to glucose and ceramide. Heterozygous GBA mutations have been associated with the development of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). We generated two induced pluripotent stem cell (iPSC) lines from PD patients carrying heterozygous GBA W378G or N370S mutations and subsequently produced isogenic control lines using CRISPR/Cas9 genome editing. The patient-derived iPSCs and isogenic control lines maintained full pluripotency, normal karyotypes, and differentiation capacity. All iPSC lines could be differentiated into dopaminergic neurons, thus providing valuable tools for studying PD pathogenesis.

Dynamic gelatin-based hydrogels promote the proliferation and self-renewal of embryonic stem cells in long-term 3D culture

Long-term maintenance of embryonic stem cells (ESCs) in the undifferentiated state is still challenging. Compared with traditional 2D culture methods, 3D culture in biomaterials such as hydrogels is expected to better support the long-term self-renewal of ESCs by emulating the biophysical and biochemical properties of the extracellular matrix (ECM). Although prior studies showed that soft and degradable hydrogels favor the 3D growth of ESCs, few studies have examined the impact of the structural dynamics of the hydrogel matrix on ESC behaviors. Herein, we report a gelatin-based structurally dynamic hydrogel (GelCD hydrogel) that emulates the intrinsic structural dynamics of the ECM. Compared with covalently crosslinked gelatin hydrogels (GelMA hydrogels) with similar stiffness and biodegradability, GelCD hydrogels significantly promote the clonal expansion and viability of encapsulated mouse ESCs (mESCs) independent of MMP-mediated hydrogel degradation. Furthermore, GelCD hydrogels better maintain the pluripotency of encapsulated mESCs than do traditional 2D culture methods that use MEF feeder cells or medium supplementation with GSK3β and MEK 1/2 inhibitors (2i). When cultured in GelCD hydrogels for an extended period (over 2 months) with cell passaging every 7 days, mESCs preserve their normal morphology and maintain their pluripotency and full differentiation capability. Our findings highlight the critical role of the structural dynamics of the hydrogel matrix in accommodating the volume expansion that occurs during clonal ESC growth, and we believe that our dynamic hydrogels represent a valuable tool to support the long-term 3D culture of ESCs.

Acquired semi-squamatization during chemotherapy suggests differentiation as a therapeutic strategy for bladder cancer.

Cisplatin-based chemotherapy remains the primary treatment for unresectable and metastatic muscle-invasive bladder cancers (MIBCs). However, tumors frequently develop chemoresistance. Here, we established a primary and orthotopic MIBC mouse model with gene-edited organoids to recapitulate the full course of chemotherapy in patients. We found that partial squamous differentiation, called semi-squamatization, is associated with acquired chemoresistance in both mice and human MIBCs. Multi-omics analyses showed that cathepsin H (CTSH) is correlated with chemoresistance and semi-squamatization. Cathepsin inhibition by E64 treatment induces full squamous differentiation and pyroptosis, and thus specifically restrains chemoresistant MIBCs. Mechanistically, E64 treatment activates the tumor necrosis factor pathway, which is required for the terminal differentiation and pyroptosis of chemoresistant MIBC cells. Our study revealed that semi-squamatization is a type of lineage plasticity associated with chemoresistance, suggesting that differentiation via targeting of CTSH is a potential therapeutic strategy for the treatment of chemoresistant MIBCs.

Eisenia arborea (Areschoung) domestication and mariculture development on the Pacific coast of Baja California, México

Eisenia arborea (sensu Ecklonia arborea) is the kelp with the greatest latitudinal distribution on the Northeastern Pacific Coast. It is harvested in Mexico in small amounts for abalone farm fodder and occasionally exported to Asia for human consumption. Because the high-energy environment where it naturally grows limits its sustainable harvest, we explored the domestication and cultivation of this kelp on the west coast of Baja California, Mexico. The life history of E. arborea was completed in the laboratory following traditional methods for kelp cultivation. Gametophytes became reproductive approximately 50 days after spore release and sporophytes were visible to the naked eye after 80 days. When sporophytes reached 2–3 cm, they were transplanted to two sites on long-line systems: in an open bay (Todos Santos Bay) and in a shallow coastal lagoon (San Quintín Bay). At both sites, full differentiation occurred 6 to 8 months after deployment, with fertile blades occurring within a year. Once individuals reached maximum size, blades were harvested by pruning. Monthly, regrowth of pruned blades and new blade addition were measured. Pruned individuals reached the same weight as controls in three months. On the basis of these results, a seasonal seeding program was implemented at a third site where the presence of natural Eisenia is rare due to the lack of hard substrate but was hypothesized to provide appropriate conditions for kelp cultivation (Santa María Bay). Cultures were deployed at three different depths and growth was correlated with season, depth, and temperature. Only cultures seeded in winter developed successfully at this site. This study shows that the culture of E. arborea in a variety of ocean conditions is possible; however, site selection and seasonality are important considerations. Because E. arborea is perennial, it can be harvested periodically during the year thereby increasing yield, and because it is the warmest tolerant kelp species, it is an ideal target for ocean farming and commercial cultivation, considering climate change.