Glycogen synthase kinase 3 (GSK3)-inhibitor SB216763 promotes the conversion of human umbilical cord mesenchymal stem cells into neural precursors in adherent culture

The ability to generate neural progenitor cells from human umbilical cord mesenchymal stem cells (hUC-MSCs) has provided an option to treat neurodegenerative diseases. To establish a method for this purpose, we characterized the early neural markers of hUC-MSCs-derived cells under different conditions. We found that neither the elimination of signals for alternative fate nor N2 supplement was sufficient to differentiate hUC-MSCs into neural precursor cells, but the GSK3 inhibitor SB216763 could promote an efficient neural commitment of hUC-MSCs. The results indicated that Wnt/β-catenin might play an important role during the early neural differentiation of hUC-MSCs. Here, we report a method for hUC-MSCs to commit efficiently into a neural fate within a short period of time. This protocol provides an efficient method for hUC-MSCs-based neural regeneration.     

A novel GSK3 inhibitor that promotes self-renewal in mouse embryonic stem cells

ABSTRACT

Small molecules that regulate cell stemness have the potential to make a major contribution to regenerative medicine. In the course of screening for small molecules that affect stemness in mouse embryonic stem cells (mESCs), we discovered that NPD13432, an aurone derivative, promoted self-renewal of mESCs. Normally, mESCs start to differentiate upon withdrawal of 2i/LIF. However, cells treated with the compound continued to express endogenous Nanog, a pluripotency marker protein essential for sustaining the undifferentiated state, even in the absence of 2i/LIF. Biochemical characterization revealed that NPD13432 inhibited GSK3α and GSK3β with IC₅₀ values of 92 nM and 310 nM, respectively, suggesting that the compound promotes self-renewal in mESCs by inhibiting GSK3. The chemical structure of the compound is unique among known molecules with this activity, providing an opportunity to develop new inhibitors of GSK3, as well as chemical tools for investigating cell stemness.     

P24, a glycogen synthase kinase 3 (GSK 3) inhibitor.

A heat resistant glycogen synthase kinase 3 (GSK 3) binding protein, p24, that inhibits its kinase activity at a low magnesium concentration (in a way similar to that of lithium) was found in microtubules from adult rat brains. This protein associates with GSK 3 in microtubules and corresponds to one previously described in the literature as p25, although it has a relative molecular weight of 23472. p24 is a poor substrate for GSK 3 but it could be phosphorylated by other protein kinases such as cAMP dependent protein kinase and cdk 5. Since p24 could form complexes with GSK 3, it may not only regulate GSK 3 activity but also it might act as an anchoring protein for the kinase.     

Glycogen synthase kinase-3 (Gsk-3) plays a fundamental role in maintaining DNA methylation at imprinted loci in mouse embryonic stem cells

Glycogen synthase kinase-3 (Gsk-3) is a key regulator of multiple signal transduction pathways. Recently we described a novel role for Gsk-3 in the regulation of DNA methylation at imprinted loci in mouse embryonic stem cells (ESCs), suggesting that epigenetic changes regulated by Gsk-3 are likely an unrecognized facet of Gsk-3 signaling. Here we extend our initial observation to the entire mouse genome by enriching for methylated DNA with the MethylMiner kit and performing next-generation sequencing (MBD-Seq) in wild-type and Gsk-3α^−/−;Gsk-3β^−/− ESCs. Consistent with our previous data, we found that 77% of known imprinted loci have reduced DNA methylation in Gsk-3-deficient ESCs. More specifically, we unambiguously identified changes in DNA methylation within regions that have been confirmed to function as imprinting control regions. In many cases, the reduced DNA methylation at imprinted loci in Gsk-3α^−/−;Gsk-3β^−/− ESCs was accompanied by changes in gene expression as well. Furthermore, many of the Gsk-3–dependent, differentially methylated regions (DMRs) are identical to the DMRs recently identified in uniparental ESCs. Our data demonstrate the importance of Gsk-3 activity in the maintenance of DNA methylation at a majority of the imprinted loci in ESCs and emphasize the importance of Gsk-3–mediated signal transduction in the epigenome.     

Delayed cardioprotection afforded by the glycogen synthase kinase 3 inhibitor SB-216763 occurs via a KATP- and MPTP-dependent mechanism at reperfusion

Previous studies in our laboratory suggest that an acute inhibition of glycogen synthase kinase 3 (GSK3) by SB-216763 (SB21) is cardioprotective when administered just before reperfusion. However, it is unknown whether the GSK inhibitor SB21 administered 24 h before ischemia is cardioprotective and whether the mechanism involves ATP-sensitive potassium (K(ATP)) channels and the mitochondrial permeability transition pore (MPTP). Male Sprague-Dawley rats were administered the GSK inhibitor SB21 (0.6 mg/kg) or vehicle 24 h before ischemia. Subsequently, the rats were acutely anesthetized with Inactin and underwent 30 min of ischemia and 2 h of reperfusion followed by infarct size determination. Subsets of rats received either the sarcolemmal K(ATP) channel blocker HMR-1098 (6 mg/kg), the mitochondrial K(ATP) channel blocker 5-hydroxydecanoic acid (5-HD; 10 mg/kg), or the MPTP opener atractyloside (5 mg/kg) either 5 min before SB21 administration or 5 min before reperfusion 24 h later. The infarct size was reduced in SB21 compared with vehicle (44 +/- 2% vs. 61 +/- 2%, respectively; P < 0.01). 5-HD administered either before SB21 treatment or 5 min before reperfusion the following day abrogated SB21-induced protection (54 +/- 4% and 61 +/- 2%, respectively). HMR-1098 did not affect the SB21-induced infarct size reduction when administered before the SB21 treatment (43 +/- 1%); however, HMR-1098 partially abrogated the SB21-induced infarct size reduction when administered just before reperfusion 24 h later (52 +/- 1%). The MPTP opening either before SB21 administration or 5 min before reperfusion abrogated the infarct size reduction produced by SB21 (61 +/- 2% and 62 +/- 2%, respectively). Hence, GSK inhibition reduces infarct size when given 24 h before the administration via the opening K(ATP) channels and MPTP closure.     

