Revisiting oversight and regulation of molecular-based laboratory-developed tests: a position statement of the Association for Molecular Pathology.

Since 2006, the US Food and Drug Administration, Congress, and other policymakers have explored the appropriate way to guarantee the clinical and analytical validity of laboratory-developed tests. In the past, the Association for Molecular Pathology has publicly urged the Food and Drug Administration to exercise caution in implementing regulatory changes that could potentially hinder innovation or interfere with the practice of medicine. In 2012, the Association for Molecular Pathology Professional Relations Committee chose to develop this paper with the goal of outlining the best methods for ensuring appropriate oversight and validation of molecular diagnostic procedures. At the conclusion of this process, the workgroup reaffirmed the Association's previous position that the Centers for Medicare and Medicaid Services Clinical Laboratory Improvement Amendments program can provide the appropriate level of oversight for the vast majority of diagnostic tests.

Dissection of the osteogenic effects of laminin-332 utilizing specific LG domains: LG3 induces osteogenic differentiation, but not mineralization.

The overall mechanisms governing the role of laminins during osteogenic differentiation of human mesenchymal stem cells (hMSC) are poorly understood. We previously reported that laminin-332 induces an osteogenic phenotype in hMSC and does so through a focal adhesion kinase (FAK) and extracellular signal-related kinase (ERK) dependent pathway. We hypothesized that this is a result of integrin-ECM binding, and that it occurs via the known alpha3 LG3 integrin binding domain of laminin-332. To test this hypothesis we cultured hMSC on several different globular domains of laminin-332. hMSC adhered best to the LG3 domain, and this adhesion maximally activated FAK and ERK within 120 min. Prolonged culturing (8 or 16 days) of hMSC on LG3 led to activation of the osteogenic transcription factor Runx2 and expression of key osteogenic markers (osterix, bone sialoprotein 2, osteocalcin, alkaline phosphatase, extracellular calcium) in hMSC. LG3 domain binding did not increase matrix mineralization, demonstrating that the LG3 domain alone is not sufficient to induce complete osteogenic differentiation in vitro. We conclude that the LG3 domain mediates attachment of hMSC to laminin-332 and that this adhesion recapitulates most, but not all, of the osteogenic differentiation associated with laminin-5 binding to hMSC.

Proteomics reveals multiple routes to the osteogenic phenotype in mesenchymal stem cells.

BACKGROUND: Recently, we demonstrated that human mesenchymal stem cells (hMSC) stimulated with dexamethazone undergo gene focusing during osteogenic differentiation (Stem Cells Dev 14(6): 1608-20, 2005). Here, we examine the protein expression profiles of three additional populations of hMSC stimulated to undergo osteogenic differentiation via either contact with pro-osteogenic extracellular matrix (ECM) proteins (collagen I, vitronectin, or laminin-5) or osteogenic media supplements (OS media). Specifically, we annotate these four protein expression profiles, as well as profiles from naïve hMSC and differentiated human osteoblasts (hOST), with known gene ontologies and analyze them as a tensor with modes for the expressed proteins, gene ontologies, and stimulants. RESULTS: Direct component analysis in the gene ontology space identifies three components that account for 90% of the variance between hMSC, osteoblasts, and the four stimulated hMSC populations. The directed component maps the differentiation stages of the stimulated stem cell populations along the differentiation axis created by the difference in the expression profiles of hMSC and hOST. Surprisingly, hMSC treated with ECM proteins lie closer to osteoblasts than do hMSC treated with OS media. Additionally, the second component demonstrates that proteomic profiles of collagen I- and vitronectin-stimulated hMSC are distinct from those of OS-stimulated cells. A three-mode tensor analysis reveals additional focus proteins critical for characterizing the phenotypic variations between naïve hMSC, partially differentiated hMSC, and hOST. CONCLUSION: The differences between the proteomic profiles of OS-stimulated hMSC and ECM-hMSC characterize different transitional phenotypes en route to becoming osteoblasts. This conclusion is arrived at via a three-mode tensor analysis validated using hMSC plated on laminin-5.

Mechanical strain enhances extracellular matrix-induced gene focusing and promotes osteogenic differentiation of human mesenchymal stem cells through an extracellular-related kinase-dependent pathway.

