Method for evaluation of human induced pluripotent stem cell quality using image analysis based on the biological morphology of cells

Wakui, Takashi; Matsumoto, Tsuyoshi; Matsubara, Kenta; Kawasaki, Tomoyuki; Yamaguchi, Hiroshi; Akutsu, Hidenori

doi:10.1117/1.jmi.4.4.044003

Cited by 36 publications

(34 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For this study, we used an approach based on identified image features and algorithms developed to capture the characteristics indicated by experts as important for evaluating pluripotent stem cell colony qualities. Many of the features that our experts identified are similar to features identified for similar studies [8,15,16]. The experts of Wakui et al [15] identified intercellular spacing, round, well defined margin, and smooth margin as important criteria.…”

Section: Discussionsupporting

confidence: 65%

“…Many of the features that our experts identified are similar to features identified for similar studies [8,15,16]. The experts of Wakui et al [15] identified intercellular spacing, round, well defined margin, and smooth margin as important criteria. Maddah et al [8] identified biologically relevant features that included colony margin character, as did Marx et al [16], who also used a feature associated with heterogeneity within the colony (that is likely similar to our features of local std).…”

Section: Discussionsupporting

confidence: 65%

“…In this work, the 14 expert-inspired features were sufficient to capture most of the experts' perceptions, and the addition of Haralick and wavelet features did not improve the performance of the models for comparing expert scores. By developing algorithms that are intended to quantify what the experts look for, we are able to assess the criteria that they each apparently use for visual evaluation.Inconsistencies between expert ratings and molecular markers of pluripotency have been observed by others [1,15,20]. In this work, we acknowledge that neither expert represented ground truth and allowed each expert's ratings to represent a range of values.…”

Section: Discussionmentioning

confidence: 98%

See 2 more Smart Citations

A Latent Variable Model for Evaluation of Disparate Ratings of Stem Cell Colonies by Two Experts

Halter

Lund

Peskin

et al. 2019

Preprint

View full text Add to dashboard Cite

The visual inspection of pluripotent stem cell colonies by microscopy is widely used as a primary method to assess the quality of the preparations and degree of pluripotency. The lack of ground truth and the possible inconsistency of evaluations from multiple experts within and between stem cell laboratories are sources of uncertainty about the state of the cells, the reproducibility of preparations, and the efficiency of expansion protocols. To examine how to evaluate the level of confidence one has in disparate rating from experts, we explored a statistical method for assessing the differences in ratings of pluripotent stem cells by two different experts. Two experts rated phase contrast microscope images of human embryonic stem cell (hESC) colonies on a scale of 1 (poor) to 5 (maximum pluripotency character) but agreed with one another only 48% of the time. To assess whether experts used similar criteria to rate colonies, we developed custom image feature algorithms based on the stated visual criteria provided by the experts for selection of colonies. These features, plus others, were then used to develop pluripotency scoring algorithms trained to reflect ratings of both experts. We treated expert ratings as inexact indicators of a continuous pluripotency score and considered the inconsistency between expert ratings in developing our models. The model suggests that the two experts use somewhat different scales for discriminating between colony quality. Covariance analysis indicated that both experts use features that are not included in the model. Two image features, colony perimeter and a feature based on texture, were the most important for both experts for predicting the ratings. Interestingly, colony perimeter was not one of the expert-provided criteria for rating colonies, showing that this modeling approach allowed identification of features that the experts were not aware they were using. A linear model based on both experts identified each expert's top-rated colonies as well as, or better than, the ratings of the other expert, as indicated by receiver operator characteristic curve 2 analysis. By providing an understanding of the differences and similarities in disparate sets of expert ratings, this analysis helps to establish confidence in the ratings and the criteria for ratings, even when the experts disagree.

show abstract

Section: Discussionsupporting

confidence: 65%

Section: Discussionsupporting

confidence: 65%

Section: Discussionmentioning

confidence: 98%

See 1 more Smart Citation

A Latent Variable Model for Evaluation of Disparate Ratings of Stem Cell Colonies by Two Experts

Halter

Lund

Peskin

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…; for details see Wakui et al. ). Successful generation of transduced hiPSCs depends further on the efficiency of the third‐generation lentivirus and the ratio between cells and virus.…”

