Self-renewal and differentiation of mouse embryonic stem cells as measured by Oct4 expression: the role of the cAMP/PKA pathway.

In this study we examined the role of the cAMP/protein kinase A (PKA) pathway in affecting IOUD2 ES cell self-renewal and differentiation, Oct4 expression, and cell proliferation. Forskolin, the adenylate cyclase agonist, alone had no effect on ES cell self-renewal. However, when cells were treated with the differentiation-inducing agent retinoic acid, forskolin significantly promoted ES cell self-renewal. Effectively, forskolin rescued cells from a pathway of differentiation. Culturing ES cells in the presence of the phosphodiesterase inhibitor IBMX had no effect on ES cell self-renewal but did increase cell proliferation. In the presence of 100 muM IBMX without LIF, 10 muM forskolin significantly increased ES cell self-renewal. The cell permeable cAMP analog 8-Br-cAMP (1 and 5 mM) promoted ES cell differentiation in the presence of LIF, while in the absence of LIF, it promoted ES cell self-renewal. The effect of the PKA specific inhibitors H89 and KT5720 on Oct4 expression was, again, LIF-dependent. In the presence of LIF, these inhibitors decreased Oct4 expression, while they increased Oct4 expression in the absence of LIF. In general, ES cells maintained on a self-renewal pathway through the presence of LIF show little effect from altered cAMP signaling except at higher levels. However, in strict contrast, when ES cell are on a differentiation pathway through exposure to retinoic acid or the removal of LIF, altering cAMP levels can rescue the self-renewal process promoting Oct4 expression. This study clearly shows that the cAMP/PKA pathway plays a role in ES cell self-renewal pathways.

Self-renewal and differentiation of mouse embryonic stem cells as measured by Oct 4 gene expression: effects of lif, serum-free medium, retinoic acid, and dbcAMP.

In this study we examined the interplay between serum, leukemia inhibitory factor (LIF), retinoic acid, and dibutyrl cyclic adenosine monophosphate (dbcAMP) in affecting IOUD2 embryonic stem cell self-renewal and differentiation as assessed by Oct 4 expression, and cell proliferation as measured by total cell protein. Removal of LIF, reduced levels of fetal calf serum (FCS), and addition of retinoic acid all induced embryonic stem cell differentiation as measured by reduced Oct 4 expression. Lower levels of retinoic acid (0.1-10 nM) promoted the formation of epithelial-like cells, whereas higher levels (100-10,000 nM) favored differentiation into fibroblastic-like cells. The effects of dbcAMP varied with the presence or absence of FCS and LIF and the concentration of dbcAMP. In FCS-containing media, a low level of dbcAMP (100 microM) increased self-renewal in the absence of LIF, but it had no effect in its presence. In contrast, at higher concentrations (1,000 microM dbcAMP), regardless of LIF, differentiation was promoted. A similar effect of dbcAMP was seen in the presence of retinoic acid. In media without FCS but with serum replacement supplements, there was no effect of dbcAMP. This study shows that the Oct 4 expression system of IOUD2 cells provides a novel, simple method for quantifying cellular differentiation.

Case report. Pediatric carpal fracture dislocation.

Transcarpal fractures in children are rare in the orthopaedic literature. This is a case report of a 10-year-old boy who sustained fractures across the distal radius, scaphoid, lunate, and triquetrum with gross displacement. Treatment consisted of open reduction with internal fixation of the fractures and ligamentous repair through a combined dorsal and palmar approach. The injury healed with good wrist function but abnormal carpal development. This unusual pattern of injury is described so that it may be more readily appreciated in the future.

Phospholipase C and protein kinase C involvement in mouse embryonic stem-cell proliferation and apoptosis.

Activation of the phosphatidylinositol (PtdIns) signal transduction system involves stimulation of phospholipase C (PLC) by hormones and other agonists to produce two second messengers, the inositol phosphate, Ins(1,4,5)P3 which releases calcium from intracellular stores, and diacylglycerol which activates protein kinase C (PKC). This study, using activators or inhibitors of PLC and PKC and a calcium ionophore, examined the role of the PtdIns system in mouse embryonic stem (ES) cells. The PLC inhibitor, U-73122, inhibited ES-cell proliferation and also inhibited PLC activation as evidenced by a decrease in inositol phosphate formation in response to fetal calf serum stimulation. The two PKC activators, the diacylglycerol analogue 1,2, dioctanoyl-sn-glycerol (DOG) and the phorbol ester 12-O-tetra-decanoyl phorbol 13-acetate (TPA), increased cell proliferation in a dose-dependent manner, as did the calcium ionophore, ionomycin. However, co-stimulation with either ionomycin and DOG or ionomycin and TPA resulted in a reduced number of cells. The PKC inhibitor, bisindolylmaleimide II (Bis II), significantly decreased the number of ES cells, mainly due to increased apoptosis. The possible feedback effect of PKC on PLC was examined by preincubating ES cells with either the PKC inhibitor Bis II or the activator TPA before stimulation of inositol phosphate production with fetal calf serum; preincubation with Bis II increased inositol phosphate formation whereas preincubation with TPA decreased inositol formation. These results indicate that the PtdIns system is involved in the control of ES-cell proliferation and apoptosis.