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Characterization of stem cells

This document discusses characterization of stem cells. It describes stem cell characteristics like being undifferentiated, capable of self-renewal, and having potential to differentiate. Key methods of characterization discussed are genetic analysis like karyotyping and SNP analysis, proteomic analysis of cell surface markers and transcription factors using flow cytometry and FACS, and morphological analysis. Characterization is important for applications like regenerative medicine, drug testing, and disease modeling.

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Stem Cells•
CHARACTERIZATION OF STEM CELLS Rumana Tamboli M.Sc.II (Biotechnology)
Index  What is stem cells?????  Stem Cell Characteristics  Types of stem cells  Need of stem cell characterization  Methods of stem cell characterization 1. Genetic analysis 2. Proteomic analysis 3. Morphological analysis  Flow Cytometry  Fluorescence Activated Cell Sorting (FACS)  Applications  References
What is stem cells????? Stem cells are rare, undifferentiated special kind of cell that have the ability to divide indefinitely and have the potential to give rise to specialized cells (that is, any cell of the body).
Stem Cell Characteristics • ‘Blank cells’ (unspecialized) • Capable of dividing and renewing themselves for long periods of time (proliferation and renewal) • Have the potential to give rise to specialized cell types (differentiation) • Potency
Types of stem cells e.g. The fertilized egg e.g.ESCs e.g. Adult Stem Cells e.g.Somatic cells to pluripotent cells
Need of stem cell characterization Tissue engineering – To generate healthy cells to replace diseased cells (regenerative medicine). Adult cells altered to have properties of embryonic stem cells (induced pluripotent stem cells). Test new drugs for safety and effectiveness. Bypass immune rejection. Patient specific cell therapies. Demonstration of absence of cross contamination Conformation of species of origin determination of whether the cell line is transformed or not
Factors affecting stem cell culture A variety of factors affect stem cell cultures Factors Media composition Cell density Feeder cell type/density Growth factors/additives Feeder free culture Passage method Number of passages Freezing and thawing protocols Microbial contamination Possible changes Chromosomal Phenotype/morphology Differentiation Pluripotency loss Epigenetic changes Tumorigenesis Loss of self-renewal ability
Methods of stem cell characterization • Genomic analysis  It is necessary to ensure stem cells maintained in culture have not undergone chromosomal changes .  To Monitoring the genomic integrity. • Proteomic analysis  Tracking the expression of proteins associated with pluripotency we ensures that the cells are expressing the factors necessary to maintain pluripotency. • Morphological analysis  By observing through electron microscope.
Characterization of stem cells by genetic analysis 1) Karyotyping 2) Fluorescence in situ hybridization (FISH) 3) Single nucleotide polymorphism (SNP) analysis
Karyotyping •It is the examination of chromosome number and morphology- differences in appearance include size, position of centromeres, and changes in banding patterns. •Traditional karyotyping uses dye to stain the chromosomes of a metaphase cell. The most common method is Giemsa staining, known as G-banding; •Changes in banding patterns are used to identify abnormalities. •It is generally recommended that a stem cell line be karyotyped every 10–15 passages to ensure that chromosomal duplications, insertions, deletions, translocations, or centromere loss have not occurred. Normal karotype of a human iPSC clone. Image courtesy of Dr. Miguel Esteban.
Fluorescence In Situ Hybridization (FISH) •Used for a precise assessment of complex chromosomal rearrangements. •Fluorescently labeled DNA fragments are hybridized to metaphase chromosomes. •Each chromosome is labeled with a different combination of fluorophores specific for that chromosome, giving a unique spectral signature for each chromosome. Advantage •Can detect translocations within and between chromosomes more accurately than traditional karyotyping Disadvantage •They cannot detect inversions or duplications and deletions of less than approximately 5 Mb. •Probes are required for the length of all the chromosomes.(Chromosome painting) Representative M-FISH karyotype of K562 cell.
Single nucleotide polymorphism (SNP) analysis • SNPs most common type of genetic variation . • Single base-pair mutations within a region of DNA can lead to phenotypic changes that influence survival or growth. • May result in loss of pluripotency or gain of tumorigenicity. • SNPs can be identified using PCR , microarrays, or DNA sequencing. Advantage • Aneuploidy, unbalanced translocations, deletions, and duplications can be detected with SNP arrays. Disadvantage • Inversions are not detected.
