Embryonic stem cells are obtained by isolating a small cluster of cells from the interior of a very early-stage embryo, typically around five to six days after fertilization. At that point, the embryo is a hollow ball of roughly 100 to 200 cells called a blastocyst, and the target is a group of cells on one side called the inner cell mass. Those cells are separated from the rest of the embryo, placed in a dish with nutrients and growth factors, and coaxed into multiplying indefinitely as a stem cell line. The process is technically straightforward but biologically delicate, and the details of how those inner cells are freed, fed, and kept in their undifferentiated state have evolved considerably since the first human lines were created in the late 1990s.
What a Blastocyst Looks Like and Why It Matters
By the time a fertilized egg reaches the blastocyst stage, it has divided enough times to sort itself into two distinct cell populations. The outer shell, called the trophectoderm, will eventually form the placenta and other support tissues. Tucked against one wall of the interior cavity is the inner cell mass, or ICM, which contains the cells that would go on to build the actual body of an organism. In animal studies, the proportion of ICM cells is relatively low early on and shifts as the blastocyst expands. Work in pig embryos, for instance, showed that inner cells first appear around the 12-to-16-cell stage but remain a small fraction of the total until the blastocyst begins to differentiate further.1PubMed. The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro In ferret blastocysts, ICM counts averaged around 25 cells at an early time point and grew exponentially over the following days.2Journal of Experimental Zoology. Allocation of inner cell mass and trophectoderm cells to the preimplantation blastocyst of the domestic ferret, Mustela putorius furo Human blastocysts follow a similar pattern, with the ICM making up a modest portion of the total cell count. It is this small clump of cells that researchers extract and cultivate.
Where the Embryos Come From
Nearly all human embryonic stem cell (hESC) lines have been derived from embryos created through in vitro fertilization. During IVF, clinics typically fertilize more eggs than will be transferred to a patient’s uterus, and the extras are frozen. When patients complete their families or decide not to use remaining embryos, those embryos can be donated for research with informed consent. Surveys of IVF patients show a surprisingly high willingness to donate to science: in one study, about 57 percent agreed to donating surplus embryos for stem cell research, while only 29 percent preferred donating them to other infertile couples.3Oxford Academic. Surplus embryo donation: terminology and ethico-legal perspectives
The original human embryonic stem cell lines illustrate this pathway. In the late 1990s, frozen human embryos donated for research in Israel were shipped to James Thomson’s laboratory at the University of Wisconsin. Five hESC lines, designated H1, H7, H9, H13, and H14, were established by early 1998 and announced in a landmark publication in Science that November.4PubMed Central. 20th Anniversary of Isolation of Human Embryonic Stem Cells: A Personal Perspective The H9 line from that original batch is still one of the most widely used hESC lines in the world, more than 25 years later.
Regulations governing this process vary widely by country. Many jurisdictions restrict research to embryos that would otherwise be discarded and impose a time limit: the embryo must be no more than 14 days past fertilization, a boundary known as the 14-day rule.3Oxford Academic. Surplus embryo donation: terminology and ethico-legal perspectives Some countries ban the creation of embryos specifically for research purposes, while others permit it under strict oversight. The ethical tension over embryo status is the reason so much effort has gone into finding alternative approaches, which we will get to shortly.
How the Inner Cell Mass Is Isolated
Once a donated blastocyst is thawed and confirmed to be developing normally, researchers need to separate the ICM from the surrounding trophectoderm. This is the critical technical step, and there are several ways to do it.
Immunosurgery
The classic method, used for the original Thomson lines and many since, is called immunosurgery. The blastocyst is briefly exposed to antibodies that bind to the outer trophectoderm cells, then placed in a solution containing complement proteins. These complement proteins punch holes in the antibody-tagged trophectoderm cells, destroying them while leaving the interior ICM intact. The surviving ICM is then picked out and placed onto a growth surface.5PubMed Central. Derivation of human embryonic stem cells by immunosurgery Immunosurgery works well, but it relies on animal-derived antibodies and complement, which raises concerns about contamination with animal proteins when the goal is producing cells for human therapies.
