Osteogenesis Cell-Based Assay
Studies of the mechanisms underlying osteogenesis have been greatly facilitated by the use of the undifferentiated mesenchymal cell line (Vitro Biopharma, Cat. No. SC00A1). This cell line was derived from umbilical cord tissue and under defined culture conditions can be induced to undergo a developmental sequence leading to the formation of multilayered bone nodules. This sequence is characterized by the replication of differentiation induced early activation of alkaline phosphatase (ALP) and increased expression of Runx-2, and osteopontin (OPN), followed by growth arrest and expression of mature osteoblastic characteristics such as matrix maturation, osteocalcin (OC) gene expression and eventual formation of multilayered nodules with a mineralized extracellular matrix.
Cell-based Osteogenic Assays for Drug Discovery and Metastatic Diseases
A cell-based assay for human osteogenesis is described. The assay is based on the differentiation of human MSCs into osteoblasts. The assay demonstrates differentiation induced early activation of alkaline phosphatase (ALP) and increased expression of Runx-2, and osteopontin (OPN) at 3 to 6 days followed by suppression of Runx-2 and OPN gene expression while osteocalcin (OC) gene expression was not elevated until 24 days of continuous exposure to differentiation agents. Differentiating agents included dexamethazone (10 nM & 100 nM) Lithium (0.5 mM & 1 mM) for Wnt pathway activation together with 200 ng/ml parathyroid hormone (PTH). Cytokine analysis of conditioned media showed differentiation induced secretion of GM-CSF, a well-known marker of osteogenesis. Also, differentiation induced secretion of MMP-2, TGF-β1 and TNF-α. This may reflect auto-regulation of this cell-based assay system. Smart Flare® probes were used to demonstrate live cell expression of β-actin. This MSC-based assay system is suitable for high throughput screening of specific molecular targets including Runx-2 and controlling microRNAs, especially since its expression is a key fate determinant in osteoblast differentiation from MSCs. Other molecular targets may also be analyzed using high throughput readout using this MSC-based assay.
Cell-Based Functional Assays of Stem Cells
Quantitative Analysis of Stem Cell Activating Agents
Tiana Torney & Jim Musick Ph.D.
Abstract: Bone marrow-derived stem cell therapies have been used for over 50 years in the treatment of blood disorders including leukemia, lymphoma and auto-immune disorders. Embryonic stem cells were discovered in the late 1990’s by Evans, Kauffman and Martin. J Thompson developed procedures to isolate and expand ESCs from human embryos. Since ESCs could differentiate into any cell in the body this expanded applications of stem therapy to include broad areas in disease treatment and regenerative medicine. Use of ESCs was also associated with ethical/religious issues since their derivation from an embryo also destroyed the embryo. Recent research has shown that ESCs tend to form tumors following transplantation while adult stem cells have gained significant support for efficacy in the treatment of skeletomuscular disorders and several other indications. There are limitations using adult stem cell transplants because of cost, potential safety issues and the necessity of autologous transplants. Biological products activating endogenous hematopoietic stem cells have been used clinically in the treatment of anemia (recombinant human erythropoietin) and immuno-suppression (recombinant human G-CSF) resulting from chemotherapy for over 20 years. Recently, the endogenous cytokine BMP11 aka GDF11 has been shown to activate adult stem cells including Satellite muscle stem cells and possibly NSCs. Additional studies have indicated that combined inhibition of GSK3-beta and HDAC-I may induce activation of NSCs, MSCs and possibly other adult stem cell populations. We have developed a series of stem cell functional and activation assays measuring proliferation, migration and epigenetic reprogramming. We describe initial results and validation data in this report. These assays have application in quality control testing of stem cells for cell therapy, quality control of stem cell activation agents, i.e., nutraceuticals, pharmaceuticals, and pathway-specific small molecule drug candidates.
Stem Cell Potency Assays