Characterization of stem cells by proteomic analysis 1) Alkaline Phosphatase live staining. 2) Cell surface markers & Transcription Factor analysis by- I. Flow Cytometry II. Florescence Activated Cell Sorting (FACS)
Alkaline phosphatase live staining •AP activity is higher in pluripotent cells but is greatly decreased in differentiated cell types. •Rapid method for screening pluripotent cells. •Alkaline phosphatase is toxic to the cells and once cells are stained, they change cell morphology and cannot be propagated again. Overcoming the issue, live alkaline phosphatase stain have been developed which specifically stains the pluripotent stem cell while preserving the cell integrity. Staining of mouse induced pluripotent stem cells using the Red-Color AP staining kit
Immunocytochemistry • Immunocytochemistry is a technique used to assess the presence of a specific protein or antigen in cells by use of a specific antibody, which binds to it, thereby allowing their visualization and examination under a microscope. • It is used to confirm the presence or absence of protein expression in stem cells. • There are many special molecules that can affect the pluripotency and self- renewal. • Identification, characterization, and classification of these molecules will provide useful tools for the isolation and verification of stem cells. • Cell surface markers & Transcription factors are used as marker for characterization of stem cells.
Cell Surface Markers • Some of the proteins are uniquely present or secreted in particular cell types can serve as cell markers. • Membrane proteins are the most important marker types for identifying pluripotent stem cells without destroying the cell membrane  SSEA : Stage-specific embryonic antigens  SSEA-1 is expressed on the surface of preimplantation-stage murine embryos and has been found on the surface of teratocarcinoma stem cells, but not on their differentiated derivatives.  SSEA-3 is synthesized during oogenesis and is present in the membranes of oocytes, zygotes and early cleavage-stage embryos.  It controls cell surface interactions during development.  TRA :  TRA-1-81 and TRA-1-60 are cell surface proteins involved in cell differentiation.
SSEAs markers Characteristics Classification SSEA-1 Murine embryos, mouse ES cells, mouse and human germ cells, embryonal carcinoma (EC) cells Carbohydrate- associated molecules SSEA-3 Primate ES cells, human embryonic germ cells, human ES cells, embryonal carcinoma (EC) cells Carbohydrate- associated molecules SSEA-4 Primate ES cells, human embryonic germ cells, human ES cells, embryonal carcinoma (EC) cells Carbohydrate- associated molecules TRA-1-60 Human ES cells, teratocarcinoma, embryonic germ cells, embryonal carcinoma (EC) cells Surface antigen TRA-1-81 Human ES cells, teratocarcinoma, embryonic germ cells, embryonal carcinoma (EC) cells Surface antigen Table 1: Marker and the cells in which they are present
Table 2:Representative surface markers of selected stem cells and derivatives.
Transcription Factors • Unique genes appear and do functions Therefore, tracking the expression of these genes can be used as a marker. • Oct4, Sox2, and Nanog are the core factors responsible for pluripotent stem cell self- renewal, and maintenance of pluripotency. • These factors will be rapidly down-regulated during cell differentiation.  OCT 4 :  It is expressed in totipotent embryonic stem and germ cells.  A critical level of Oct-4 expression is required to sustain stem cell self-renewal and pluripotency.  SOX :  The SOX (SRY-box containing gene) gene family and are involved in the regulation of embryonic development and in the determination of cell fate.  Nanog:  Nanog is a molecular marker that recognizes the undifferentiated state of stem cells in mouse and human.  It is homeodomain protein that directs propagation of undifferentiated ES cells.  Nanog-deficient embryonic stem cells lose pluripotency and differentiate into extraembryonic endoderm lineage.
Core transcription factors Characteristics Classific ation Oct-3/4 Mouse ES cells, human ES cells, embryonal carcinoma (EC) cells POU family transcrip tion factors Sox2 Mouse ES cells, human ES cells, embryonal carcinoma (EC) cells, neural stem (NS) cells POU family binder transcrip tion factors KLF4 Mouse ES cells, human ES cells, embryonal carcinoma (EC) cells Zinc- finger transcrip tion factors Nanog Mouse ES cells, human ES cells, embryonal carcinoma (EC) cells Transcrip tion factors Table 4: Representative intracellular markers of selected stem cells and derivatives. Table 3: Transcription Factors and the cell in which they are present.