Laser-Assisted Dissection
To avoid animal products entirely, some labs use a precision infrared laser to physically cut the blastocyst apart. The embryo is held in place with tiny pipettes, the ICM is positioned to one side, and a series of rapid laser pulses slices through the cell layer separating the ICM from the trophectoderm.6PubMed Central. Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: applications and preliminary results in a murine model This approach has been used successfully with human embryos that have undergone preimplantation genetic diagnosis, and it avoids the need for any animal reagents, making it attractive for clinical-grade cell lines.7PubMed. Laser-assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis
Mechanical Dissection
A simpler, lower-tech version involves manually cutting the blastocyst apart using fine glass needles or surgical blades under a microscope. This method requires skilled hands but no specialized equipment beyond a standard micromanipulation setup. All three approaches aim for the same result: a clean ICM free from trophectoderm contamination, ready to be plated onto a culture surface.
Growing the Cells Into a Stable Line
Extracting the ICM is only half the challenge. The isolated cells need the right environment to survive, multiply, and remain in their undifferentiated, pluripotent state instead of spontaneously maturing into specialized cell types. Early protocols placed the ICM on a layer of inactivated mouse feeder cells, which secrete growth factors and provide a physical scaffold, bathed in medium containing fetal bovine serum or serum-replacement proteins.8PubMed Central. Human embryonic stem cell cultivation: historical perspective and evolution of xeno-free culture systems Those conditions worked for establishing lines but introduced animal-derived components, which is a problem if the cells are intended for transplantation into patients.
Over the past two decades, the field has steadily moved toward chemically defined, animal-free (xeno-free) culture systems. The key biochemical signals keeping human embryonic stem cells in their pluripotent state are now fairly well understood. Two signaling pathways do most of the heavy lifting. FGF2 (also called basic FGF) activates a signaling cascade that helps keep the cells from differentiating.9PubMed Central. bFGF-mediated pluripotency maintenance in human induced pluripotent stem cells is associated with NRAS-MAPK signaling Activin and Nodal, members of a separate signaling family, are also necessary; blocking either pathway causes the cells to start differentiating. Researchers showed that a combination of Activin (or Nodal) plus FGF2 can maintain human embryonic stem cells long-term without feeder cells, conditioned medium, or serum replacer.10PubMed. Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells This finding was pivotal because it meant labs could grow the cells in a fully defined cocktail of recombinant proteins, with no mysterious animal-derived ingredient in the mix.
At the molecular level, the cells’ “stemness” is maintained by a trio of transcription factors, SOX2, OCT4, and NANOG, which keep the genes for differentiation turned off while the cell continues to divide.11PubMed Central. SOX2, OCT4 and NANOG: The core embryonic stem cell pluripotency regulators in oral carcinogenesis As long as culture conditions sustain expression of these factors, the cells remain pluripotent and can theoretically be expanded indefinitely.
Can Embryonic Stem Cells Be Obtained Without Destroying an Embryo?
This is the question that has driven some of the most creative work in the field. The standard method does destroy the blastocyst, which is why researchers have explored alternatives.
One approach borrows a technique from preimplantation genetic testing, where a single cell (a blastomere) is removed from a very early embryo, typically at the eight-cell stage, for genetic analysis. The remaining embryo can continue developing normally and be transferred to a uterus. In 2006, a team demonstrated that human embryonic stem cell lines could be derived from single blastomeres removed this way, without destroying the source embryo.12PubMed. Human embryonic stem cell lines derived from single blastomeres This technique addresses the core ethical objection, though it has its own complications: the efficiency is lower than working with a full blastocyst, and the biopsy itself carries a small risk to the embryo.
