The autonomous cell fate specification of basal cell lineage: the initial round of cell fate specification occurs at the two‐celled proembryo stage

In angiosperms, the first zygotic division usually gives rise to two daughter cells with distinct morphologies and developmental fates, which is critical for embryo pattern formation; however, it is still unclear when and how these distinct cell fates are specified, and whether the cell specification is related to cytoplasmic localization or polarity. Here, we demonstrated that when isolated from both maternal tissues and the apical cell, a single basal cell could only develop into a typical suspensor, but never into an embryo in vitro. Morphological, cytological and gene expression analyses confirmed that the resulting suspensor in vitro is highly similar to its undisturbed in vivo counterpart. We also demonstrated that the isolated apical cell could develop into a small globular embryo, both in vivo and in vitro, after artificial dysfunction of the basal cell; however, these growing apical cell lineages could never generate a new suspensor. These findings suggest that the initial round of cell fate specification occurs at the two‐celled proembryo stage, and that the basal cell lineage is autonomously specified towards the suspensor, implying a polar distribution of cytoplasmic contents in the zygote. The cell fate transition of the basal cell lineage to the embryo in vivo is actually a conditional cell specification process, depending on the developmental signals from both the apical cell lineage and maternal tissues connected to the basal cell lineage.     

Snai1 regulates cell lineage allocation and stem cell maintenance in the mouse intestinal epithelium

Snail family members regulate epithelial‐to‐mesenchymal transition (EMT) during invasion of intestinal tumours, but their role in normal intestinal homeostasis is unknown. Studies in breast and skin epithelia indicate that Snail proteins promote an undifferentiated state. Here, we demonstrate that conditional knockout of Snai1 in the intestinal epithelium results in apoptotic loss of crypt base columnar stem cells and bias towards differentiation of secretory lineages. In vitro organoid cultures derived from Snai1 conditional knockout mice also undergo apoptosis when Snai1 is deleted. Conversely, ectopic expression of Snai1 in the intestinal epithelium in vivo results in the expansion of the crypt base columnar cell pool and a decrease in secretory enteroendocrine and Paneth cells. Following conditional deletion of Snai1, the intestinal epithelium fails to produce a proliferative response following radiation‐induced damage indicating a fundamental requirement for Snai1 in epithelial regeneration. These results demonstrate that Snai1 is required for regulation of lineage choice, maintenance of CBC stem cells and regeneration of the intestinal epithelium following damage.     

A Lox/CHOP‐10 crosstalk governs osteogenic and adipogenic cell fate by MSCs

Accelerated marrow adipogenesis has been associated with ageing and osteoporosis and is thought to be because of an imbalance between adipogenic and osteogenic differentiation of mesenchymal stem cell (MSCs). We have previously found that lysyl oxidase (Lox) inhibition disrupts BMP4‐induced adipocytic lineage commitment and differentiation of MSCs. In this study, we found that lox inhibition dramatically up‐regulates BMP4‐induced expression of CCAAT/enhancer binding protein (C/EBP) homologous protein 10 (CHOP‐10), which then promotes BMP4‐induced osteogenesis of MSCs both in vitro and in vivo. Specifically, Lox inhibition or CHOP‐10 up‐regulation activated Wnt/β‐catenin signalling to enhance BMP4‐induced osteogenesis, with pro‐adipogenic p38 MAPK and Smad signalling suppressed. Together, we demonstrate that Lox/CHOP‐10 crosstalk regulates BMP4‐induced osteogenic and adipogenic fate determination of MSCs, presenting a promising therapeutic target for osteoporosis and other bone diseases.     

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.     

Effect of BMP4 preceded by retinoic acid and co‐culturing ovarian somatic cells on differentiation of mouse embryonic stem cells into oocyte‐like cells

Bone morphogenetic protein 4 (BMP4) and retinoic acid (RA) signaling are the key regulators for germ cell and meiosis induction, respectively. Gonadal tissue also provides an appropriate microenvironment for oocyte differentiation in vivo. The current study aimed to determine whether mimicking in vivo niche is more efficient for oocyte differentiation from embryonic stem (ES) cells. Here, differentiation of mouse ES cells toward oocyte‐like cells using embryoid body (EB) and monolayer protocols was induced in the presence (+BMP4) or absence (‐BMP4) of BMP4. On day 5, each group was co‐cultured with ovarian somatic cells in the presence or absence of RA (+RA or –RA) for an additional 14 days. Our results showed a significant increase in expression of meiotic markers in the +BMP4 condition in EB differentiation protocol. Further differentiation with ovarian somatic cells led to a subpopulation of oocyte‐like cell formation. Compared to the controls, the +RA condition resulted in a significant elevation of the meiotic gene expression in contrast to Oct4 that significantly decreased in both protocols. In the cells pre‐treated with BMP4 and then exposed to RA in the monolayer differentiation protocol, the gene expression levels of germ cell, Mvh, and maturation markers, Cx37, Zp2, and Gdf9, were also upregulated significantly. Therefore, it can be concluded that +BMP4 and +RA along with ovarian somatic cell co‐culture improved the rate of in vitro oocyte differentiation.     