Cell surface staining  Not require permeabilization  Provides live cell populations for further analysis.  Sample preparation  Adherent cell type  Single-cell suspension is prepared by either enzymatic digestion or mechanical scraping  Monoclonal antibodies  Cells are harvested  Dissociation buffer is removed  Cells are ready to be stained with antibodies conjugated to a variety of fluorochromes  Analyze on a flow cytometer OR FACS Intracellular staining  Require the cells to be fixed and permeabilized so that antibodies can access the cytoplasm and nucleus.  Since fixation effectively kills the cells, intracellular staining is not compatible with live-cell sorting.  Sample preparation  Adherent cell type  Single-cell suspension is prepared by either enzymatic digestion or mechanical scraping  Monoclonal antibodies  Cells are harvested  Dissociation buffer is removed  Cells must be fixed and permeabilized to enable antibodies to enter. The cells are then stained with fluorescent-labeled antibodies to intracellular antigens.  Analyze on a flow cytometer OR FACS
Flow Cytometry • Heterogeneous nature of differentiating cultures remains a primary challenge in stem cell research. • Flow cytometry is a commonly used technique used for the analysis of stem cells. Fluorescently tagged monoclonal antibodies can be used to distinguish specific cell populations on the basis of their size and complexity. • Flow cytometry is unique in its ability to investigate large cell populations at the single-cell level. • Researchers can use fluorochromeconjugated antibodies to either cell surface or intracellular biomarkers to verify that stem cells have maintained pluripotency. • Analysis based on cell surface markers can preserve cell viability for use in additional experiments.
How Flow cytometer works???
Flowcytometer
Fluorescence Activated Cell Sorting (FACS) • Cell sorting can be used to physically separate different stem cell populations. For example, when a population of somatic cells is being reprogrammed, iPSCs can be separated from cells either not reprogrammed or not fully reprogrammed. • It is nothing but the advanced version of flowcytometer. • Cell surface markers are used for sorting. • As stem cells differentiate, the loss of these markers and expression of new markers can be used to track the lineages and level of differentiation of the population. • Sorting can be used to remove unwanted cell types and retain homogeneity of the population.
FACS Cell sorting on the basis on charge Cell sorting on the basis of fluoresence
How to do analysis?? Q 1 Q 2 Q 3 Q 4
Human fibroblast cell line IMR90 expressing the human ESC surface markers TRA1-60, TRA-1-81, SSEA-3, SSEA-4, and the transcription factor NANOG; nuclei are stained blue. Image courtesy of Dr. Miguel Esteban.
Characterization of stem cells by morphological analysis iPSCs are morphologically examined to be round shape, having large nucleolus and scant cytoplasm. These cells are morphologically similar to ESCs. Morphological transformation of dermal fibroblast cells into iPSCs
Applications of stem cell characterization 1. Regenerative medicine -Tissue engineering 2. Test new drugs for safety and effectiveness. 3. Disease modelling 4. Modeling cancer pathogenesis & drug screening
References • Methods of Isolation and Characterization of Stem Cells from Different Regions of Oral Cavity Using Markers: A Systematic Review Kavarthapu Avinash, Sankari Malaippan, Jayakumar Nadathur Dooraiswamy • Alkaline Phosphatase in Stem Cells :Katelina Štefková,1 Jilina Procházková,2,3 and Jilí Pacherník1,4 • Novel Live Alkaline Phosphatase Substrate for Identification of Pluripotent Stem Cells : Upinder Singh & Rene H. Quintanilla & Scott Grecian & Kyle R. Gee & Mahendra S. Rao & Uma Lakshmipathy • https://www.bio-rad.com/en-in/applications-technologies/analysis-stem-cells • https://www.bio-rad.com/en-in/applications-technologies/analysis-stem- cells?ID=LUSR41KSY • https://www.creative-bioarray.com/Services/Stem-Cell-Characterization.htm • https://stemcells.nih.gov/info/2001report/appendixE.htm • https://www.iitk.ac.in/che/pdf/resources/Flow-Cytometry-reading-material.pdf
Characterization of stem cells