Morphological features of IFN-γ-stimulated mesenchymal stromal cells predict overall immunosuppressive capacity.

Human mesenchymal stromal cell (MSC) lines can vary significantly in their functional characteristics, and the effectiveness of MSC-based therapeutics may be realized by finding predictive features associated with MSC function. To identify features associated with immunosuppressive capacity in MSCs, we developed a robust in vitro assay that uses principal-component analysis to integrate multidimensional flow cytometry data into a single measurement of MSC-mediated inhibition of T-cell activation. We used this assay to correlate single-cell morphological data with overall immunosuppressive capacity in a cohort of MSC lines derived from different donors and manufacturing conditions. MSC morphology after IFN-γ stimulation significantly correlated with immunosuppressive capacity and accurately predicted the immunosuppressive capacity of MSC lines in a validation cohort. IFN-γ enhanced the immunosuppressive capacity of all MSC lines, and morphology predicted the magnitude of IFN-γ-enhanced immunosuppressive activity. Together, these data identify MSC morphology as a predictive feature of MSC immunosuppressive function.

Identification of Predictive Gene Markers for Multipotent Stromal Cell Proliferation.

Multipotent stromal cells (MSCs) are known for their distinctive ability to differentiate into different cell lineages, such as adipocytes, chondrocytes, and osteocytes. They can be isolated from numerous tissue sources, including bone marrow, adipose tissue, skeletal muscle, and others. Because of their differentiation potential and secretion of growth factors, MSCs are believed to have an inherent quality of regeneration and immune suppression. Cellular expansion is necessary to obtain sufficient numbers for use; however, MSCs exhibit a reduced capacity for proliferation and differentiation after several rounds of passaging. In this study, gene markers of MSC proliferation were identified and evaluated for their ability to predict proliferative quality. Microarray data of human bone marrow-derived MSCs were correlated with two proliferation assays. A collection of 24 genes were observed to significantly correlate with both proliferation assays (|r| >0.70) for eight MSC lines at multiple passages. These 24 identified genes were then confirmed using an additional set of MSCs from eight new donors using reverse transcription quantitative polymerase chain reaction (RT-qPCR). The proliferative potential of the second set of MSCs was measured for each donor/passage for confluency fraction, fraction of EdU+ cells, and population doubling time. The second set of MSCs exhibited a greater proliferative potential at passage 4 in comparison to passage 8, which was distinguishable by 15 genes; however, only seven of the genes (BIRC5, CCNA2, CDC20, CDK1, PBK, PLK1, and SPC25) demonstrated significant correlation with MSC proliferation regardless of passage. Our analyses revealed that correlation between gene expression and proliferation was consistently reduced with the inclusion of non-MSC cell lines; therefore, this set of seven genes may be more strongly associated with MSC proliferative quality. Our results pave the way to determine the quality of an MSC population for a particular cellular therapy in lieu of an extended in vitro or in vivo assay.

High Content Imaging of Early Morphological Signatures Predicts Long Term Mineralization Capacity of Human Mesenchymal Stem Cells upon Osteogenic Induction.

Human bone marrow-derived multipotent mesenchymal stromal cells, often referred to as mesenchymal stem cells (MSCs), represent an attractive cell source for many regenerative medicine applications due to their potential for multi-lineage differentiation, immunomodulation, and paracrine factor secretion. A major complication for current MSC-based therapies is the lack of well-defined characterization methods that can robustly predict how they will perform in a particular in vitro or in vivo setting. Significant advances have been made with identifying molecular markers of MSC quality and potency using multivariate genomic and proteomic approaches, and more recently with advanced techniques incorporating high content imaging to assess high-dimensional single cell morphological data. We sought to expand upon current methods of high dimensional morphological analysis by investigating whether short term cell and nuclear morphological profiles of MSCs from multiple donors (at multiple passages) correlated with long term mineralization upon osteogenic induction. Using the combined power of automated high content imaging followed by automated image analysis, we demonstrated that MSC morphology after 3 days was highly correlated with 35 day mineralization and comparable to other methods of MSC osteogenesis assessment (such as alkaline phosphatase activity). We then expanded on this initial morphological characterization and identified morphological features that were highly predictive of mineralization capacities (>90% accuracy) of MSCs from additional donors and different manufacturing techniques using linear discriminant analysis. Together, this work thoroughly demonstrates the predictive power of MSC morphology for mineralization capacity and motivates further studies into MSC morphology as a predictive marker for additional in vitro and in vivo responses.

Chromosomal stability of mesenchymal stromal cells during in vitro culture.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) are being investigated for use in cell therapy. The extensive in vitro expansion necessary to obtain sufficient cells for clinical use increases the risk that genetically abnormal cells will arise and be propagated during cell culture. Genetic abnormalities may lead to transformation and poor performance in clinical use, and are a critical safety concern for cell therapies using MSCs. METHODS: We used spectral karyotyping (SKY) to investigate the genetic stability of human MSCs from ten donors during passaging. RESULTS: Our data indicate that chromosomal abnormalities exist in MSCs at early passages and can be clonally propagated. The karyotypic abnormalities observed during our study diminished during passage. CONCLUSIONS: Karyotyping of MSCs reveals characteristics which may be valuable in deciding the suitability of cells for further use. Karyotypic analysis is useful for monitoring the genetic stability of MSCs during expansion.

