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Phinney Lab Research

MSCs and Oxidative Stress 

MSCs are known to reside in a low oxygen environment in bone marrow.  Therefore, our lab studies how MSCs manage oxidative stress and if such stress contributes to disease pathophysiology by adversely impacting MSC function.  Our studies show that primary MSCs are highly sensitive to oxygen-induced stress and this stress negatively impacts their growth, survival, tri-lineage differentiation, and hematopoiesis-supporting activity.  Furthermore, cellular responses to oxygen-induced stress are P53 dependent and MSCs that lack a functional P53 protein are insensitive to such stress but also show defects in their stem/progenitor functions.  These findings have important implications with respect to the methods routinely used to isolate mouse MSCs from bone marrow.  For example, our data clearly demonstrate that standard culture conditions (21% oxygen) are growth restrictive, and long-term exposure to such conditions drives selection for cells harboring loss-of-function mutations in P53, which allows escape from oxygen-induced growth inhibition.  Therefore, while the emergence of rapidly dividing cells from these cultures has been reported to represent expansion of the stem cell pool, it actually is a consequence of cell immortalization.  Therefore, cell lines that are widely accepted as MSC surrogates including those generated from embryonic mesoderm (NIH 3T3, C3H10T1/2), calvaria (MC3T3), or by long-term expansion of marrow cells in 21% oxygen do not accurately recapitulate the normal biology of primary mouse MSCs.  This likely explains why translational studies conducted in rodents poorly predict the activity of human MSCs in clinical trials.   Ongoing studies in the lab are examining the molecular pathways that regulate oxidative stress responses in MSCs, the specific role of P53, JNK and FANC proteins in this process, and whether stress-induced defects in MSC function contribute to the pathophysiology of osteosarcoma and bone marrow failure syndromes, such as Fanconi Anemia. 

 p53 Schematic showing how prolonged exposure to atmospheric oxygen selects for MSCs that lack a functional p53 protein, which results in cellular immortalization, escape from oxygen-induced growth arrest, and alters the stem/progenitor properties of cells. 

Improved Methods for Large-Scale Production of Primary MSCs from Mouse Bone Marrow

Our laboratory has developed a reliable method based on immuno-depletion to isolate primary MSCs from the bone marrow of mice.  This method produces highly purified cell populations in relatively high yield.  (Phinney DG, Methods Mol. Biol. 2008; 449:171).  These cultures are obtained by purging the marrow from the long bones of 3-4 week old mice.  A single cell suspension of marrow cells is then expanded in a closed system in 5% oxygen for 7-10 days, after which the attached cells are subjected to three rounds of immuno-depletion using anti-CD11b, anti-CD34, and anti-CD45 antibodies. These antibodies are conjugated to magnetic beads, which are used to remove contaminating hematopoietic cell lineages.  The immune-depleted MSCs are then cultured for 24-48 h after which they are frozen for preservation and distribution.   All preparations of cells are characterized by FACS analysis to verify their surface phenotype.  In addition, their CFU-F activity and tri-lineage differentiation potential are evaluated using quantifiable assays.  Click here to learn about how you can obtain primary MSCs from our laboratory.

AdipocytesEtc 
Left, Representative photomicrographs of plastic adherent cells obtained from bone marrow (upper) and primary mouse MSCs (lower) following purification by immunodepletion.  Right, Flow cytometric analysis of the surface phenotype of immuno-depleted MSCs obtained from the bone marrow of FVB/n mice.  Bottom, Representative high powered photomicrographs of immune-depleted primary MSCs differentiated into adipocytes (Adipo Red stain), chondrocytes and osteoblasts (micromass sections stained with toluidine blue and H&E, respectively), and hematopoiesis-supporting stroma (anti-CD11b antibody staining of granulocyes).

Functional Heterogeneity of MSCs

MSCs were first characterized as skeletal stem cells based on their capacity to generate ectopically a miniature bone organ with the proper architecture to support hematopoiesis.  More recently, MSCs have been shown to possess potent effector (angiogenic, anti-inflammatory, immuno-modulatory) functions that are largely mediated via paracrine action and are thought to underlie most of their therapeutic potential.  These effector functions have been exploited in a large number of clinical trials to treat various non-skeletal disorders, and while some patients have shown a clear benefit from such treatments, most trials have yielded suboptimal results.  Moreover, data from these trials are largely incongruent with respect to effects of cell dose and route of administration on patient outcome.  One impediment toward achieving efficacious MSC-based therapies is the fact that MSC populations are heterogeneous and show significant donor-to-donor variability.  However, few criteria have been established to predict which populations are best suited for treating a given disease.  A principle focus of our lab is to delineate the molecular mechanisms that regulate stem/progenitor and effector functions in MSCs, and determine how these functions are specified at the population level.  Our goal is to use this information to establish a framework that reliably predicts the potency of different MSC donor populations pre- and post-manufacturing.  This information can then be used to inform clinical trials to produce more predictable and efficacious treatments.   

MicroRNAs in Cancer

In an effort to identify microRNAs (miRNAs) regulating MSC survival and lineage specification, our laboratory identified a miRNA cluster within the imprinted DLK1-DIO3 locus that targets proteins regulating the epithelial-to-mesenchymal transition (EMT), which is a major driver of tumor metastasis.  We further showed that this microRNA cluster is epigenetically silenced in early stage human breast cancers, thereby implicating them in tumorigenesis.  Using molecular and cell-based approaches, we have found that one microRNA in the cluster is highly induced in response to hypoxia and regulates metabolic adaptation of tumor cells to hypoxic stress.  In collaboration with the Disney lab at Scripps Florida, we have designed small molecules that block biogenesis of this microRNA and found that these molecules sensitize tumor cells to hypoxia-induced killing in vitro and in vivo.  Ongoing studies are aimed at further studying the role played by this microRNA in cancer progression and developing second generation small molecules with improved selectivity and potency.