The establishment of permanent human embryonic stem cell lines (hESCs) was first reported in 1998. Due to their pluripotent nature and ability to differentiate to all cell types in the body, they have been considered as a cell source for regenerative medicine. Since then, intensive studies have been carried out regarding factors regulating pluripotency and differentiation. hESCs are obtained from supernumerary human IVF (in vitro fertilization) embryos that cannot be used for the couple’s infertility treatment. Today, we can establish and expand these cells in animal substance-free conditions, even from single cells biopsied from eight-cell stage embryos. There are satisfactory tests for the demonstration of genetic stability, absence of tumorigenic mutations, functionality, and safety of hESCs. Clinical trials are ongoing for age-related macular degeneration (AMD) and spinal cord injury (SCI). This review focuses on the present state of these techniques.
Highlights
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Human embryonic stem cell (hESC) can be derived from a single eight-cell embryo blastomere.
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hESC can be derived, expanded, and maintained in defined, xeno-free conditions.
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The safety, functionality, and stability of hESC lines can be experimentally studied.
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hESC-based clinical therapies for several conditions are ongoing.
Establishment of human embryonic stem cell lines
For quite some time, human embryonic stem cells (hESCs) were derived from the inner cell mass (ICM) of blastocysts, which required destruction of the embryo. This has been considered a major ethical problem in many countries. In particular, many religious groups have adopted the view that cells from a human preimplantation embryo cannot be used for any purpose. Hence, derivation and even culture of hESC are forbidden by law in some European countries . In the USA, there are limitations for making hESCs when human in vitro fertilization (IVF) embryos are destroyed. However, it has recently become possible to effectively derive hESC lines from single biopsied blastomeres of an IVF embryo in xeno-free chemically defined conditions without destroying the embryo . The use of the new human recombinant laminin 521, an adhesive matrix component present in the stem cell niche, which supports ESC growth, has made the procedure easily feasible.
Murine embryonic fibroblasts (MEFs) were initially used as feeder cells, in the presence of fetal bovine serum-containing culture medium. We initiated the use of human neonatal fibroblasts as feeder cells in the derivation of new hESC lines , and then serum replacement (SR) instead of bovine serum in order to develop a better-defined culture medium , and managed in establishing several new hESC lines in these more defined conditions . The use of human feeder cells was then adopted worldwide. To remove one additional xeno-component from the cultures, we replaced immunosurgery, a method in where a mouse antibody against human trophectoderm and guinea pig complement are used in the isolation of the ICM, by mechanical isolation of the ICM .
The impractical use of feeder cells has been replaced in many laboratories by adding conditioned medium from feeder cells instead of having two cell types in the cultures . The most commonly used culture substrate was previously Matrigel, a mouse Engelbreth–Holm–Swarm (EHS) sarcoma tumor extract . It is a complex undefined mixture of basement membrane and cellular proteins as well as growth factors. It displays extensive batch-to-batch variation, and it is far from being chemically defined. The International Stem Cell Initiative (ISCI) has carried out studies aiming at standardized cultures using chemically defined media . A major step forward came with a specific cell culture coating of human recombinant laminin-511 (LN-511) that had been originally identified in the early embryo and produced as recombinant protein by Karl Tryggvason’s group . The cultures became chemically defined, when a xeno-free chemically defined medium was used in addition to the laminin as growth support. Importantly, this protocol allowed the cells to be cultured as monolayers and passaged as a single cell suspension. Another closely related human laminin, LN-521, an adhesion protein also present in the ICM and other in vivo stem cell niches, was then successfully produced . The result was another robust chemically defined, xeno-free cell culture matrix that now allows the maintenance of highly stable hESC cultures . With this system, it is now possible to derive new hESC lines from a single blastomere biopsied from an eight-cell IVF embryo by culturing it on a mixture of LN-521 and E-cadherin, which provides cell–cell contact inducing signals. The cells grow as a homogeneous monolayer ( Fig. 1 ) such that each cell can be individually inspected. An important aspect is that this procedure does require the destruction of the IVF embryo . The blastomere biopsy is similar to that normally carried out to obtain a single cell for preimplantation genetic diagnostics (PGD). In our IVF unit, we regularly get pregnancies and infants from such embryos from which one cell has been removed.
The splitting ratio of the hESC in these cultures is 1:30 instead of 1:3 in the conventional cell clump cultures. This means that we can obtain large numbers of hESCs for regenerative medicine with fewer passages, which is faster and safer.
If the line is family specific, the need for immunosuppression in regenerative medicine is much smaller than when using completely allogenic hESC lines. As the new cell derivation and expansion methods are effective, it has become feasible to establish an hESC bank of clinical quality hESC lines. For such a bank, cell lines of about 150 haplotypes are estimated to be sufficient to provide cell therapy of the majority of the human population .