Another category of sources involves embryos that have already stopped developing on their own. During IVF, a substantial fraction of fertilized eggs arrest before reaching the blastocyst stage. These embryos are not viable and would never result in a pregnancy, yet many of them still contain living cells that express the same pluripotency markers found in healthy blastocysts.13PubMed. Derivation of human embryonic stem cells from developing and arrested embryos Work has shown that most arrested embryos still have viable cells on day six of culture, suggesting they could serve as a practical source of starting material.14PubMed. Non-viable human embryos as a source of viable cells for embryonic stem cell derivation One group even derived a stem cell line from a single blastomere of an arrested four-cell-stage embryo, though that particular line turned out to have chromosomal abnormalities.15PubMed. Derivation of the first Swiss human embryonic stem cell line from a single blastomere of an arrested four-cell stage embryo Still, the principle that non-viable embryos can yield pluripotent cells is well established and sidesteps some of the ethical concerns about using embryos that could otherwise develop into a pregnancy.
Unconventional Routes to Embryonic-Like Stem Cells
Beyond traditional IVF embryos, researchers have created embryonic stem cells from embryos that never involved fertilization at all. In parthenogenesis, an unfertilized egg is chemically triggered to begin dividing as though it had been fertilized. The resulting embryo has only maternal DNA and cannot develop into a viable organism, but it can reach the blastocyst stage and yield stem cell lines. Two groups independently demonstrated this with human eggs, producing lines that were genetic matches to the egg donors.16PubMed. Derivation of human embryonic stem cell lines from parthenogenetic blastocysts17PubMed Central. Derivation and long-term culture of human parthenogenetic embryonic stem cells using human foreskin feeders Because the embryo has no developmental potential, parthenogenesis raises fewer ethical objections. It also creates cells with a simpler immune profile, which could make them easier to match to potential transplant recipients.
Somatic cell nuclear transfer, or SCNT, takes a different approach entirely. A patient’s own body cell nucleus is transferred into a donated egg that has had its nucleus removed, creating an embryo that is a near-genetic match to the patient. This was long considered a distant goal in humans, but in 2013 a team succeeded, deriving human embryonic stem cell lines from as few as two donor eggs using optimized protocols.18PubMed Central. Human embryonic stem cells derived by somatic cell nuclear transfer The resulting cells had normal chromosomes and inherited their nuclear DNA exclusively from the donor’s body cells, meaning they could theoretically provide patient-matched tissue for transplantation. SCNT remains technically demanding and ethically complex because it requires human eggs, but it demonstrated that the biology works.
Primed Versus Naive Pluripotency
Most human embryonic stem cell lines exist in what is called a “primed” pluripotent state, meaning they are already slightly biased toward certain developmental paths compared to the very earliest embryonic cells. Mouse embryonic stem cells, by contrast, are typically captured in a “naive” state that more closely resembles the cells of a freshly formed blastocyst. This difference has practical consequences: naive cells may be more versatile for certain research applications. Researchers have shown that human naive embryonic stem cells can be derived directly from blastocysts under specific culture conditions, meeting the criteria previously established only in mouse cells, including distinct growth characteristics, gene expression patterns, and metabolic features.19PubMed Central. Derivation of naive human embryonic stem cells Capturing truly naive human cells remains tricky and requires precise culture adjustments, but the fact that it is possible means the field is not limited to primed-state cells.
