Phinney Lab Research

Basic Biology of Mesenchymal Stem Cells (MSCs)

A main focus of the laboratory is to delineate the molecular mechanisms that regulate self renewal and lineage specification of MSCs. To gain insight into these processes our laboratory cataloged the transcriptome of mouse and human MSCs via serial analysis of gene expression (SAGE). Interrogation of this data using a comparative genomics approach identified several putative regulatory proteins expressed by cells that are known inhibitors of cellular differentiation. Further analysis revealed that expression of these proteins is up regulated in MSCs by FGF2, which was shown to reversibly inhibit multi-lineage differentiation of mouse MSCs. In collaboration with researches at Tulane University, we have developed high throughput assays to quantify the differentiation potential of clonally-derived populations of human MSCs, providing a means to identify clones of all possible potencies (uni-, bi-, and tri-potent). Our analyses indicate that regulatory proteins identified by SAGE are highly enriched in tri-potent human MSC clones. A variety of molecular-based approaches are being used to study the expression of these proteins and their role in modulating lineage specification in MSCs.

Hierarchy of MSC Lineage Specification.

High throughput quantitative assays were used to analyze the multi-lineage differentiation potential of clonally-derived human MSCs.  This analysis identified clones of all possible potencies (A, C, O, AO, AC, OA, ACO).  Data indicate that FGF2 induces expression of repressors in tri-potent clones that block further specification of cells along the hierarchy of differentiation.  A, adipogenic; C, chondrogenic; O, osteogenic.

Anti-Inflammatory Properties of MSCs and Use as Vectors to Treat Chronic Inflammatory Diseases.

Our laboratory was one of the first to demonstrate that primary mouse MSCs ameliorate inflammation and fibrosis when administered systemically to mice following acute lung injury. Interrogation of the MSC transcriptome revealed that cells express high levels of interleukin 1 receptor antagonist (Il1rn / Il1f3), and secretion of this protein was shown to contribute significantly to their anti-inflammatory effects. Our recent studies indicate that human MSCs express a much larger repertoire of anti-inflammatory proteins as compared to their mouse counterparts and many of these factors belong or are related to the IL-1 family. Therefore, species-specific differences exist with respect to the anti-inflammatory properties of human vs. rodent MSCs. Currently, we are exploring the anti-inflammatory and anti-fibrotic effects of human MSCs in a SCID mouse model of bleomycin-induced lung injury and dissecting the intracellular signaling pathways that regulate expression of IL-1 family members in response to various extra-cellular stimuli.  To date these studies have identified a novel link between pathways that regulate MSC self-renewal and cytokine signaling, thereby demonstrating that paracrine functions may also be specified hierarchically within MSC populations.

Therapeutic Effect of Mouse MSCs in Acute Lung Injury

A-C) Photomicrographs of lung tissue harvested from mice 14 days post-exposure to saline (A), bleomycin (B), and bleomycin + mouse MSCs (C).  Saline/bleomycin was delivered via the intra-tracheal route and MSCs were injected into the jugular vein.  D). Changes in the concentration of leukocyte populations in bronchoalveolar lavage (BAL) from mice at 7 days post-exposure.  E) Concentration of hydroxyproline in lung tissue from mice harvested at 14 days post-exposure.  Note that MSC administration at the time of bleomycin exposure (Day 0) but not 7 days later (Day 7) significant inhibited fibrosis.  

MSCs as Vectors for Treating Neuro-Degenerative Diseases.

Our laboratory was the first to demonstrate that MSCs injected intra-cranially into newborn mice durably engraft in brain and adopted characteristics of neural cells.  Thereafter, we developed real-time PCR assays to quantify male DNA levels in female tissues and used these to analyze the engraftment kinetics and anatomical distribution of male MSCs injected intra-cranially into newborn and adult female mice and rhesus macaques.  These studies revealed that MSCs persistently engraft in brain and that overall levels were significantly affected by the age of the transplant recipient.   In addition, MSCs were found to localize to similar anatomical structures in both species, indicating that cell migration is regulated by a conserved mechanism.  Interrogation of the MSC transcriptome revealed that cells expressed transcripts encoding several neural adhesion molecules and guidance receptors known to regulate tangential migration of interneurons in the brain. Expression of these proteins was conserved in MSCs from rodents, non-human primates, and humans.  Currently, MSCs are being transplanted to the CNS of newborn rhesus macaques afflicted with Krabbe disease.  These studies are aimed at evaluating if MSCs can ameliorate neuro-inflammation and prevent neuronal cell loss in this model.  In addition, host allo-reactivity against the donor MSCs is also being studied in a comprehensive manner.

Improved Methods for Large-Scale Production of Primary Mouse MSCs

Over a decade ago our laboratory developed a method based on immuno-depletion to isolate MSCs from mouse bone marrow. This method yields primary cells with the capacity to differentiate into various connective tissue lineages in vitro, as well as contribute to bone repair in vivo. We have recently shown that primary, immuno-depleted MSCs from bone marrow but not adipose tissue are highly sensitive to oxygen-induced toxicity and exposure to atmospheric oxygen induced rapid growth arrest of these cells.  Moreover, we have shown that the growth inhibitory effect of oxygen is p53 dependent.  These findings indicate that widely established protocols that rely on long-term culture expansion of marrow in atmospheric oxygen to isolate mouse MSCs in actuality select for clones with reduced or absent p53 activity, which allows escape from oxygen-induced growth inhibition.  Therefore, these methods produce immortalized clones with defects in p53 function, which likely explains why outcomes from mouse MSC experiments vary significantly between labs.  By using a closed oxygen system we can now generate very large numbers of primary mouse MSCs that retain sensitivity to atmospheric oxygen even after serial passage.   

Enrichment of MSCs from Mouse Bone Marrow via Immuno-depletion.

Top panel: Mouse bone marrow expanded for 7-10 days in vitro yields a heterogeneous cell population. Immuno-depletion using antibodies against CD11b, CD34 and CD45 removed contaminating hematopoietic lineages yielding a morphologically homogeneous cell population.  Bottom panel:  Immuno-depleted MSCs differentiate into adipocytes, chondrocytes, osteoblasts, myoblasts and hematopoiesis-supporting stroma.

MicroRNA Regulation of Tumor Metastasis

In an effort to evaluate the role of microRNAs (miRNAs) in lineage specification of MSCs, our laboratory identified a miRNA cluster that appears to function as an intrinsic regulator of the epithelial-to-mesenchymal transition (EMT).  Each miRNAs within the cluster has been shown to regulate a large number of molecules including transcription factors, adhesion molecules, and other miRNAs that are known to play critical roles in the EMT process.  Gain- and loss-of-function studies are underway to evaluate the specific role played by each miRNA in regulating the EMT process.  Collaborations with area hospitals have also been established to evaluate if expressed levels of these miRNAs have diagnostic values with respect to staging human breast, ovary, and prostate cancers and predicting their likelihood for metastases.  Efforts are also underway to determine the anti-metastatic activity of these miRNAs in tumor xeno-graft models.  Future studies will be directed at exploiting the function of these miRNAs to develop novel compounds that prevent tumor metastasis by inhibiting the EMT process.