An advantage with hESCs is that they are normal cells existing in the human embryo for 3–6 days after fertilization. In that respect, they differ from the induced pluripotent stem cells (iPSC) that are genetically modified adult cells. The pluripotent stem cells differ from cord blood cells in being capable of dividing without limits. Large amounts of cells for several recipients and treatments can be obtained, and all human cell types for which proper differentiation protocols have been developed can be generated. iPSCs generated by the incorporation of certain transcription factor genes into the genome of somatic cells are likely to be less immunogenic to the original host than other foreign cells, but they can also be immunogenic to the previous host . More problematic is that the transcription factor genes that have been transferred to their genome have caused genomic changes that may make the cells tumorigenic. For the time being, they are not considered as safe as hESCs. The newer techniques based on integration-free transient expression of pluripotency factors may be safer for future cell therapy purposes .
Characterization of hESC lines
Pluripotent stem cells are important as a cell source for regenerative medicine because they are able to proliferate indefinitely (self-renew) and differentiate into any cell type of the human body. As culturing of hESCs in vitro is a relatively challenging task, technical errors may lead to spontaneous differentiation, and there is a need to characterize the cells to confirm their pluripotency. One more important feature of pluripotent stem cells is that they may accumulate genetic changes during prolonged culturing in vitro . Some of those changes resemble the ones found in tumors. Therefore, for safety issues, there is also a need to characterize hESCs to confirm their genetic integrity.
Characterization of hESC lines
Pluripotent stem cells are important as a cell source for regenerative medicine because they are able to proliferate indefinitely (self-renew) and differentiate into any cell type of the human body. As culturing of hESCs in vitro is a relatively challenging task, technical errors may lead to spontaneous differentiation, and there is a need to characterize the cells to confirm their pluripotency. One more important feature of pluripotent stem cells is that they may accumulate genetic changes during prolonged culturing in vitro . Some of those changes resemble the ones found in tumors. Therefore, for safety issues, there is also a need to characterize hESCs to confirm their genetic integrity.
Testing of genetic stability
Previously, karyotyping was used as a standard method to ensure the genetic integrity of cultured hESCs. The method is easy and fast, but it does not provide sufficient resolution. A small genetic change leading to the multiplication of locus BCL2L1 has been detected in many human pluripotent stem cell lines with a normal karyotype . The mutation may lead to overexpression of anti-apoptotic factor BCL2L1 providing the cells with abnormal properties, and it is often too small to be detected by karyotyping. Therefore, there is a need for systematic high-resolution testing the genetic integrity of cultured hESC. Sequencing of the whole genome is an ideal method for that, but it is still expensive for routine use. A cheaper alternative is a whole-genome genotyping analysis with sufficient number of probes to detect genetic abnormalities equal or larger than 50 kb . It is a useful and affordable alternative to sequencing. As genotyping has certain limitations such as inability to detect balanced translocations and inversions, it should be used in combination with karyotyping. It is agreed that the genetic integrity of cultured hESCs should be monitored for every 10 passages (2 months) in culture.
Testing of pluripotency in vitro
The characterization of hESCs should also confirm their identity. Cells that express markers of pluripotency (such as Oct-4, Nanog, and Sox-2) do not express significant amounts of differentiation markers (Sox7, Pax6, alpha-fetoprotein, or others), and they can give rise to cell lineages of all three germ layers in in vitro and in vivo tests, defined as pluripotent. A typical characterization of hESCs includes immunostaining of the fixed cells for markers of pluripotency and differentiation. Although the method is easy and fast, it is only qualitative. It has been shown that the level of pluripotency marker expression may affect the differentiation of the potential of mouse ES cells . Therefore, the expression of the markers should also be studied using methods allowing quantitation, for instance, quantitative real time polymerase chain reaction (RT-PCR) (qRT-PCR). Expression levels of pluripotency markers Oct-4, Nanog, and Sox2 should be monitored for every 10 passages, and they should be stable at different time points. Expression levels of differentiation markers should not increase with time. One more important parameter of robust hESC cultures is the homogeneity of cell populations. It is not enough to estimate overall levels of pluripotency marker expression by qRT-PCR because high levels can be achieved by overexpression in a subpopulation of cells. Fluorescence-activated cell-sorting analysis for pluripotency markers Oct-4 and SSEA-4 is usually used to confirm homogeneity of cells. A homogeneous population of hESCs should contain not less than 90% of Oct4- and SSEA-4-positive cells.