Human mesenchymal stem cells (hMSCs) are a population of multipotent bone marrow cells capable of differentiating along multiple lineages, including bone. Our recently published proteomics studies suggest that focusing of gene expression is the basis of hMSC osteogenic transdifferentiation, and that extracellular matrix proteins play an important role in controlling this focusing. Here, we show that application of a 3-5% tensile strain to a collagen I substrate stimulates osteogenesis in the attached hMSCs through gene focusing via a MAP kinase signaling pathway. Mechanical strain increases expression levels of well-established osteogenic marker genes while simultaneously reducing expression levels of marker genes from three alternate lineages (chondrogenic, adipogenic, and neurogenic). Mechanical strain also increases matrix mineralization (a hallmark of osteogenic differentiation) and activation of extracellular signal-related kinase 1/2 (ERK). Addition of the MEK inhibitor PD98059 to reduce ERK activation decreases osteogenic gene expression and matrix mineralization while also blocking strain-induced down-regulation of nonosteogenic lineage marker genes. These results demonstrate that mechanical strain enhances collagen I-induced gene focusing and osteogenic differentiation in hMSCs through the ERK MAP kinase signal transduction pathway.

Activation of FAK is necessary for the osteogenic differentiation of human mesenchymal stem cells on laminin-5.

Human mesenchymal stem cell (hMSC) differentiation into osteoblasts and the signaling events involved are poorly understood. We recently established that contact with specific extracellular matrix (ECM) proteins, in particular laminin-5, is sufficient to induce an osteogenic phenotype in hMSC through an extracellular signal-related kinase (ERK)-dependent pathway. Activation of ERK 1/2 by laminin-5 induces phosphorylation of the runx2/cbfa-1 transcription factor that controls osteogenic gene expression. We hypothesized that focal adhesion kinase (FAK) mediated signaling pathways supply a link between cell surface integrin-ECM binding and activation of ERK 1/2, and that laminin-5 promotes its osteogenic effects through this pathway. To test this hypothesis, we plated hMSC on a laminin-5 matrix in the presence or absence of FAK-specific small inhibitory RNAs (siRNA), and assayed for phosphorylation of runx2/cbfa-1 as well as expression of established osteogenic differentiation markers (bone sialoprotein, osteocalcin, alkaline phosphatase, calcium deposition, and mineral:matrix ratio). We found that siRNA treatment reduced total endogenous FAK protein by approximately 40%, and reduced FAK phosphorylation on Y397 by approximately 33% in cells plated on laminin-5 for 30 min. SiRNA treated cells exhibited a decrease in ERK 1/2 phosphorylation after 1 h, and reduced serine/threonine phosphorylation of Runx2/Cbfa-1 after 8 days. Finally, FAK inhibition blocked osteogenic differentiation of hMSC, as assessed by lowered expression of osteogenic genes (RT-PCR), decreased alkaline phosphatase activity, greatly reduced calcium deposition, and a lower mineral:matrix ratio after 28 days in culture. These results establish FAK as an important mediator of laminin-5-induced osteogenic differentiation of hMSC.

Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells.

The intracellular signaling events controlling human mesenchymal stem cells (hMSC) differentiation into osteoblasts are not entirely understood. We recently demonstrated that contact with extracellular matrix (ECM) proteins is sufficient to induce osteogenic differentiation of hMSC through an ERK-dependent pathway. We hypothesized that FAK signaling pathways provide a link between activation of ERK1/2 by ECM, and stimulate subsequent phosphorylation of the Runx2/Cbfa-1 transcription factor that controls osteogenic gene expression. We plated hMSC on purified collagen I (COLL-I) and vitronectin (VN) in the presence or absence of FAK-specific siRNA, and assayed for phosphorylation of Runx2/Cbfa-1 as well as expression of established osteogenic differentiation markers (bone sialoprotein-2, osteocalcin, alkaline phosphatase, calcium deposition, and spectroscopically determined mineral:matrix ratio). We found that siRNA treatment reduced FAK mRNA levels by >40% and decreased ECM-mediated phosphorylation of FAK Y397 and ERK1/2. Serine phosphorylation of Runx2/Cbfa-1 was significantly reduced after 8 days in treated cells. Finally, FAK inhibition blocked osterix transcriptional activity and the osteogenic differentiation of hMSC, as assessed by lowered expression of osteogenic genes (RT-PCR), decreased alkaline phosphatase activity, greatly reduced calcium deposition, and a lower mineral:matrix ratio after 28 days in culture. These results suggest that FAK signaling plays an important role in regulating ECM-induced osteogenic differentiation of hMSC.