Discovering pluripotency: 30 years of mouse embryonic stem cells

Embryonic stem (ES) cells are pluripotent cells isolated from an early embryo and grown as a cell line in tissue culture. Their discovery came from the conjunction of studies in human pathology, mouse genetics, early mouse embryo development, cell surface immunology and tissue culture. ES cells provided a crucial tool for manipulating mouse embryos to study mouse genetics, development and physiology. They have not only revolutionized experimental mammalian genetics but, with the advent of equivalent human ES cells, have now opened new vistas for regenerative medicine.     

TRPC3 is required for the survival,pluripotency and neural differentiation of mouse embryonic stem cells(mESCs)

Transient receptor potential canonical subfamily member 3(TRPC3) is known to be important for neural development and the formation of neuronal networks. Here, we investigated the role of TRPC3 in undifferentiated mouse embryonic stem cells(mESCs) and during the differentiation of mESCs into neurons. CRISPR/Cas9-mediated knockout(KO) of TRPC3 induced apoptosis and the disruption of mitochondrial membrane potential both in undifferentiated mESCs and in those undergoing neural differentiation. In addition, TRPC3 KO impaired the pluripotency of mESCs. TRPC3 KO also dramatically repressed the neural differentiation of mESCs by inhibiting the expression of markers for neural progenitors, neurons, astrocytes and oligodendrocytes.Taken together, our new data demonstrate an important function of TRPC3 with regards to the survival, pluripotency and neural differentiation of mESCs.     

Glycogen synthase kinase (GSK) 3beta directly phosphorylates Serine 212 in the regulatory loop and inhibits microtubule affinity-regulating kinase (MARK) 2

MARK/Par-1, a kinase family with diverse functions particularly in inducing cell polarity, can phosphorylate microtubule-associated proteins in their repeat domain and cause their detachment from microtubules, and thereby microtubule destabilization. Because of its role in abnormal phosphorylation of the Tau protein in Alzheimer disease, we searched for regulatory kinases. MARK family kinases can be activated by phosphorylation of a conserved threonine (Thr-208 in MARK2), and inactivated by phosphorylation of a serine (Ser-212), both in the activation loop of the catalytic domain. Activation is achieved by the kinases MARKK/TAO1 or LKB1, although the inactivating kinase was unknown. We show here that GSK3beta serves the role of the inhibitory kinase. Because GSK3beta can also phosphorylate Tau at sites outside the repeat domain, the activation of GSK3beta, and concomitant inactivation of MARK can shift the pattern of pathological phosphorylation of Tau protein in Alzheimer disease.     

Maintenance of pluripotency in mouse embryonic stem cells cultivated in stirred microcarrier cultures

The development of efficient and reproducible culture systems for embryonic stem (ES) cells is an essential pre‐requisite for regenerative medicine. Culture scale‐up ensuring maintenance of cell pluripotency is a central issue, because large amounts of pluripotent cells must be generated to warrant that differentiated cells deriving thereof are transplanted in great amounts and survive the procedure. This study aimed to develop a robust scalable cell expansion system, using a murine embryonic stem cell line that is feeder‐dependent and adapted to serum‐free medium, thus representing a more realistic model for human ES cells. We showed that high concentrations of murine ES cells can be obtained in stirred microcarrier‐based spinner cultures, with a 10‐fold concentration of cells per volume of medium and a 5‐fold greater cell concentration per surface area, as compared to static cultures. No differences in terms of pluripotency and differentiation capability were observed between cells grown in traditional static systems and cells that were replated onto the traditional system after being expanded on microcarriers in the stirred system. This was verified by morphological analyses, quantification of cells expressing important pluripotency markers (Oct‐4, SSEA‐1, and SOX2), karyotype profile, and the ability to form embryoid bodies with similar sizes, and maintaining their intrinsic ability to differentiate into all three germ layers. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Nucleostemin maintains self-renewal of embryonic stem cells and promotes reprogramming of somatic cells to pluripotency

Nucleostemin (NS) is a nucleolar GTP-binding protein that was first identified in neural stem cells, the functions of which remain poorly understood. Here, we report that NS is required for mouse embryogenesis to reach blastulation, maintenance of embryonic stem cell (ESC) self-renewal, and mammary epithelial cell (MEC) reprogramming to induced pluripotent stem (iPS) cells. Ectopic NS also cooperates with OCT4 and SOX2 to reprogram MECs and mouse embryonic fibroblasts to iPS cells. NS promotes ESC self-renewal by sustaining rapid transit through the G1 phase of the cell cycle. Depletion of NS in ESCs retards transit through G1 and induces gene expression changes and morphological differentiation through a mechanism that involves the MEK/ERK protein kinases and that is active only during a protracted G1. Suppression of cell cycle inhibitors mitigates these effects. Our results implicate NS in the maintenance of ESC self-renewal, demonstrate the importance of rapid transit through G1 for this process, and expand the known classes of reprogramming factors.