Determine the quality of human embryonic stem colonies with laser light scattering patterns
© Chen et al.; licensee BioMed Central Ltd. 2013
Received: 23 August 2012
Accepted: 5 January 2013
Published: 14 January 2013
With the prompt developments of regenerative medicine, the potential clinical applications of human embryonic stem cells have attracted intense attention. However, the labor-intensive and complex manual cell selection processes required during embryonic stem cell culturing have seriously limited large-scale production and broad applications. Thus, availability of a label-free, non-invasive platform to replace the current cumbersome manual selection has become a critical need.
A non-invasive, label-free, and time-efficient optical platform for determining the quality of human embryonic stem cell colonies was developed by analyzing the scattering signals from those stem cell colonies. Additionally, confocal microscopy revealed that the cell colony morphology and surface structures were correlated with the resulting characteristic light scattering patterns. Standard immunostaining assay (Oct-4) was also utilized to validate the quality-determination from this light scattering protocol. The platform developed here can therefore provide identification accuracy of up to 87% for colony determination.
Our study here demonstrated that light scattering patterns can serve as a feasible alternative approach to replace conventional manual selection for human embryonic stem cell cultures.
KeywordsLight-scattering Human embryonic stem cell Pluripotency Label-free detection
There have been increasing interests in the applications of human embryonic stem cells (hESCs). These hESCs present almost unlimited applications and opportunities for future advances in biotechnology and regenerative medicine. Through the research of regenerative medicine, specific functional cell types have been differentiated from hESCs for stem cell-based therapies [1, 2]. For instance, advanced protocols have been developed to enrich the functional cardiomyocytes differentiated from hESCs for the treatment of irreversible cardiac tissue damage . In addition, ESCs-derived cardiomyocytes were implanted into adult dystrophic mice which formed intracardiac grafts after 7-weeks . Stem cells can furthermore be used to provide functional neuronal cells for potential treatments of neurodegenerative disorders such as Alzheimer’s disease, Huntington’s’ disease, and spinal cord injury [1, 5]. Specifically, dopaminergic neurons differentiated from ESCs have shown to offer partial Parkinson’s disease recovery in animal models [6, 7]. Moreover, functional islet-like cells derived from ESCs have been demonstrated to respond to glucose and produce insulin, which hold promising potential for treatments of diabetes . In addition to regenerating tissue replacements, human stem cells and/or differentiated cells can serve as promising platforms for drug discovery and toxicity testing within the pharmaceutical industry. These human cell-based platforms not only provide human pathology models, but are also important platforms for evaluating new drug compounds in human physiological environments. For example, differentiated hepatocytes have been used for drug metabolism studies in preclinical drug discovery; additionally, cardiomyocytes differentiated from hESCs have been utilized for cardiac drug discovery and cardiac safety assessments . Likewise, undifferentiated hESCs can provide a model for embryotoxicity testing .
To fully develop these promising industrial applications, one of the most crucial issue is the maintenance and expansion of the self-renewing undifferentiated hESCs that are able to retain the capacity to differentiate into desired cell types . In order to maintain the pluripotency of hESCs in cultures, manual microdissection is broadly used for cell passaging, which requires laborious protocols and quality-controlled manual selection . Typically, according to the protocols established by National Stem Cell Bank (NSCB) and biotechnology companies [11, 12], undifferentiated hESC cell colonies need to be manually selected and transferred to new plates every 7 ~ 10 days. The quality of undifferentiated hESC colonies is determined with bright-field/phase contrast light microscopy based on human-experience/assessment. Various rating scales/criteria have been established mainly based on the morphology of individual colonies . Chiefly, good quality (good) hESCs, without stacking nor undesired differentiations, grow as uniform flat colonies with clear colony edges; low quality (bad) hESC colonies show various shapes with visible surface structures and irregular edges. The quality of hESCs plays a critical role in the downstream applications. Consequently, determining the undifferentiated hESC cell colonies by this manual selection process serves as an essential step in subculture; only high-quality undifferentiated colonies should be transferred; conversely, transferring poor-quality colonies results in the lost of pluripotency . Nonetheless, this time-consuming and labor–intensive manual selection can hinder the development of large-scale culture for practical/clinical applications. Moreover, lack of standard criteria for rapid determination of stem cell qualities may lead to the quality control/assurance issues of industrial-scale reproducibility for therapeutic applications .