Laminin-5 activates extracellular matrix production and osteogenic gene focusing in human mesenchymal stem cells.

We recently reported that laminin-5, expressed by human mesenchymal stem cells (hMSC), stimulates osteogenic gene expression in these cells in the absence of any other osteogenic stimulus. Here we employ two-dimensional liquid chromatography and tandem mass spectrometry, along with the Database for Annotation, Visualization and Integrated Discovery (DAVID), to obtain a more comprehensive profile of the protein (and hence gene) expression changes occurring during laminin-5-induced osteogenesis of hMSC. Specifically, we compare the protein expression profiles of undifferentiated hMSC, hMSC cultured on laminin-5 (Ln-5 hMSC), and fully differentiated human osteoblasts (hOST) with profiles from hMSC treated with well-established osteogenic stimuli (collagen I, vitronectin, or dexamethazone). We find a marked reduction in the number of proteins (e.g., those involved with calcium signaling and cellular metabolism) expressed in Ln-5 hMSC compared to hMSC, consistent with our previous finding that hOST express far fewer proteins than do their hMSC progenitors, a pattern we call "osteogenic gene focusing." This focused set, which resembles an intermediate stage between hMSC and mature hOST, mirrors the expression profiles of hMSC exposed to established osteogenic stimuli and includes osteogenic extracellular matrix proteins (collagen, vitronectin) and their integrin receptors, calcium signaling proteins, and enzymes involved in lipid metabolism. These results provide direct evidence that laminin-5 alone stimulates global changes in gene/protein expression in hMSC that lead to commitment of these cells to the osteogenic phenotype, and that this commitment correlates with extracellular matrix production.

Focal adhesion kinase signaling controls cyclic tensile strain enhanced collagen I-induced osteogenic differentiation of human mesenchymal stem cells.

Focal adhesion kinase (FAK) is a key integrator of integrin-mediated signals from the extracellular matrix to the cytoskeleton and downstream signaling molecules. FAK is activated by phosphorylation at specific tyrosine residues, which then stimulate downstream signaling including the ERK1/2 pathway, leading to a variety of cellular responses. In this study, we examined the effects of FAK point mutations at tyrosine residues (Y397, Y925, Y861, and Y576/7) on osteogenic differentiation of human mesenchymal stem cells exposed to collagen I and cyclic tensile strain. Our results demonstrate that FAK signaling emanating from Y397, Y925, and to a lesser extent Y576/7, but not from Y861, controls osteogenic differentiation through an ERK1/2 pathway, as measured by expression levels of key osteogenesis marker genes and subsequent matrix mineralization. These data indicate that FAK is a critical decision maker in extracellular matrix/strain-enhanced osteogenic differentiation.

Apocynin derivatives interrupt intracellular signaling resulting in decreased migration in breast cancer cells.

Cancer cells are defined by their ability to divide uncontrollably and metastasize to secondary sites in the body. Consequently, tumor cell migration represents a promising target for anticancer drug development. Using our high-throughput cell migration assay, we have screened several classes of compounds for noncytotoxic tumor cell migration inhibiting activity. One such compound, apocynin (4-acetovanillone), is oxidized by peroxidases to yield a variety of oligophenolic and quinone-type compounds that are recognized inhibitors of NADPH oxidase and may be inhibitors of the small G protein Rac1 that controls cell migration. We report here that while apocynin itself is not effective, apocynin derivatives inhibit migration of the breast cancer cell line MDA-MB-435 at subtoxic concentrations; the migration of nonmalignant MCF10A breast cells is unaffected. These compounds also cause a significant rearrangement of the actin cytoskeleton, cell rounding, and decreased levels of active Rac1 and its related G protein Cdc42. These results may suggest a promising new route to the development of novel anticancer therapeutics.

Comparing the protein expression profiles of human mesenchymal stem cells and human osteoblasts using gene ontologies.