Testing of the differentiation potential of hESCs is an essential part of a characterization procedure. Usually, in vitro and in vivo tests are used to confirm that cells may give rise to cell lineages of all three embryonic cell lineages. Spontaneous differentiation of hESCs into embryoid bodies (EBs) is used to test the differentiation potential in vitro. EB development somewhat resembles that of embryos in vivo. There are several slightly different methods of EB formation . We collect hESCs in large cell aggregates, culture them in suspension in a medium without basic fibroblast growth factor (bFGF) for 7–14 days, plate the aggregates on gelatin-coated dishes, and analyze all three germ layer marker expression using immunostaining or RT-PCR after 7 days of culturing on gelatin. After 2–3 days in suspension cultures, the hESC cell aggregates should appear as small round and solid clumps of cells with sharp edges. Interestingly, it is sometimes complicated to induce EB formation in very homogeneous cultures of hESCs. Epiblast differentiation that is crucial for the three germ layer formations depends on interaction with laminin-111 . To facilitate EB development, we culture hESCs on plates coated with laminin-111 and laminin-521 taken at equal w/w ratios for one passage before EB formation . Although useful, EB test is insufficient to confirm pluripotency. As of today, additional in vivo experiments are also needed to prove the pluripotency of cultured cells.
Testing of pluripotency in vivo
The ultimate test of bona fide pluripotency would be the incorporation of the cell line to a morula or a blastocyst stage embryo, and proven contribution of the cells to all different germ layers in the embryo/fetus. This protocol is often used in the case of mouse embryonic stem cells, but it cannot be applied as such to hESC pluripotency testing in humans due to obvious ethical problems. A modified version, where hESC or iPSC is injected to mouse embryos, can be applied . However, the efficiency of the model and the contribution of the human cells to the embryos are low. Instead, the teratoma assay is widely used for the in vivo pluripotency testing of hESC . This assay relies on the spontaneous growth and differentiation of hESC to a tumor, teratoma, when injected to immunodeficient mice. Typically, up to 1 million hESCs are injected into the animals, and the histology of the formed tumors is studied. If tissue structures stemming from endoderm, mesoderm, and ectoderm are discovered, the cell line is considered as pluripotent.
There is no standardized guideline for the teratoma assay, but the ISCI is working on the establishment of consensus protocols. Currently, several different variations of the teratoma assay exist, for example, the injection site, number of injected cells, and injection vehicle vary, as well as the breed and gender of the animals. The stem cells can be injected to the testis or kidney capsule, muscle, liver, subcutaneously, or even intraperitoneally . The number of injected cells can vary from a few hundred to a million, and different protocols recommend using single cell solutions or cell aggregates. Most protocols utilize Matrigel in the injection solution, and some even co-inject inactivated feeder cells with the hESC . The testicular and subcutaneous models have the advantage that the tumor growth can be followed by palpations, which is not possible when the cells are injected for instance to the kidney capsule. The time from injection to tumor growth can vary markedly between different cell lines, and even between different animals receiving the same cell line. When the mice are examined by weekly palpations, the progression of the experiment can be managed at the level of an individual animal. This minimizes the risk of terminating some animals prematurely before the tumors have grown, and, on the other hand, it eliminates the risk of letting animals suffer due to too large tumors. An additional advantage of the subcutaneous model is the possibility to inject the cells without anesthesia. The cell injection under the skin to the flank of the animal can be easily carried out within seconds, which minimizes the stress to the animals.
In our hands, the subcutaneous model has been the easiest to set up and manage, and the most reliable in terms of tissue growth. The formation of large, liquid-filled cysts is sometimes reported in hESC teratoma models , but this has not been a problem in our laboratory with the subcutaneous model. All obtained tumors have contained significant amount of well-differentiated solid tissue, although small cysts regularly form ( Fig. 2 A). We use immunodeficient female mice of the SCID/Beige breed, and we start the experiments at the age of 6–10 weeks. We have three alternative strategies in use, and every strategy is based on injecting the cells as aggregates in a vehicle consisting of 30% Matrigel in culture medium. We either (i) inject 1 million stem cells, (ii) 0.5 million stem cells together with 0.5 million inactivated human foreskin fibroblasts feeders, or (iii) 1 million stem cells as 24-h pre-differentiated spheres. We have tested over 30 new stem cell lines created in our laboratory using these protocols. Both hESC and iPSC lines can be successfully tested, as well as cell lines grown on feeder cells, laminins, or spider silk. The overall tumor incidence has been 43%. When all experiments are summarized, the average lag time from injection to tumor appearance has been 6.2 weeks (standard deviation (SD) 2.0, range 3–11), and the average tumor growth time is 27 days (SD 15, range 4–72 days). Slow tumor growth is desirable to reach good cell differentiation within the tumor. Altogether, with the subcutaneous teratoma model, results can be obtained 2–3 months after the start of the experiments.