A critical requirement for hESC research and technological developments is the efficient assessment of cell differentiation status. To address this issue, several approaches have been developed [13, 14]. For instance, laser flow cytometers have been broadly used to provide comprehensive information of stem cell differentiation with the use of fluorescent cell surface antigens or protein markers such as Oct-4 and Nanog, or SSEA-3 in hESCs . To further simplify specimen preparation and lower the immunohistochemistry costs, the microfluidic dielectrophoresis (DEP) platform have been introduced. Based on different cellular dielectric properties, without cell-type specific markers, neurons and astrocytes differentiated from mouse neural precursor cells can be detected and isolated in microfluidic channels (500 μm in width and 50 μm in height) . However, these current technologies may not be appropriate for hESC colony quality evaluation, since hESC colonies would be squeezed within sheath flow and broken into undesired small pieces while they are transported in the cytometry pipe line or microfluidic channels. As a result, the dispersion of cell colonies may further complicate the subculture process.
To maintain the colonies’ integrity during the selection process, advanced image analysis systems have been developed to analyze the cell colonies (STEMvision, STEMCELL technologies, Vancouver, Canada). With methylcellulose-labeled assay, images of hematopoietic colonies can be acquired and scored automatically. However, excessive labeling assays and the requirement of specific cultureware may hinder the large-scale hESC technological developments and have an unknown influence on hESC differentiation capacities.
Results and discussion
Conventionally, one of the important criterion for evaluating the quality of hESCs has been the determination of colony morphology by manual evaluation with light microscopy. Since colony morphology will likewise influence the scattering patterns of hESC colonies, we aim to utilize this connection to determine colony quality from scattering patterns. In order to evaluate these key attributes, we used laser scanning confocal microscopy to investigate the morphology of hESCs colonies with different qualities. Optical-sliced images of DAPI stained cells were collected and reconstructed to reveal detailed 3-dimensional (3-D) hESC colonies. Results of 3-D z-stack measurements showed the average height was 53 ± 4.2 μm of good colonies and was 44 ± 13.9 μm of bad colonies. Student t-test was applied to compare the different heights and indicated the significant height difference (p-value < 0.01) between good/bad colonies. The larger variations of height measurements also indicate the non-uniform stacking microstructures within bad colonies, rather than the homogenous spatial distribution of cells within good colonies. Our 3-D colony image analysis provided consistent quantified assessments that were supported by the outcomes from the human experience-based evaluation.
As in the diffraction patterns created when light propagates through optical apertures, the amplitudes and phases of light are modulated when light passes through the biological specimens . In previous studies [17, 20], it has been shown that the central thickness and radius of bacterial colonies may dominate the scattering pattern formations. Served as the superposition of various apertures, the observed non-even spatial colony variations can lead to the non-uniform scattering patterns.
Distinguish rate (%) of good/bad colonies
Good hESCs colonies
Bad hESCs colonies
Good hESCs colonies
Bad hESCs colonies
In this study, we demonstrate the feasibility in determining the quality of hESC cell colonies with optical forward-scattering technology. Integrating with SVM machine learning, this technology provides a critical module for automatic hESC cell colony selection. Without any biochemistry labeling processes or manual labor, the determination results from our protocol showed high correlation with the results of standard immunohistochemistry assay. In addition, this new label-free light scattering process significantly reduces the cost and time for specimen preparation. Though only two fundamental categories of the hESC database were assessed here, our results demonstrated that the light scattering patterns could provide unique signature standards for hESC categorization. Additionally, this non-invasive optical protocol demonstrates its potential capacity for applications on other various stem cell colony types with specific differentiation lineages, such as iPS (induced pluripotent stem) cells, neuronal stem cells, cells grown on Matrigel, etc. Due to the unique simplicity of this non-destructive technology, the hESC cell colonies scoring protocol developed in this study is expected to provide a general calibrator to facilitate industrial scale productivity of consistent quality stem cells for applications of regenerative medicine.
Human stem cell culture
(Madison, WI, passage 32–60) were cultured following the NSCB protocols. Briefly, undifferentiated hESC were maintained on feeder cell layers, which were mitomycin C treated mouse embryonic fibroblasts (MEF). Serum-free medium was used in this experiment and the medium was composed of DMEM-F-12, Knockout Serum Replacement, basic fibroblast growth factor, L-glutamine, and MEM non-essential amino acid . Medium was changed daily and hESCs were manually selected to pass every 7 days. All hESC colonies were analyzed on day 7 to acquire forward light scattering patterns in order to eliminate the variations from culture conditions.
Laser forward-scattering system and image analysis
In this study, an automated BARDOT system was modified for hESC colony scanning. This system was composed of a laser diode (0.95 mW, 632 nm), monochromatic CMOS image sensor, and an x-y scanning stage. The laser beam was illuminated on a single colony with computer-aided positioning. The resulting forward-scattering signals were collected with a CMOS image sensor. To analyze the features from colony scattering patterns, Pseudo-Zernike moments (PZMs) were applied in our current models. Support vector machine (SVM)-based algorithm was employed for recognition and classification of the images [17, 29]. In order to establish the colony characteristic database, 290 colonies were scanned with BARDOT. Their scattering patterns were then collected and analyzed.