One of the hallmark events regulating the process of osteogenesis is the transition of undifferentiated human mesenchymal stem cells (hMSCs) found in the bone marrow into mineralized-matrix producing osteoblasts (hOSTs) through mechanisms that are not entirely understood. With recent developments in mass spectrometry and its potential application to the systematic definition of the stem cell proteome, proteins that govern cell fate decisions can be identified and tracked during this differentiation process. We hypothesize that protein profiling of hMSCs and hOSTs will identify potential osteogenic marker proteins associated with hMSC commitment and hOST differentiation. To identify markers for each cell population, we analyzed the expression of hMSC proteins and compared them to that of hOST by two-dimensional gel electrophoresis and two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS). The 2D LC-MS/MS data sets were analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Only 34% of the spots in 2D gels were found in both cell populations; of those that differed between populations, 65% were unique to hOST cells. Of the 755 different proteins identified by 2D LCMS/ MS in both cell populations, two sets of 247 and 158 proteins were found only in hMSCs and hOST cells, respectively. Differential expression of some of the identified proteins was further confirmed by Western blot analyses. Substantial differences in clusters of proteins responsible for calcium- based signaling and cell adhesion were found between the two cell types. Osteogenic differentiation is accompanied by a substantial change in the overall protein expression profile of hMSCs. This study, using gene ontology analysis, reveals that these changes occur in clusters of functionally related proteins. These proteins may serve as markers for identifying stem cell differentiation into osteogenic fates because they promote differentiation by mechanisms that remain to be defined.

Focusing of gene expression as the basis of stem cell differentiation.

In a prior report (Stem Cells Dev 14(4):354-366, 2005), we employed two-dimensional gel electrophoresis followed by advanced proteomics and the Database for Annotation, Visualization and Integrated Discovery (DAVID) to compare the protein expression profiles of mesenchymal stem cells to that of fully differentiated osteoblasts. These data were reported to advance technical approaches to define the basis of differentiation, but also led us to suggest that osteogenic differentiation of stem cells may result from the focusing of gene expression in functional clusters (e.g., calcium-regulated signaling proteins or adherence proteins) rather than simply from the induced expression of new genes, as many have assumed. Here, we have employed these analytical techniques to compare protein expression by mesenchymal stem cells directly with that of cells derived from them after induced osteogenic differentiation. Our results support the concept of gene focusing as the basis of differentiation. Specifically, induced differentiation results in a decrease in the number of mesenchymal cell markers and calcium-mediated signaling molecules expressed by their differentiated progeny. This effect was seen in parallel to increased expression of specific extracellular matrix (ECM) molecules and their receptors. These results strongly imply that changes in the ECM have a direct impact on stem cell differentiation, and that osteogenic differentiation of stem cells directed by matrix clues results from focusing of the expression of genes involved in Ca2+-dependent signaling pathways.

Laminin-5 induces osteogenic gene expression in human mesenchymal stem cells through an ERK-dependent pathway.

The laminin family of proteins is critical for managing a variety of cellular activities including migration, adhesion, and differentiation. In bone, the roles of laminins in controlling osteogenic differentiation of human mesenchymal stem cells (hMSC) are unknown. We report here that laminin-5 is found in bone and expressed by hMSC. hMSC isolated from bone synthesize laminin-5 and adhere to exogenous laminin-5 through alpha3beta1 integrin. Adhesion to laminin-5 activates extracellular signal-related kinase (ERK) within 30 min and leads to phosphorylation of the osteogenic transcription factor Runx2/CBFA-1 within 8 d. Cells plated on laminin-5 for 16 d express increased levels of osteogenic marker genes, and those plated for 21 d deposit a mineralized matrix, indicative of osteogenic differentiation. Addition of the ERK inhibitor PD98059 mitigates these effects. We conclude that contact with laminin-5 is sufficient to activate ERK and to stimulate osteogenic differentiation in hMSC.

ERK signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells on collagen I and vitronectin.

Adhesion to the extracellular matrix (ECM) proteins collagen I and vitronectin is sufficient to drive human mesenchymal stem cells (hMSCs) into an osteogenic differentiation pathway, but the mechanisms underlying this stimulation are not well understood. We found that addition of beta1 and alpha(v)beta3 integrin blocking antibodies inhibited ECM-induced ERK activation, while addition of the MEK inhibitor PD98059 blocked ERK activation, serine phosphorylation of the osteogenic transcription factor runx2/cbfa-1, osteogenic gene expression, and calcium deposition. These results suggest that ERK plays an important role in driving the ECM-induced osteogenic differentiation of hMSC.