Reversible protein phosphorylation is an essential component in the transduction of signals associated with normal and cancer cell proliferation as well as cell cycle arrest and exit into myogenic differentiation. Our research uses cell biology approaches on primary and established mammalian cultured cells and adult stem cells isolated from skeletal muscle to examine the role of protein post-translational modifications such as phosphorylation in the signaling pathways regulating mammalian cell proliferation, and the multi-lineage differentiation potential of adult stem cells.
Microneedle microinjection and real time single cell analysis.
Our lab specializes in microneedle microinjection with over 38 years of experience using this approach. The technique offers a unique opportunity to introduce a wide range of biochemically active materials into cells including siRNAs, miRNAs, RNA, linear and circular plasmid DNA, cosmids, BACs and a wide variety of purified proteins, kinases, phosphatases, regulatory and structural proteins. Microinjection enables single cell live analysis with precise temporal acuity using both fluorescent and non fluorescent components as well as real time and time lapse investigation. Effects in injected cells can be directly compared to adjacent cells uninjected cells or cells injected with control minimizing the need for statistical compensation. Alternatively, cells analysis can be scaled up by microinjection of several hundred cells to measure biochemical events combining metabolic labeling and microinjection to follow changes in protein phosphorylation and expression in real time. Overall, the methodology is highly tuned to the analysis of temporal and structural events in living cells particularly those involved in initiation and control of specific cellular processes linked to cell cycle progression and cellular transformation.
Muscle Derived Stem Cells, MDSC
Our ongoing major research theme involves the isolation and characterization of a non-adherent population of skeletal muscle-derived stem cells, MDSC, capable of multipotent differentiation particularly into spontaneously beating cardiac muscle cells, neuronal lineages and pancreatic alpha and beta cells.
Cell Therapy Potential of MDSC
In collaboration with IGF teams, we are analyzing the in vivo multi-lineage differentiation and physiological repair potential of MDSC using mouse models of targeted diseases and lineage-specific tracking of MDSC differentiation in particular towards cardiac and beta-pancreatic differentiation.
Cardiac Rhythm disorders.
Beating myocytes differentiated from MDSC in vitro are shown to be fully functional pacemaker cells such as those in the sino-atrial node (SAN) of the heart and systemic transplantation experiments, in mutant mice, revealed that multipotent MDSC engraft into the SAN of severely bradycardic mice and improved heart rhythm thus proving a very promising repair and regeneration potential.
Type I Diabetes.
We are also investigating the capacity of MDSC to restore normal glycemia in type 1 diabetic mice. We have recently shown the in vitro differentiation of MDSC into lineages that show expression of several markers consistent with differentiation into insulin expressing beta pancreatic precursor cells. Our transplantation studies further showed that MDSC can differentiate into beta islet-like cells in vivo by engraftment and differentiation into insulin-expressing cells in pancreatic islets of streptozotocin-treated mice.
MDSC show the capacity to differentiate into several other lineages that we are currently investigating particularly neuronal, vascular and hepatic.
Isolation and characterization of MDSC from human muscle.
The isolation of MDSC from human skeletal muscle (hMDSC) is an ongoing theme in our group paying particular attention to the multipotent potential of hMDSC. Our current isolation protocols from mice, rat and microcebe demonstrate that a broad range of stem cell types can be isolated including myogenic, neuronal, pancreatic, cardiac and vascular. This is coherent with the inherent plasticity of stem cells and diversity of cell types present in the initial isolates which are derived from the entire leg muscle. When moving to human samples, while the quantity of material derived (2 g / animal) will be similar, the diversity of stem cell types in human biopsies is likely to be more restricted. We are currently examining the diversity of cell lineages obtained from human cells in vitro and investigating the capacity of hMDSC to repair type 1 diabetes in streptozotocin treated nude mice.
Teratogenic potential and cancer stem cells.
Importantly for the future therapeutic use of MDSC are the observations that they can be directly transplanted without prior induction into any differentiation lineage, and their non tumorigenic behavior up to several months after subcutaneous xenografting or intra peritoneal injection into immunodeficient mice. Without the need for induction into a pre-differentiated stage, the direct usability of MDSC preserves the high plasticity, survival and migratory potential of transplanted cells. This is in sharp contrast with iPSC or ESC which develop fast growing lethal teratoma and tumors 2-3 weeks after engraftment if used without per-differentiation. MDSC thus represent a valuable multivalent low risk source of autologous stem cells for regenerative therapy approaches. One of the major priorities of our lab is to understand the molecular basis of this absence of tumorigenic behavior of MDSC.
Cell Cycle Regulation and Cancer Signaling
We have identified key points in the crosstalk of major multi-tasking enzymes, such as cAMP-dependent Protein Kinase (PKA), Akt/PKB family kinases and phosphatase 2A (PP2A) in the modulation Cyclin-Dependent Kinases (CDKs) during cell cycle progression. This crosstalk is the target of specific checkpoints that are bypassed in transformed cells and we are analyzing these bypass mechanisms by comparative analysis of cell cycle regulation in adult versus embryonic stem cells and in normal fibroblasts versus transformed human cell lines.
Insulin Signaling and Akt kinase family.
The Akt (protein kinase B) family of protein kinases are an integral part of insulin and other signaling cascades and have been implicated in essential cellular pathways and processes including cell differentiation and transformation. Our studies are focusing on differentiating potential interacting partners, such as p21 and CTMP, and the specific action of Akt1 and Akt2 isoforms in proliferating normal or transformed cells. Our objectives are to identify key events and substrates for Akt involved in tumorigenesis and the epithelial-mesenchymal transition which, in addition to better understanding how Akt instigates tumorigenic changes, will allow development of isoform-specific tools with potential value as diagnostic markers.
Protein phosphatase 2A
Phosphatase type 2A is implicated in many aspects of cell growth and metabolism. The catalytic activity of PP2A is modulated by several regulatory subunits the precise function of which has yet to be fully understood. We are interested in two aspects of PP2A regulation: the differential roles of the different subunits at specific points in cell cycle progression and secondly the specific interaction of PP2A PR55 with assembled microtubules. For the former, we have purified to homogeneity each subunit with or without fluorescent fusion moiety for microinjection into synchronized human fibroblasts. For the other, we are analyzing the nature of the complexes associated with polymerized microtubules in vivo to examine how PP2A modulates microtubule dynamics both at mitosis and during cell migration and the epithelial/mesenchymal transition.
Our old phosphatase pages and the BH domain work are here
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