In order to quantify pluripotency, after culturing in medium for 7 days, hESCs were fixed with 4% wt paraformaldehyde for 20 min at room temperature. Paraformaldehyde was removed with 3 subsequent PBS rinses. Triton-X (0.1%, Sigma) was used to penetrate cell membranes. The sample was then incubated in 2% BSA for 40 min to block non-specific binding. Primary antibody anti-Oct-4 (Millipore, 1:100) was used to identify pluripotent stem cells within colonies . Alexafluor-488 conjugated antibody was used for secondary antibody staining. All samples were stained with 4′, 6-diamdino-2-phenylindole (DAPI, Invitrogen, 1:3000) for quantifying cell numbers.
Fluorescent imaging of hESC colonies
Colony images for Oct-4 quantities analysis were collected with fluorescent microscopy. Fluorescence of Alexafluor-488 was excited at 488 nm and the emission was collected between 500 nm and 560 nm. DAPI staining signals were collected to identify the cell numbers. In this study, images of colonies were categorized into two separated groups by BARDOT collection (N = 18 in each classification). Laser scanning confocal microscopy was utilized to investigate the surface morphology and colony homogeneity of good/bad colonies.
Bacterial Rapid Detection using Optical scattering Technology
Bovine serum albumin
Human embryonic stem cells
Mouse embryonic fibroblasts
Phosphate buffered saline
Support vector machine.
This study was supported in part by a grant from the Muscular Dystrophy Association (MDA). CSC was supported by the UC Merced GRC summer fellowship and the Jane Vilas Fellowship. EYC was in part supported by NIH (1R15HL095039).
- Volarevic V, Ljujic B, Stojkovic P, Lukic A, Arsenijevic N, Stojkovic M: Human stem cell research and regenerative medicine—present and future. British Medical Bulletin. 2011, 99 (1): 155-168. 10.1093/bmb/ldr027.View ArticlePubMed
- Wobus AM, Boheler KR: Embryonic Stem Cells: Prospects for Developmental Biology and Cell Therapy. Physiol Rev. 2005, 85 (2): 635-678. 10.1152/physrev.00054.2003.View ArticlePubMed
- Xu C, Police S, Rao N, Carpenter MK: Characterization and Enrichment of Cardiomyocytes Derived From Human Embryonic Stem Cells. Circ Res. 2002, 91 (6): 501-508. 10.1161/01.RES.0000035254.80718.91.View ArticlePubMed
- Klug MG, Soonpaa MH, Koh GY, Field LJ: Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts. J Clin Invest. 1996, 98 (1): 216-224. 10.1172/JCI118769.PubMed CentralView ArticlePubMed
- Keller G: Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes Dev. 2005, 19 (10): 1129-1155. 10.1101/gad.1303605.View ArticlePubMed
- Björklund LM, Sánchez-Pernaute R, Chung S, Andersson T, Chen IYC, McNaught KSP, Brownell A-L, Jenkins BG, Wahlestedt C, Kim K-S: Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model. Proc Natl Acad Sci. 2002, 99 (4): 2344-2349. 10.1073/pnas.022438099.PubMed CentralView ArticlePubMed
- Kim J-H, Auerbach JM, Rodriguez-Gomez JA, Velasco I, Gavin D, Lumelsky N, Lee S-H, Nguyen J, Sanchez-Pernaute R, Bankiewicz K: Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease. Nature. 2002, 418 (6893): 50-56. 10.1038/nature00900.View ArticlePubMed
- Jiang J, Au M, Lu K, Eshpeter A, Korbutt G, Fisk G, Majumdar AS: Generation of Insulin-Producing Islet-Like Clusters from Human Embryonic Stem Cells. STEM CELLS. 2007, 25 (8): 1940-1953. 10.1634/stemcells.2006-0761.View ArticlePubMed
- Sartipy P, Björquist P, Strehl R, Hyllner J: The application of human embryonic stem cell technologies to drug discovery. Drug Discovery Today. 2007, 12 (17–18): 688-699.View ArticlePubMed
- Améen C, Strehl R, Björquist P, Lindahl A, Hyllner J, Sartipy P: Human embryonic stem cells: Current technologies and emerging industrial applications. Critical Reviews in Oncology/Hematology. 2008, 65 (1): 54-80. 10.1016/j.critrevonc.2007.06.012.View ArticlePubMed
- WiCell: [http://www.wicell.org/]
- Viacyte: [http://www.viacyte.com/]
