Members of the MAPK superfamily are known as key regulators of ciliogenesis. Long flagellar (LF) 4, a MAPK‐related kinase in Chlamydomonas, is the first kinase that was implicated in ciliary assembly and length. However, little is known about its cellular properties, regulation, and molecular functions. LF4 is localized both in the flagella and cell body with enrichment at the 2 basal bodies, shown by super‐resolution microscopy. LF4 is constitutively phosphorylated at T159 at the kinase activation loop and remains at the basal bodies during flagellar assembly. Gene mutations that affect the kinase activity or T159 phosphorylation alter the localization of LF4 at the basal bodies, and the mutants fail to rescue lf4‐3, a null mutant. LF4 does not affect the velocities of intraflagellar transport (IFT). However, LF4 null mutation induces accumulation of IFT proteins in the flagellum and reduces the phosphorylation of the kinesin‐II subunit FLA8/KIF3B, indicating that LF4 negatively regulates IFT entry. Furthermore, LF2, a cell cycle–related kinase, and LF3, a novel protein, are required for LF4 phosphorylation. Our study demonstrates that LF4 is likely a constitutively active kinase that is regulated by LF2 and regulates IFT entry at the basal bodies to control flagellar assembly and length.—Wang, Y., Ren, Y., Pan, J. Regulation of flagellar assembly and length in Chlamydomonas by LF4, a MAPK‐related kinase. FASEB J. 33, 6431–6441 (2019). http://www.fasebj.org

Preparation of mouse embryonic fibroblast (MEF) feeder cells to maintain pluripotent stem cells (PSCs) is time consuming and involved in animal issues. Here, we demonstrated a novel method to prepare feeder cells with high efficiency, timesaving, and low costs. MEFs in 3 × 104 cell/cm2 were fixed by methanol for 5 min and air drying for 5 min. Thereafter, the methanol fixed MEF cells (MT-MEF) were able to be used directly to culture PSCs or stored at room temperature for the future usage. PSCs cultured on MT-MEF could be continuously expanded for over 40 passages with the naïve pluripotency. MT-MEFs could also be used to maintain human and pig iPSCs. Moreover, methanol fixed MEFs’ culture dish was able to be reused for at least 4 times, and to be applied for antibiotic resistant screening assay to establishing stable transfected PSC lines. Alternatively, the immortalized cell lines, for instance NIH3T3 cells, could also be fixed by methanol and used as feeder cells to maintain PSCs. Thus, this novel means of methanol fixed feeder cells can completely replace the mitomycin C and gamma radiation treated MEF feeder cells, and be used to maintain PSCs derived from mouse as well as other animal species.

The estrogen-related receptor b (ESRRB) is an orphan nuclear receptor and targets many genes involved in self-renewal and pluripotency. In mouse ES cells, overexpression of ESRRB can maintain LIF-independent self-renewal in the absence of Nanog. However, the fundamental features of porcine ESRRB remain elusive. In this study, we revealed the expression profiles of ESRRB in both porcine pluripotent stem cells and early stage embryos and dissected the functional domains of ESRRB protein to prove that ESRRB is a key transcription factor that enhanced porcine pluripotent gene activation. Addition of ESRRB into the cocktail of core pluripotent factors Oct4, Sox2, Klf4, and c-Myc (OSKM + E) could significantly enhance the reprograming efficiency and the formation of alkaline phosphatase positive colonies. Conversely, knockdown of ESRRB in piPSCs significantly reduced the expression level of pluripotent genes, minimized the alkaline phosphatase activity, and initiated the porcine induced pluripotent stem cell differentiation. Therefore, porcine ESRRB is a crucial transcription factor to improve the self-renewal of piPSCs.

Initiation of meiosis is the most difficult aspect of inducing competent oocytes differentiation from human stem cells in vitro. Human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) were cultured with follicle fluid, cytokines and small molecule to induced oocyte-like cells (OLCs) formation through a three-step induction procedure. Expression of surface markers and differentiation potential of germ cells were analyzed in vitro by flow cytometry, gene expression, immunocytochemistry, western blotting and RNA Sequencing. To induce the differentiation of hiPSCs into OLCs, cells were firstly cultured with a primordial germ cell medium for 10 days. The cells exhibited similar morphological features to primordial germ cells (PGCs), high expressing of germ cell markers and primordial follicle development associated genes. The induced PGCs were then cultured with the primordial follicle-like cell medium for 5 days to form the induced follicle-like structures (iFLs), which retained both primordial oocytes-like cells and granulosa-like cells. In the third step, the detached iFLs were harvested and transferred to the OLC-medium for additional 10 days. The cultured cells developed cumulus-oocyte-complexes (COCs) structures and OLCs with different sizes (50–150 μm diameter) and a zona pellucida. The in vitro matured OLCs had polar bodies and were arrested at metaphase II (MII) stage. Some OLCs were self-activated and spontaneously developed into multiple-cell structures similar to preimplantation embryos, indicating that OLCs were parthenogenetically activated though in vitro fertilization potential of OLCs are yet to be proved. in vitro maturation of OLCs derived from hiPSCs provides a new means to study human germ cell formation and oogenesis. Graphical Abstract