Feeder-free matrices: animal origin
conditioned medium.19 Unfortunately, most of these matrices are of animal origin and the hESCs may also be exposed to animal based proteins from an unconditioned medium20 or conditioned medium.2122 Derivation and growth of hESCs on xenosupports in the presence of xenoproteins introduces several disadvantages with respect to exploiting the therapeutic potential of hESCs. A major drawback is the risk of transmitting pathogens such as the hantavirus and lymphocytic choriomeningitis virus from the mouse feeder cells or conditioned medium to the hESCs. A xeno-free hESC line was derived and propagated by our group using human fetal muscle as a feeder in the presence of a culture medium containing ingredients of human origin (human serum, human insulin, human transferrin) or knock out serum (KO).13 We later evaluated a panel of in-house derived and commercial sources of human fetal and adult feeders (Table 2). Human fetal muscle (in-house derived), human fetal skin (D551, ATCC) and human adult skin (in-house derived) all supported hESCs well and ranked first, second and third, respectively.15 Later, other reports also showed that human foreskin fibroblasts14'23 were able to successfully support hESCs in prolonged culture. Although generally in tissue culture practice, fetal tissues perform better than adult tissues in vitro, the use of an adult skin biopsy from the same IVF patient donating embryos for hESC derivation has the advantages of autologous support as well as safety, since the patient would have been previously tested for HIV and hepatitis B before enrolling for IVF treatment. Also, commercially available human cell lines are usually exposed to
Table 2. Supportive and Non-supportive Feeders for Prolonged hESC Growth
Undifferentiated hESC colonies
Human fetal muscle +++
Human adult skin ++
Human adult fallopian tubal epithelium ++
Human foreskin (CRL-2552) ++
Human adult muscle +
Mouse embryonic fibroblasts ++ Non-supportive
Human glandular endometrium —
xenoproteins such as fetal calf serum (American Type Culture Collection, ATCC) and hence are not xeno-free. The preparation of human feeders from fetal and adult tissues is described in detail by Fong and Bongso.24 Xeno-free derivation and propagation of hESCs without feeders would be the most ideal as it will be less labor-intensive, involving lesser risks of feeder contamination and enabling easier scaling-up of hESC numbers. However, at this point in time it is extremely difficult to grow ICMs and derive hESC cell lines without feeder cell support. Transcriptome profiling of the genetic make-up of human feeder cells will shed light on possible proteins released by these cells that will hopefully allow feeder-free growth of hESCs in the future. Since all existing NIH hESC lines are derived and propagated on MEFs, it is urgently necessary to produce panels of xeno-free clinically compliant hESC lines using cGMP and GTCP conditions.25 The cells and tissues differentiated from such cell lines when grown under the same stringent quality control conditions will help take this science to clinical trials faster.
Safe and efficient freezing and storage methods are an important prerequisite for hESC clinical application. These novel methods complement the production of xeno-free clinical grade hESC lines. In most stem cell laboratories, hESCs are stored in closed cryovials in the liquid phase of liquid nitrogen (LN2) using the conventional programmeable slow freezing method and a mixture of 10% dimethylsulfoxide (DMSO) and 90% fetal calf serum (FCS) as cryoprotectant. The thaw-survival rates, plating efficiencies and undifferentiation rates after thaw and subsequent passage are quite low with this method. The vitrification (snap-freezing or ultrarapid freezing) open pulled straw (OPS) protocol using ethylene glycol as cryoprotectant which is much simpler and yields better thaw-survival rates has replaced the slow freezing method.26 Both the slow freezing and OPS vitrification protocols, however, are fraught with problems in that: 1) the slow freezing method is inefficient and not xeno-free, and hence unsafe for clinical application because of exposure of the cells to FCS; and 2) the open straw at one end in the OPS method allows the hESCs to come into direct contact with LN2. Numerous reports describe the contamination of frozen blood and cells with adventitious agents, primarily viruses, in LN2 storage tanks.27 The cross-contamination of frozen cell stock between cell lines and cell types in LN2 storage tanks has also been documented.28 Also, several viruses including HIV and hepatitis B are known to survive in LN2.29 30 Thus, an efficient xeno-free protocol to freeze and store cGMP compliant clinical grade hESCs is important. Our group recently reported a safe, xeno-free cyropreservation protocol for hESCs using vitrification in closed sealed straws (heat sealed at both ends), using human serum albumin (HSA) as opposed to FCS in the cryoprotectant solution, and long term storage in the vapor phase of liquid nitrogen.31 Post-thaw, the hESCs showed high thaw-survival rates (88%), low differentiation rates, remained pluripotent and maintained normal karyotypes throughout extended passage. The xeno-free closed straw-vitrification LN2 vapor phase storage method will prove very useful for the setting up of hESC banks throughout the world.
Nature and Properties of hESCs
The various protocols used in hESC derivation and propagation are outlined in Fig. 5. hESCs in vitro behave like established continuous cell lines exhibiting the characteristic of immortality by indefinite growth. This feature separates them from multipotent adult somatic stem cells which show senescence in culture after a certain period of time. hESCs when frozen and
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