- Loring JF, Rao MS: Establishing standards for the characterization of human embryonic stem cell lines. STEM CELLS. 2006, 24 (1): 145-150. 10.1634/stemcells.2005-0432.View ArticlePubMed
- Terstegge S, Laufenberg I, Pochert J, Schenk S, Itskovitz-Eldor J, Endl E, Brustle O: Automated maintenance of embryonic stem cell cultures. Biotechnol Bioeng. 2007, 96 (1): 195-201. 10.1002/bit.21061.View ArticlePubMed
- Flanagan LA, Lu J, Wang L, Marchenko SA, Jeon NL, Lee AP, Monuki ES: Unique Dielectric Properties Distinguish Stem Cells and Their Differentiated Progeny. STEM CELLS. 2008, 26 (3): 656-665. 10.1634/stemcells.2007-0810.View ArticlePubMed
- Weber DC, Hirleman ED: Light-Scattering Signatures of Individual Spheres on Optically Smooth Conducting Surfaces. Appl Optics. 1988, 27 (19): 4019-4026. 10.1364/AO.27.004019.View Article
- Bae E, Bai N, Aroonnual A, Bhunia AK, Hirleman ED: Label-free identification of bacterial microcolonies via elastic scattering. Biotechnol Bioeng. 2011, 108 (3): 637-644. 10.1002/bit.22980.View ArticlePubMed
- Bae E, Bai N, Aroonnual A, Robinson JP, Bhunia AK, Hirleman ED: Modeling light propagation through bacterial colonies and its correlation with forward scattering patterns. J Biomed Opt. 2010, 15 (4):
- Scott CT, McCormick JB, Owen-Smith J: And then there were two: use of hESC lines. Nat Biotechnol. 2009, 27 (8): 696-697. 10.1038/nbt0809-696.PubMed CentralView ArticlePubMed
- Banada PP, Guo S, Bayraktar B, Bae E, Rajwa B, Robinson JP, Hirleman ED, Bhunia AK: Optical forward-scattering for detection of Listeria monocytogenes and other Listeria species. Biosens Bioelectron. 2007, 22 (8): 1664-1671. 10.1016/j.bios.2006.07.028.View ArticlePubMed
- Khotanzad A, Hong YH: INVARIANT IMAGE RECOGNITION BY ZERNIKE MOMENTS. IEEE Trans Pattern Anal Mach Intell. 1990, 12 (5): 489-497. 10.1109/34.55109.View Article
- Bayraktar B, Banada PP, Hirleman ED, Bhunia AK, Robinson JP, Rajwa B: Feature extraction from light-scatter patterns of Listeria colonies for identification and classification. J Biomed Opt. 2006, 11 (3):
- Rajwa B, Venkatapathi M, Ragheb K, Banada PP, Hirleman ED, Lary T, Robinson JP: Automated classification of bacterial particles in flow by multiangle scatter measurement and support vector machine classifier. Cytometry Part A. 2008, 73A (4): 369-379. 10.1002/cyto.a.20515.View Article
- Burges CJC: A tutorial on Support Vector Machines for pattern recognition. Data Mining and Knowledge Discovery. 1998, 2 (2): 121-167. 10.1023/A:1009715923555.View Article
- Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A: Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol. 2000, 18 (4): 399-404. 10.1038/74447.View ArticlePubMed
- Loh YH, Wu Q, Chew JL, Vega VB, Zhang WW, Chen X, Bourque G, George J, Leong B, Liu J: The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet. 2006, 38 (4): 431-440. 10.1038/ng1760.View ArticlePubMed
- Halvorsen YDC, Franklin D, Bond AL, Hitt DC, Auchter C, Boskey AL, Paschalis EP, Wilkison WO, Gimble JM: Extracellular matrix mineralization and osteoblast gene expression by human adipose tissue-derived stromal cells. Tissue Eng. 2001, 7 (6): 729-741. 10.1089/107632701753337681.View ArticlePubMed
- Jaiswal N, Haynesworth SE, Caplan AI, Bruder SP: Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem. 1997, 64 (2): 295-312. 10.1002/(SICI)1097-4644(199702)64:2<295::AID-JCB12>3.0.CO;2-I.View ArticlePubMed
- Bae EW, Aroonnual A, Bhunia AK, Robinson JP, Hirleman ED: System automation for a bacterial colony detection and identification instrument via forward scattering. Meas Sci Technol. 2009, 20 (1):
- Chen C-S, Soni S, Le C, Biasca M, Farr E, Chen E, Chin W-C: Human stem cell neuronal differentiation on silk-carbon nanotube composite. Nanoscale Research Letters. 2012, 7 (1): 126-10.1186/1556-276X-7-126.PubMed CentralView ArticlePubMed
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.