Label-Free Morphology-Based Prediction of Multiple Differentiation Potentials of Human Mesenchymal Stem Cells for Early Evaluation of Intact Cells

Abstract

Precise quantification of cellular potential of stem cells, such as human bone marrow–derived mesenchymal stem cells (hBMSCs), is important for achieving stable and effective outcomes in clinical stem cell therapy. Here, we report a method for image-based prediction of the multiple differentiation potentials of hBMSCs. This method has four major advantages: (1) the cells used for potential prediction are fully intact, and therefore directly usable for clinical applications; (2) predictions of potentials are generated before differentiation cultures are initiated; (3) prediction of multiple potentials can be provided simultaneously for each sample; and (4) predictions of potentials yield quantitative values that correlate strongly with the experimental data. Our results show that the collapse of hBMSC differentiation potentials, triggered by in vitro expansion, can be quantitatively predicted far in advance by predicting multiple potentials, multi-lineage differentiation potentials (osteogenic, adipogenic, and chondrogenic) and population doubling potential using morphological features apparent during the first 4 days of expansion culture. In order to understand how such morphological features can be effective for advance predictions, we measured gene-expression profiles of the same early undifferentiated cells. Both senescence-related genes (p16 and p21) and cytoskeleton-related genes (PTK2, CD146, and CD49) already correlated to the decrease of potentials at this stage. To objectively compare the performance of morphology and gene expression for such early prediction, we tested a range of models using various combinations of features. Such comparison of predictive performances revealed that morphological features performed better overall than gene-expression profiles, balancing the predictive accuracy with the effort required for model construction. This benchmark list of various prediction models not only identifies the best morphological feature conversion method for objective potential prediction, but should also allow clinicians to choose the most practical morphology-based prediction method for their own purposes.

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

“…Previous methods of morphological assessment have relied exclusively on unsupervised PCA and other multivariate analyses . Although inclusion of all cell morphological features in an unsupervised PCA allows for an unbiased morphological signature assessment, this includes features with high degrees of variability and with potentially poor correlation with mineralization capacity.…”

Shaping the Cell and the Future: Recent Advancements in Biophysical Aspects Relevant to Regenerative Medicine

“…This group also showed how measuring cell morphological parameters can be used in combination with experimental data in order to predict stem cell phenotypic outcomes at a later time point. Here, stem cell shape was used as predictive marker with the intention to replace measuring later time points [21][22][23]. Hence, studies have begun to increasingly and successfully use quantitative approaches to access morphological parameters and to correlate them to biological parameters and cell fate outcome.…”

“…As an example, using high content imaging, Marklein et al [25] demonstrated that nuclear morphological profiles of MSCs had distinct morphological features that were highly predictive of MSC mineralization capabilities, with >90% accuracy. Kato's group has proposed a similar theory [21][22][23]26]. Their group demonstrated that cell morphological features from the first three days of differentiation can predict osteogenic differentiation potential (i.e., alkaline phosphatase and calcium deposition).…”

Morphological Evaluation of Nonlabeled Cells to Detect Stimulation of Nerve Growth Factor Expression by Lyconadin B

“…[6][7][8][9] We applied this technique to evaluate the quality of cultured cells, including the differentiation potential of stem cells. [3][4][5] Our morphologybased evaluation method provided not only the three aforementioned advantages but also enabled daily evaluation of rapidly changing cellular conditions. In this report, we verified the ability of our method to evaluate the cellular response to potential drug lead compounds.…”