Notch signaling regulates gastric antral LGR5 stem cell function

The major signaling pathways regulating gastric stem cells are unknown. Here we report that Notch signaling is essential for homeostasis of LGR5⁺ antral stem cells. Pathway inhibition reduced proliferation of gastric stem and progenitor cells, while activation increased proliferation. Notch dysregulation also altered differentiation, with inhibition inducing mucous and endocrine cell differentiation while activation reduced differentiation. Analysis of gastric organoids demonstrated that Notch signaling was intrinsic to the epithelium and regulated growth. Furthermore, in vivo Notch manipulation affected the efficiency of organoid initiation from glands and single Lgr5‐GFP stem cells, suggesting regulation of stem cell function. Strikingly, constitutive Notch activation in LGR5⁺ stem cells induced tissue expansion via antral gland fission. Lineage tracing using a multi‐colored reporter demonstrated that Notch‐activated stem cells rapidly generate monoclonal glands, suggesting a competitive advantage over unmanipulated stem cells. Notch activation was associated with increased mTOR signaling, and mTORC1 inhibition normalized NICD‐induced increases in proliferation and gland fission. Chronic Notch activation induced undifferentiated, hyper‐proliferative polyps, suggesting that aberrant activation of Notch in gastric stem cells may contribute to gastric tumorigenesis.     

Indirect osteoblast differentiation by liposomal clodronate

Bisphosphonates impair function of osteoclasts and prevent bone resorption, the mechanism of which has been studied extensively. However, the possible effects of bisphosphonates on chondroblast differentiation and calcium deposition by osteoblasts have only been demonstrated recently. Moreover, cells from monocytic lineage are capable of stimulating osteoblast proliferation. Hence, susceptibility of osteoblasts to various factors requires further investigation. A primary culture of bone marrow‐derived stromal cells was treated with liposomal clodronate (0.1, 0.5, or 1.0 mg/ml) or conditioned medium from liposomal clodronate. Liposomal clodronate (0.25 mg) was injected into mouse femur for in vivo experiments. The effects of liposomal clodronate were examined by alkaline phosphatase staining and/or activity assay, and real‐time RT‐PCR was used for studying the effect on osteogenic gene expression. Administration of liposomal clodronate to bone marrow‐derived mesenchymal stromal cell culture enhanced alkaline phosphatase activity and mRNA levels of Runx2 and Dlx5. In addition, conditioned medium from liposomal clodronate also stimulated osteogenic characteristics similar to those of observed in vitro, and the number of exosomes in the conditioned medium was highest when pre‐treated with liposomal clodronate. Western blot analysis revealed the presence of RANK proteins in exosomes collected from conditioned medium of liposomal clodronate. Identical observations were obtained in vivo, as liposomal clodronate‐injected mouse femur showed increased alkaline phosphatase activity and Runx2 and Dlx5 mRNA expressions, even though the numbers of monocytes and macrophages were reduced. In conclusion, osteoblast differentiation was promoted via soluble RANK‐containing exosomes in response to clodronates.     

Diallyl disulfide down‐regulates calreticulin and promotes C/EBPα expression in differentiation of human leukaemia cells

Diallyl disulfide (DADS), the main active component of the cancer fighting allyl sulfides found in garlic, has shown potential as a therapeutic agent in various cancers. Previous studies showed DADS induction of HL‐60 cell differentiation involves down‐regulation of calreticulin (CRT). Here, we investigated the mechanism of DADS‐induced differentiation of human leukaemia cells and the potential involvement of CRT and CCAAT enhancer binding protein‐α (C/EBPα). We explored the expression of CRT and C/EBPα in clinical samples (20 healthy people and 19 acute myeloid leukaemia patients) and found that CRT and C/EBPα expressions were inversely correlated. DADS induction of differentiation of HL‐60 cells resulted in down‐regulated CRT expression and elevated C/EBPα expression. In severe combined immunodeficiency mice injected with HL‐60 cells, DADS inhibited the growth of tumour tissue and decreased CRT levels and increased C/EBPα in vivo. We also found that DADS‐mediated down‐regulation of CRT and up‐regulation of C/EBPα involved enhancement of reactive oxidative species. RNA immunoprecipitation revealed that CRT bound C/EBPα mRNA, indicating its regulation of C/EBPα mRNA degradation by binding the UG‐rich element in the 3′ untranslated region of C/EBPα. In conclusion, the present study demonstrates the C/EBPα expression was correlated with CRT expression in vitro and in vivo and the molecular mechanism of DADS‐induced leukaemic cell differentiation.