Mesenchymal Stem/Progenitor Cell (MSC/MPC)
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Like the lympho-hematopoietic system, the MSC/MPC system has been shown to demonstrate a similar organization in that several differentiation lineages diverge from a common stem cell compartment. However, the MSC/MPC system represents a heterogenous group of cells; in fact, it is not really known whether these are actually stem cells. Nevertheless, one of the definitive properties of MSC/MPC is their ability to differentiation into chondrocytes, bone or fat cells depending on the stimulators and other factors present. The MSC/MPC population represent adherent cells that express, at a minimum, CD105, CD73 and CD90, but not CD45, CD34, CD14, CD11beta, CD79alpha, CD19 or HLA-DR, on their surface.
Toxicity or damage to MSC/MPSs and the system as a whole can result in serious problems to the maintenance of steady-state conditions. These include, but are not limited to:
- Disruption of lympho-hematopoietic regulation, since the MSC system is responsible, in part, for the hematopoietic stroma, without which stem cell "niches" would not be present and hematopoiesis would not function correctly.
- Disruption of bone formation, since MSCs are responsible for the production of osteogenesis.
- Disruption of chondrogenesis, since MSCs are responsible for the production of cartilage.
- Disruption of adipogenesis, without which fat cells would not be formed. Fat cells are also part of the hematopoietic stroma.
- Since MSCs can also differentiate into different muscle cells and neuronal cells, it follows that damage to the MSC system can have severe effects.
For many years, the colony-forming unit - fibroblast (CFU-F) assay has been used as a test of MSC/MPC functionality and to study the effects of various compounds on MSC/MPCs. Since MSC/MPCs exhibit a finite lifetime in culture, but continuously proliferate, the CFU-F assay, which relies on the cells forming adherent colonies in vitro does not provide an accurate measurement of potential toxicity. HemoGenix® has developed two bioluminescence assay platforms to study the MSC/MPC system and its response to virtually any compound.
MSC/MPC can be obtained from numerous primary sources, for example, bone marrow, umbilical cord blood, Wharton's gelee, adipose tissue. The MSCGlo™ assays can be used to study MSC/MPCs from any source, including iPS-derived cells. MSC/MPCs have a finite lifespan in culture. This lifespan is usually measured in the number of passages or the number of doubling times. Withime in culture, the potency and quality of the cells decreases, a fact that is particularly important when MSC/MPC are used in regenerative medicine applications.
These parameters are also important in measuring potential toxicity to the system regardless of the cell source. MSCGlo™-Real Time can be used to measuring the doubling time of MSC/MPCs and determine the growth kinetics, onset of cytotoxicity and other parameters. MSCGlo™-Tox HT is a multifacetted, standardized and validated in vitro end-point assay platform designed to measured multiple parameters of toxicity. The graphs show the exponential increase, measured using MSCGlo™, of human bone marrow MSCs when passaged and expanded over a 44 day period. After 44 days of MSC culture, MSCGlo™ can detect a linear dose range from at least 500 MSC/well to 50,000 MSC/well. On days 28, 36 and 44, phenotypic analysis of the MSCs detected by multiplexing with MSCGlo™ demonstrates how the MSCs change with time in culture.
Benefits of Using MSCGlo™-Tox HT and MSCGlo™-Real Time for Predictive In Vitro MSC Toxicity Testing
- MSCGlo™-Tox HT is an end-point, lytic, ATP bioluminescence predictive toxicity assay for MSC/MPCs from any source or species.
- MSCGlo™-Real Time is a non-lytic, bioluminescence assay for MSC/MPCs from any source or species that can help optimize culture conditions, define growth kinetics, measure doubling time and determine the onset of potential toxicity.
- Both assays measure cell viability and proliferation/cytotoxicity. Under specific conditions, the onset of MSC/MPC differentiation into chondrocytes, adipocytes or osteoblasts can be determined.
- Used to analyze effects to the bone marrow stroma (microenvironment).
- Can be used with other HemoGenix® assays to study the immunomodulatory role of MSCs.
- Both assays can replace the manual CFU-F assay.
- Both assays have high-throughput capability using 96- or 384-well plate formats allowing ADME-Tox drug or compound screening, thereby significantly reducing unexpected results during pre-clinical testing.
- The assays can be used at all stages of drug development.
- Both are 3Rs Assay Platform-Reduction, Refinement, Replacement for animal testing.
- MSCGlo™-Tox HT is part of the ComparaTOX™ 1 Platform for comparing and ranking toxicity according to cell type and species.
- Both assays can use CRUXRUFA human platelet lysate or proprietary, high performance MSCGro™ medium with either low serum, serum-free or humanized formulations.
- Results are usually available in 2 to 7 days depending on the assay used, source and species.
- MSCGlo™-Tox HT has been validated according to FDA Bioanalytical Method Guidelines.
- MSCGlo™-Tox HT standardization allows results to be compared over time and between samples.
- Both assays have been designed for multiplexing with other assays using the same sample. The non-destructive MSCGlo™-Real Time assay actually allows cells to be phenotypically analyised by flow cytometry during culture.
Two types of assay procedures have been used to characterize and identify MSC from different sources. These include the subjective Colony-Forming Unit - Fibroblast (CFU-F) assay and flow cytometry. The CFU-F assay is, in many ways, similar to the CFC assay for hematopoietic cells. Unlike the CFC assay, the CFU-F assay requires that the cells are grown under adherent conditions, usually in large petri dishes (60-100mm in diameter). Since the MSCs represent a continuously proliferating system, it is important that when the colonies of fibroblasts are counted under an inverted microscope, cell confluency has not occured. The assay lacks standards and controls and has not be validated according to regulatory requirements. Although flow cytometry is usually used to charcterize MSCs by the expression (or lack of) membrane markers, flow cytometry provides no indication regarding the functional capability or capacity of the MSCs.
MSCGlo™-Tox HT is a validated MSC/MPC cytotoxicity assay that can be multiplexed with the following (and other) assays:
- Flow cytometry to characterize the cells, perform cell cycle analysis and apoptosis.
- Growth factor, cytokine, chemokine production and release.
- Apoptosis detecting caspases.
- Membrane integrity.
- Mitochondrial dysfunction.
- Oxidative stress.
- Oxidative DNA damage (OxyFLOW™, see below).
- Kinase assays.
- Protease assays.
- Lactate dehydrogenase assays.
- Cell-based drug interaction.
- Residual toxicity and drug sensitivity change assays.
Toxicity to Mesenchymal Stem/Progenitor Cells
To demonstrate how drugs, in particular anti-cancer drugs, can affect MSCs, consider the effect of doxorubicin (Adriamycin) on human bone marrow MSCs shown in the graph. The MSCs were tested at two different cell doses. In both cases, there was significant cytotoxicity.
Cellular Drug-Drug Interaction
Drug-drug interaction can activate or inhibit CYP450 enzymes. Although occuring primarily in hepatocytes as the cells responsible for detoxification, drug interactions can occur in many other cell type. Although little, if anything, is known regarding the response of MSCs to drug interactions, drug-drug interaction assays can be performed in a similar manner to those for the lympho-hematopoietic stem cells using HALO®-DDI. For more information, please contact HemoGenix® directly.