Section: Commentarymentioning

confidence: 99%

“…The culture should be regularly checked for the expression of hiPSC markers (e.g., OCT3/4, SSEA4, Nanog, and Sox2), a normal karyotype (Steichen, Hannoun, Luce, Hauet, & Dubart-Kupperschmitt, 2019), and typical stem cell morphology with high nuclei to cytoplasm ratio and prominent nucleoli ( Fig. 5; for details see Wakui et al 2017). Successful generation of transduced hiPSCs depends further on the efficiency of the third-generation lentivirus and the ratio between cells and virus.…”

Section: Critical Parameters and Troubleshootingmentioning

confidence: 99%

Efficient Modulation of TP53 Expression in Human Induced Pluripotent Stem Cells

Uhlmann

Kuhn

Tigges

et al. 2019

CP Stem Cell Biology

View full text Add to dashboard Cite

TP53 point mutations are found in 50% of all cancers and seem to play an important role in cancer pathogenesis. Thus, human induced pluripotent stem cells (hiPSCs) overexpressing mutant TP53 are a valuable tool for the generation of in vitro models of cancer stem cells or for in vivo xenograft models. Here, we describe a protocol for the alteration of gene expression in hiPSCs via overexpression of a mutant form of the TP53 (R249S) gene using lentiviral transduction. A high amount of TP53 protein is detected 1 week after transduction and antibiotic selection. Differentiation of transduced hiPSCs gives insight into better understanding cancer formation in different tissues and may be a useful tool for genetic or pharmacologic screening assays. © 2019 The Authors. Basic Protocol 1: Production and concentration of third‐generation lentivirus Support Protocol 1: Cloning of gene of interest into modulation vector Support Protocol 2: Preparation of DMEM GlutaMAX™ with 10% fetal bovine serum and 1% penicillin‐streptomycin Basic Protocol 2: Transduction of human induced pluripotent stem cells and selection of positively transfected cells Support Protocol 3: Preparation of Matrigel®‐coated plates Support Protocol 4: Preparation of mTeSR™1 medium

show abstract

Minimum criteria for defining induced mesenchymal stem cells

Choudhery

Mahmood

Harris

et al. 2022

Cell Biology International

View full text Add to dashboard Cite

Mesenchymal stem cells (MSCs) are a promising cell type for cell-based therapies.The therapeutic potential of MSCs has been verified in preclinical and clinical studies, however; low cell number in adult tissues, restricted expansion and differentiation capacity, and donor-related heterogeneity limit their use. To address these issues, there has been considerable interest in induced pluripotent stem cells (iPSCs) derived MSCs (induced mesenchymal stem cells [iMSCs]). Investigators obtain iMSCs from iPSCs of different origins, with variable methods of generation and expansion. Results of current studies have suggested iMSCs as a unique alternative source of MSCs. However, iMSCs are defined using the same criteria (proposed previously for primary MSCs by the International Society for Cellular Therapy [ISCT]) without realizing the distinct nature of iMSCs as compared to primary MSCs. To rationally define iMSCs, additional characterization is proposed along with ISCT's minimum criteria for defining primary MSCs. Minimum criteria for defining iMSCs should include (1) spindle-shaped morphology, (2) plastic adherent growth, (3) positive expression of CD29, CD44, CD73, CD90, CD105, along with negative expression of hematopoietic markers (CD45, CD34, CD14 or CD11b, CD79α or CD19, HLA-DR), (4) lack of expression of iPSCs induction factors, (5) trilineage differentiation potential, (6) lack of ability to form teratoma, and (7) release of MSC relevant paracrine factors. Defining the minimum criteria for iMSCs will be of great interest in the field and will provide a uniform description and identification of iMSCs to expedite progress in the field. Furthermore, due to increased interest in the clinical use of iMSCs, the above-mentioned additional characterization before the clinical application is important to avoid unwanted complications for recipients.

show abstract

Method for evaluation of human induced pluripotent stem cell quality using image analysis based on the biological morphology of cells

Cited by 36 publications

References 25 publications

A Latent Variable Model for Evaluation of Disparate Ratings of Stem Cell Colonies by Two Experts

A Latent Variable Model for Evaluation of Disparate Ratings of Stem Cell Colonies by Two Experts

Efficient Modulation of TP53 Expression in Human Induced Pluripotent Stem Cells

Minimum criteria for defining induced mesenchymal stem cells

Contact Info

Product

Resources

About