What Happens to the Cells Over Time in Culture
A stem cell line is only useful if it stays genetically stable as it multiplies. Unfortunately, long-term culture introduces problems. Every time cells divide, there is a chance of acquiring small mutations, and over many rounds of splitting and re-plating (called passages), those mutations accumulate. One longitudinal study found that the number of single-nucleotide changes in human embryonic stem cells increased roughly 1.8-fold as cells were passaged from early to later stages, with a significant rise in mutations affecting protein-coding regions.20Experimental & Molecular Medicine. Longitudinal analysis of genetic and epigenetic changes in human pluripotent stem cells in the landscape of culture-induced abnormality Some of the chromosomal changes seen in long-term culture resemble those found in cancer, including gains of specific chromosome segments that give cells a growth advantage.21PubMed. Tumor progression of culture-adapted human embryonic stem cells during long-term culture
This does not mean every old cell line is dangerous, but it means quality control is critical. Labs regularly check the chromosomes (karyotype) of their lines and compare the genetic profile at different passage numbers. For any clinical application, regulatory agencies require extensive genomic characterization to ensure the cells have not picked up worrisome mutations. The broader lesson is that human pluripotent stem cells of all kinds, whether embryonic or reprogrammed, can acquire genetic and epigenetic changes in culture, and vigilance is non-negotiable.22PubMed. The consequences of recurrent genetic and epigenetic variants in human pluripotent stem cells
Freezing and Banking Cell Lines
Because genetic drift is a real concern, labs do not simply keep growing cells continuously. Instead, they freeze large batches at early passage numbers and thaw fresh vials when needed. The freezing method matters. Traditional slow-freezing protocols work but can damage cells as ice crystals form. Vitrification, an ultra-rapid cooling technique that turns the liquid into a glass-like state without crystal formation, produces much higher survival rates after warming and preserves pluripotency markers and the ability to form all three tissue layers in functional tests.23PubMed. Cryopreservation of human embryonic stem cells with a new bulk vitrification method Newer vitrification solutions designed specifically for stem cells have further improved the process.24PubMed Central. StemCell Keep™ Is Effective for Cryopreservation of Human Embryonic Stem Cells by Vitrification
Several national and international stem cell banks maintain characterized, quality-controlled lines that researchers can order. Banking ensures that a well-validated line derived decades ago can still be used by labs worldwide without each group having to derive new lines from scratch, reducing both the number of embryos needed and the risk of working with poorly characterized cells.
How Embryonic Stem Cells Compare to Induced Pluripotent Stem Cells
Since 2006, researchers have been able to create cells that closely resemble embryonic stem cells by reprogramming ordinary adult cells. These induced pluripotent stem cells (iPSCs) bypass the need for embryos entirely, which is a huge ethical advantage. But the two cell types are not identical. Several studies have found that iPSCs tend to carry more epigenetic and genetic irregularities as a consequence of the reprogramming process itself.25PubMed Central. Embryonic stem cells or induced pluripotent stem cells? A DNA integrity perspective Whether those differences matter in practice depends on the application. For basic research and drug screening, iPSCs are often perfectly adequate and have the advantage of being patient-specific. For some clinical applications, however, the “gold standard” comparison remains embryonic stem cells, because they arise from a natural developmental process rather than an artificial reprogramming event.26PubMed. Embryonic stem cells versus induced pluripotent stem cells: the game is on
In practice, the two types of cells increasingly coexist. Many labs use embryonic stem cells as a reference standard while doing their day-to-day experiments with iPSCs. Clinical trials have launched using both. The existence of iPSCs has not made embryonic stem cells obsolete; rather, it has expanded the toolkit and reduced the pressure to derive new embryonic lines for every new research question.
From Dish to Patient
The whole point of obtaining embryonic stem cells is to use them, and the most advanced clinical applications involve turning them into specific cell types for transplantation. One area where this has progressed furthest is retinal disease. Researchers showed that human embryonic stem cells can be directed to become retinal pigment epithelium (RPE), the supportive cell layer behind the retina that degenerates in age-related macular degeneration. Nicotinamide and members of the TGF-beta signaling family guide the differentiation, and the resulting cells rescued retinal function in animal models of RPE-based blindness.27PubMed. Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells An optimized version of this protocol converted roughly 80 percent of starting cells into RPE in just 14 days, which is fast enough to be practical for manufacturing transplant-ready cells.28PubMed Central. Rapid and efficient directed differentiation of human pluripotent stem cells into retinal pigmented epithelium
Clinical trials using hESC-derived RPE cells for macular degeneration have been underway for years, and similar approaches are being explored for spinal cord injury, type 1 diabetes, Parkinson’s disease, and heart failure. Each application requires its own differentiation protocol, its own safety testing, and its own regulatory pathway, so progress is uneven. But the pipeline from blastocyst to bedside, while long, is no longer theoretical.