The ultimate goal of stem cell research is to cure patients with diseases caused by the loss of functional tissue, such as myocardial infarction (Pekkanen-Mattila et al., 2009). Before the clinical use of differentiated cardiomyocytes becomes a reality, it is likely that stem cell-derived pluripotent cardiomyocytes will be useful for disease modeling, drug discovery, and safety pharmacology applications (Pekkanen-Mattila et al., 2009). The aim of this study was to compare the effect of different stem cell culture conditions on cardiac differentiation.
Three pluripotent stem cell lines were used in this study: human embryonic stem cell line H7 and human induced pluripotent stem cell lines UTA.00112.hFF and UTA.04602.WT.
Stem cells
Fetal and adult stem cells
Within the last decade, adult stem cells have also been identified in other organs and tissues such as the brain, dental pulp and heart (Mimeault et al., 2007). Adult and fetal stem cells are both multipotent stem cells, but adult stem cells have an even more limited differentiation potential. Adult stem cells are thought to be restricted to differentiating into different cell types of their tissue of origin (Doss et al., 2004).
Tissues derived from embryonic and adult stem cells may differ in their likelihood of rejection after transplantation.
Human embryonic stem cells
The basic characteristics are a high nuclear-to-cytoplasmic ratio, prominent nucleoli and distinct colony morphology (Carpenter et al., 2009). Human ESCs can be used to treat diseases such as Parkinson's disease, spinal cord injury, and diabetes (Thomson et al., 1998). Teratoma formation can be tested in vivo after transplantation into severe combined immunodeficiency (SCID) mice (Thomson et al., 1998).
Also the immunosuppressive capacity of the adult stem cells can favor the growth of the tumor cells (Carpenter et al., 2009).
Human induced pluripotent stem cells
Such induced pluripotent human cell lines could be useful in addition to the production of new disease models and in drug development, also for applications in transplant medicine, once technical limitations (for example, mutation due to viral integration) are eliminated (Yu et al. ., 2007). A general method for generating an iPS cell is to use a circular DNA plasmid carrying reprogramming factors (1.). This somatic cell is then reprogrammed and changes from an elongated fibroblast-like shape to a rounded, immature cell type (3-4).
Image modified from the photo of Terese Winslow, 2008, The Promise of Induced Pluripotent Stem Cells (iPSCs) by Charles A.
Culture of stem cells
Feeder cell-dependent culture conditions
The first animal-free culture system for hESCs replaced the traditionally used MEFs with human feeder cells of fetal or adult tubal epithelial cell origin. Feeder cells support self-renewal by providing necessary factors or by removing inhibitory factors. Human mast cells secrete transforming growth factor beta 1 (TGFβ1), activin A, basic fibroblast growth factor (bFGF) and low levels of bone morphogenic protein 4 (BMP4), whereas mouse mast cells secrete comparable levels of TGFβ1 and BMP4, higher levels of activin A and without bFGF (Prowse et al., 2007).
Culture medium containing fetal bovine serum (FBS) or replacement serum (KO-SR) is used in the culture of MEFs or other feeder cells such as SNLs (Lu et al., 2006).
Feeder cell-free maintenance method
Exposure to animal pathogens through MEF-conditioned medium or Matrigel matrix is an option; human monolayer-based culture systems require simultaneous growth of both monolayers and hES cells; and the culture system cannot be precisely defined due to differences between the different feed layer lines or the use of conditioned medium (Amit et al., 2003). The feeder cells must be mitotically inactivated prior to addition of ES or iPS cells, such as by treatment with mitomycin C (2-4 h, 10 µg/ml) or by irradiation. The original publication describes the use of cell support matrix composed of four human components (collagen IV, fibronectin, laminin and vitronectin).
However, using TeSR1 or mTeSR1, cells must be observed daily and split when the appropriate density is reached, usually every 4–5 days (Ludwig et al., 2006).
Cardiac differentiation
Characterization of differentiated cardiomyocytes
In addition, pluripotent stem cell-derived cardiomyocytes show multinucleation below 1% frequency compared to 20% of adult human cardiomyocytes (Snir et al., 2003). The cardiac phenotype of iPS cell-derived cardiomyocytes appears to be comparable to hESC-derived cardiomyocytes (Takahashi et al., 2007). Differentiated beating cells exhibit spontaneous APs and contractile activity and therefore express cardiac structural proteins and ion currents ( Mummery et al., 2003 ).
Microelectrode array (MEA) technology provides another useful platform for studying cellular electrophysiology, particularly EC-derived cardiomyocytes (Reppel et al., 2004).
Ethical approval
Culture conditions
MEF and SNL feeder cells were prepared by first coating a 6-well plate with 0.1% gelatin (1 ml/well) and incubating for one hour at room temperature (RT). In passaging, the differentiated regions and feeder cells surrounding the stem cell colonies were first removed by scraping with a pipette tip. MEF feeder cells were removed manually or enzymatically with 1 mg/ml collagenase IV (Invitrogen), and source cells were transferred onto new layers of MEF feeder cells.
Stem cells were ready to be passaged when colonies were large, began to coalesce, and had centers that were dense and phase-bright compared to their edges, usually 5–7 days after inoculation.