Gene markers of cellular aging in human multipotent stromal cells in culture.

INTRODUCTION: Human multipotent stromal cells (MSCs) isolated from bone marrow or other tissue sources have great potential to treat a wide range of injuries and disorders in the field of regenerative medicine and tissue engineering. In particular, MSCs have inherent characteristics to suppress the immune system and are being studied in clinical studies to prevent graft-versus-host disease. MSCs can be expanded in vitro and have potential for differentiation into multiple cell lineages. However, the impact of cell passaging on gene expression and function of the cells has not been determined. METHODS: Commercially available human MSCs derived from bone marrow from six different donors, grown under identical culture conditions and harvested at cell passages 3, 5, and 7, were analyzed with gene-expression profiling by using microarray technology. RESULTS: The phenotype of these cells did not change as reported previously; however, a statistical analysis revealed a set of 78 significant genes that were distinguishable in expression between passages 3 and 7. None of these significant genes corresponded to the markers established by the International Society for Cellular Therapy (ISCT) for MSC identification. When the significant gene lists were analyzed through pathway analysis, these genes were involved in the top-scoring networks of cellular growth and proliferation and cellular development. A meta-analysis of the literature for significant genes revealed that the MSCs seem to be undergoing differentiation into a senescent cell type when cultured extensively. Consistent with the differences in gene expression at passage 3 and 7, MSCs exhibited a significantly greater potential for cell division at passage 3 in comparison to passage 7. CONCLUSIONS: Our results identified specific gene markers that distinguish aging MSCs grown in cell culture. Confirmatory studies are needed to correlate these molecular markers with biologic attributes that may facilitate the development of assays to test the quality of MSCs before clinical use.

Automated microscopy as a quantitative method to measure differences in adipogenic differentiation in preparations of human mesenchymal stromal cells.

BACKGROUND AIMS: Multipotent stromal cells, also called mesenchymal stromal cells (MSCs), are potentially valuable as a cellular therapy because of their differentiation and immunosuppressive properties. As the result of extensive heterogeneity of MSCs, quantitative approaches to measure differentiation capacity between donors and passages on a per-cell basis are needed. METHODS: Human bone marrow-derived MSCs were expanded to passages P3, P5 and P7 from eight different donors and were analyzed for colony-forming unit capacity (CFU), cell size, surface marker expression and forward/side-scatter analysis by flow cytometry. Adipogenic differentiation potential was quantified with the use of automated microscopy. Percentage of adipogenesis was determined by quantifying nuclei and Nile red-positive adipocytes after automated image acquisition. RESULTS: MSCs varied in expansion capacity and increased in average cell diameter with passage. CFU capacity decreased with passage and varied among cell lines within the same passage. The number of adipogenic precursors varied between cell lines, ranging from 0.5% to 13.6% differentiation at P3. Adipogenic capacity decreased significantly with increasing passage. MSC cell surface marker analysis revealed no changes caused by passaging or donor differences. CONCLUSIONS: We measured adipogenic differentiation on a per-cell basis with high precision and accuracy with the use of automated fluorescence microscopy. We correlated these findings with other quantitative bioassays to better understand the role of donor variability and passaging on CFU, cell size and adipogenic differentiation capacity in vitro. These quantitative approaches provide valuable tools to measure MSC quality and measure functional biological differences between donors and cell passages that are not revealed by conventional MSC cell surface marker analysis.

Global proteomic signature of undifferentiated human bone marrow stromal cells: evidence for donor-to-donor proteome heterogeneity.

The clinical application of human bone marrow stromal cells (hBMSCs) largely depends on their capacity to expand in vitro. We have conducted a comprehensive comparative proteomic analysis of culture-expanded hBMSCs obtained from different human donors. The data reveal extensive donor-to-donor proteomic heterogeneity. Processing and database-searching of the tandem MS data resulted in a most comprehensive to date proteomic dataset for hBMSC. A total of 7753 proteins including 712 transcription and translation regulators, 384 kinases, 248 receptor proteins, and 29 cytokines were confidently identified. The proteins identified are mainly nuclear (43.2%) and the share of proteins assigned to more than one subcellular location constitutes 10% of the identified proteome. Bioinformatics tools (IPA, DAVID, and PANTHER) were used to annotate proteins with respect to cellular locations, functions, and other physicochemical characteristics. We also compared the proteomic profile of hBMSCs to recently compiled datasets for human and mouse pluripotent stem cells. The result shows the extent of similarity between the three cell populations and also identified 253 proteins expressed uniformly by all lines of hBMSCs but not reported in the proteomic datasets of the two pluripotent stem cells. Overall, the proteomic database reported in this paper can serve as a reference map for extensive evaluation of hBMSC to explain their biology as well as identify possible marker candidates for further evaluation.