The mechanisms controlling human mesenchymal stem cells (hMSC) differentiation are not entirely understood. We hypothesized that the contact with extracellular matrix (ECM) proteins normally found in bone marrow would promote osteogenic differentiation of hMSC in vitro. To test this hypothesis, we cultured hMSC on purified ECM proteins in the presence or absence of soluble osteogenic supplements, and assayed for the presence of well-established differentiation markers (production of mineralized matrix, osteopontin, osteocalcin, collagen I, and alkaline phosphatase expression) over a 16-day time course. We found that hMSC adhere to ECM proteins with varying affinity (fibronectin>collagen I≥collagen IV≥vitronectin>laminin-1) and through distinct integrin receptors. Importantly, the greatest osteogenic differentiation occurred in cells plated on vitronectin and collagen I and almost no differentiation took place on fibronectin or uncoated plates. We conclude that the contact with vitronectin and collagen I promotes the osteogenic differentiation of hMSC, and that ECM contact alone may be sufficient to induce differentiation in these cells.

Numerous procedures for data analysis of seed germination responses are scattered throughout the literature. Here an attempt has been made to summarize the existing methods, to compare the information they provide and to examine their strengths and weaknesses. The methods reviewed include the percent germination, germination index, coefficient of velocity, median response time, probit analysis, curve‐fitting of cumulative germination, heat sums, survival analysis with life tables, logistic regression, proportional hazards regression and accelerated failure time analysis. Comparisons among these method are discussed and illustrated with data from germination responses in tomato (Lycopersicon eseulentum Mill.). Seed germination involves not only qualitative responses of individuals, but also population responses which are distributed over time. Standard analysis of variance or regression methods are appropriate for a data analysis where germination of all viable seed is observed, but they are inappropriate when some viable seeds fail to germinate. Such missing (censored) data complicates statistical analysis and subsequent interpretation. Germinations tests should be designed to determine the nature of censored responses, which can be subsequently accommodated by several statistical procedures referred to as survival analysis.

The intracellular signaling events controlling human mesenchymal stem cell (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 ERKdependent pathway. We hypothesized that FAK signaling pathways provide a link between activation of ERK 1/2 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 ERK 1/2. 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 ECMinduced osteogenic differentiation of hMSC.

No abstract

No abstract

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 ␣3␤1 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. INTRODUCTIONHuman mesenchymal stem cells (hMSC) are multipotent cells found within the bone marrow and periosteum (Barry and Murphy, 2004). Typically they differentiate into chondrogenic, adipogenic, or osteogenic lineages, but recent evidence suggests that hMSC can also express phenotypic characteristics of endothelial, neural, smooth muscle, skeletal myoblast, and cardiac myocyte cells (Pittenger and Martin, 2004). The mechanisms governing hMSC differentiation are not well understood, but the ability of these cells to self renew and develop into numerous tissues makes their potential use in clinical applications quite promising.Extracellular matrix (ECM) proteins are well-known regulators of multiple cellular functions, including differentiation. The laminin (Ln) family of ECM proteins are ubiquitously expressed but are especially abundant in the basement membrane of many epithelial and endothelial tissues, where they mediate cell attachment, migration, and tissue organization in conjunction with other ECM proteins (Malinda and Kleinman, 1996). Each laminin molecule is a heterotrimer, composed of an ␣-, ␤-, and ␥-subunit. The subunits share homology with one another and upon combining through disulfide bonds form an asymmetric crosslike structure with one long and three short arms (Colognato and Yurchenco, 2000). The Ln-5 isoform is composed of ␣3, ␤3, and ␥2 subunits. Expression of the ␥2 subunit has only been found in Ln-5, whereas the ␣3 subunit is found in both Ln-6 and Ln-7. Ln-5 is bound by ␣2␤1, ␣3␤1, ␣6␤1, and ␣6␤4 integrin receptors (Decline and Rousselle, 2001), all of which are found in hMSC (Pittenger et al., 1999). The role of Ln family members in osteogenic differentiation is not known (Roche et al., 1999), though expression of the ␥2 chain has been previously detected in bone marrow (Siler et al., 2002).Ln-5 is expressed in distinct temporal and spatial patterns in developing epithelial tissues and influences tissue compartmentalization and cellular phenotypes from early embryonic development onward (Aberdam et al., 1994;Ti...

No abstract

Shading of underlying leaves by maize tassels probably reduces photosynthetic rates in the community and hence the grain yields. The shading effect is small at low plant populations but increases with plant density. Tassel size varies with plant population and variety. The area of shade cast by a single tassel varies with sun angle. Possible effects of tassel shading on grain yield are estimated by computer simulation.