Cardiac differentiation
Above the line there are the days after the start of differentiation and below the line there are the tasks made at each time point.
Cytospin
Dissociation protocol of beating areas of cardiomyocytes
Immunocytochemical staining
Sample collection and RNA isolation
In the columns are the binding targets to which the antibodies attach, the animal of origin, the dilution used in this study, and the production. In the column is the color of the antibody under fluorescent light and the wavelength, dilution used in this study and production. Total RNA was extracted using the NucleoSpin RNA II - total RNA purification kit (Macherey-Nagel) according to the manufacturer's instructions.
Silica membrane was desalted by adding 350 ul Membrane Desalting Buffer and membrane was dried by centrifugation for 1 minute. DNA digestion was made by applying 95 ul of rDNase reaction mixture, which hydrolyzes the phosodiester bonds of DNA strands. Washing of the silica membrane was done by first adding 200 ul of Buffer RA2 which inactivated the rDNase and centrifuging for 30 seconds.
Second and third washes were done by adding 600 µl and 250 µl of buffer RA3 and centrifuged for 30 seconds and 2 minutes. The RNA concentration and quality of the samples were measured with NanoDrop ND-1000 spectrophotometer, ND-1000V3.7.1 using nucleic acid setting and sample type RNA-40. Samples were stored in nuclease-free collection tubes at -70 oC until the complementary DNA reverse transcription step.
Complementary DNA reverse transcription
Quantitative polymerase chain reaction
The results of the q-PCR runs were analyzed with 7300 System Software and exported to Excel. RPLP0 was used as an internal control to normalize the amount of RNA added and cell line H7 from MEFs was used as a calibrator.
Morphology of undifferentiated colonies
Both MEFs and SNL feeder cells were sometimes difficult to remove without removing colonies from the bottom of the well. Also important when using MEFs and SNLs was passing colonies of the correct size, which were slightly smaller than the colonies on Matrigel.
Cardiac differentiation and beating areas
Below each picture is the cell line and culture condition used in this study. The amount of attached areas and beat areas, counted beat area average and differentiation efficiency per one well are shown in APPENDIX 1. Cell line H7 on MEFs and SNLs has the best beat area average per one well through all three passages.
Cell line H7 differentiated most efficiently in beating cardiomyocytes compared to the other two cell lines used in this study. The stem cells from MEFs were best at differentiating into palpitation cells and worse were the stem cells from Matrigel in every three passages. The cell lines UTA.00112.hFF or UTA.04602.WT from all three culture conditions had a strike area average per well below two, i.e.
The amount of attached areas at day 6 did not clearly correlate in all cell lines with the amount of pulsating areas at day 30, but % END-2 differentiation efficiency was calculated by dividing the total number of pulsating areas by the total number of attached areas from each cell line. The UTA.04602.WT cell line from Matrigel also shows quite good differentiation efficiency in every three passages.
Cytospin
Then, troponin T positive cells per well for sample 1 and 2 were counted by dividing the total cell count by 3 (ml) and then multiplied by the percentage of troponin T positive cells in that sample. show the combined results of the parallel samples 1 and 2 in the columns "average".
Immunocytochemical staining
The differentiated heart cells stained positive with several cardiac markers as shown in Figure 10, including cardiac α-actinin (red), cardiac troponin T (red) and ventricular α-myosin heavy chain (vMHC), which stained the ventricular cardiomyocytes green . Ca 2+ channels and Z-disc filaments stained red with α-actinin, and Troponin T stained all cardiomyocytes red. Connexin-43 was localized between the α-actinin- or troponin T-positive cells, indicating that the beating cells had gap junctions between them, but is not shown in Figure 10 .
Dissociated beating cells in the single cell state looked similar when stained between different cell lines.
RNA extraction
Q-RT-PCR
The aim of the study was to evaluate the influence of different culture conditions on the differentiation of pluripotent stem cells into cardiomyocytes and to characterize the differentiated cells. Pluripotent stem cells can differentiate into functional cardiomyocytes, however the differentiation potential of different cell lines differs from each other. In the culture phase, the neural cells surrounding the colonies growing on Matrigel inhibited the normal growth of the stem cells.
The cardiac differentiation of stem cells cultured on MEFs was most efficient in all three passages. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. Cardiomyocytes derived from human embryonic stem cells in pro-survival factors improve the function of infarcted rat hearts.
Differentiation of human embryonic stem cells into cardiomyocytes: role of coculture with visceral endodermal-like cells. Identification of potential pluripotency determinants for human embryonic stem cells following proteomic analysis of human and mouse fibroblast-conditioned media. Human feeders support long-term undifferentiated growth of human inner cell masses and embryonic stem cells.
Comparative evaluation of various human feeders for long-term undifferentiated growth of human embryonic stem cells. Unique gene expression signature by human embryonic stem cells cultured under serum-free conditions correlates with their enhanced and prolonged growth at an undifferentiated stage. Induction of pluripotent stem cells from mouse embryonic and mature fibroblast cultures by defined factors.
Cardiac bodies: a new culture method for the enrichment of cardiomyocytes derived from human embryonic stem cells.
