Human Embryonic Stem cells (hESC) are derived from blastocysts. They are grown in strict culture conditions and offer a promising future in applications such as cell therapy, drug development and drug discovery. It is known that hESC cells can be derived from mouse embryonic fibroblasts (mEF) or humans. When maintained in feeder layers and passaged over time this derivation becomes difficult thereby causing shortcomings in co-culture, contamination and reproducibility. In order to overcome these problems, Xeno-free culture conditions aroused. This review focuses on Invitrogen's CELLstart, which is a feeder-free culture. Using CELLstart has its pros and cons. Shef3 and Shef6 hESC cell lines are used and tested in feeder layers, other xeno-free cultures and CELLstart for various aspects. Shef3 cell lines when grown on inactivated mEF under low feeder density colonies were seen to grow outward. In due course the cells appeared to be larger and elongated. When the same were transferred onto a CELLstart matrix they attached within a short time, formed colonies and formed hESCs with the same morphology. This can be seen in various phase-contrast photographs. Shef6 when grown in CELLstart gave rise to colonies faster, yielded healthier colonies and had classic ES morphology of small cells with large nuclei.
Another positive aspect of using CELLstart is its pluripotency culturing both cell lines Shef3 and Shef6 in matrix proliferated and expanded just as the feeders, however they did not require collagenase treatment and cells attached quickly to matrix. No loss of pluripotency markers was seen and morphology was the same as first batch. hESCs proliferate indefinitely while retaining undifferentiated state. In order to determine if hESCs are in their undifferentiated state, characterization was done to determine expression of certain transcription factors. On analysis using flow cytometry it was observed that both Shef3 and Shef6 cell lines tested positive for all pluripotency markers namely OCT Â¾, SSEA 3, TRA 1-6c. Significant upregulation of SOX2 was noted in Shef3 cell lines when grown on CELLstart and this was assured to affect the cells ability to differentiate. In Shef6 there is upregulation of TERT but after 15 passages returns to normal.
hESCs can differentiate into cells of three embryonic germ layers namely ectoderm, mesoderm and endoderm. This differentiation of hESCs was studied in vitro through induction of embryoid bodies (EBs). A medium with 10% KSR (absence of bFGF) helped in formation of EBs in a week or ten days. All EBs were pooled from 3 dishes and analyzed by RT PCR, markers for all germ layers were amplified from these EBs. The expression of different markers between Shef3 and Shef6 cell lines has some differences. This suggests intrinsic difference in the competency of individual hESC cell lines to differentiate into different germ layers.
When karyology studies were done on Shef3 and Shef6 it was seen that even after 8 passages, Shef3 in CELLstart had normal 46 XY karyotype and Shef6 with 46 XX karyotype. This was an unexpected result as after free thaw cycle one would think the cells would replicate and divide fast but in this case they remained same from the start. On using time- lapse microscopy over a 72-hour period there was extensive movement of cells along matrix, expansion of colonies as well as detachment and reattachment after cell division, which was noted. There was expression of SOX2 and SSEA4 markers by these colonies indicating they do not lose phenotypic characteristics and retain morphology.
CELLstart has setbacks related to the fact that it has not been performed in vivo teratoma formation assays to assess differentiation potential of the feeder-free adapted cells which would further confirm that adapted cells stay pluripotent. There have been concerns in relation to induced pluripotent stem cell field for in vitro use of cells for research this assay is not needed to be performed on new cell lines created as invitro assays show capability of differentiation to the three germ layers. Apart from these minor setbacks CELLstart for hESCs have many advantages. To sum up they contain components only of human origin, enable expansion of hESCs and other stem cells too, maintain pluripotency and differentiation capabilities, sustain the same karyotype always, aid in clonal expansion and finally produced under cGMP always.
Invitrogen, a leading provider of stem cell products, has developed CELLstart that by far beats other feeder-free cultures for hESCs cell expansion in a reproducible manner.
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