Stem cells possess two key abilities: self-renewal, allowing them to make copies of themselves over extended periods, and differentiation, enabling them to develop into specialized cell types like muscle, nerve, or blood cells. These capabilities make stem cells a major focus of scientific research. Generating these cells in a laboratory involves manipulating cellular processes to achieve or restore their unique properties.
Deriving Embryonic Stem Cells
One method for obtaining stem cells involves deriving them from early-stage embryos. These cells, known as embryonic stem cells (ESCs), originate from the inner cell mass of a blastocyst, an embryo typically 3 to 5 days old. The inner cell mass is the cluster of cells within the blastocyst that will eventually form the fetus.
To derive ESCs, scientists isolate this inner cell mass. Once isolated, these cells are cultured in vitro (in a laboratory dish) with specific nutrients and growth factors. Under these controlled conditions, ESCs can proliferate indefinitely while maintaining their pluripotency, the ability to differentiate into any cell type of the three embryonic germ layers: ectoderm, mesoderm, and endoderm.
Reprogramming Adult Cells into Induced Pluripotent Stem Cells
Reprogramming adult somatic cells into induced pluripotent stem cells (iPSCs) bypasses the need for embryos. This method reverts specialized adult cells, such as skin or blood cells, back to a pluripotent state by introducing specific “reprogramming factors.”
The most common set, known as the Yamanaka factors, includes Oct4, Sox2, Klf4, and c-Myc. These factors can be delivered into the cells using viral vectors or non-viral techniques. Once introduced, these factors epigenetically modify the cell’s DNA, restoring pluripotency. This technique allows for the creation of patient-specific stem cells, which can avoid immune rejection for therapeutic applications and address ethical concerns associated with embryonic stem cells.
Somatic Cell Nuclear Transfer
Somatic Cell Nuclear Transfer (SCNT), also known as “therapeutic cloning,” is another method for generating stem cells. This technique begins by removing the nucleus from an unfertilized egg cell. The nucleus from an adult somatic cell, such as a skin cell, is then inserted into this enucleated egg.
The reconstructed egg is stimulated to begin dividing, often using an electric pulse or chemical treatments. If successful, it develops into an early-stage embryo, a blastocyst, which is genetically identical to the somatic cell donor. From this blastocyst’s inner cell mass, patient-specific embryonic stem cells can be derived for research or therapeutic uses, such as replacing diseased tissues. SCNT aims to produce stem cells for therapeutic purposes and is distinct from reproductive cloning, which involves implanting the embryo into a surrogate mother.
Confirming Stem Cell Identity
After generating cells, scientists must confirm they possess the defining characteristics of stem cells to ensure suitability for research or therapeutic applications. One characteristic is self-renewal, their ability to proliferate extensively in culture while remaining undifferentiated. This is assessed by observing their sustained growth over many passages.
Another characteristic is pluripotency, the capacity to differentiate into cell types representing all three embryonic germ layers. This is demonstrated through in vitro differentiation assays, where cells are exposed to specific conditions to induce specialization. An in vivo assessment is the teratoma formation assay, where the putative stem cells are injected into an immunocompromised mouse. The formation of a teratoma, a benign tumor containing tissues derived from all three germ layers, confirms their pluripotent potential.
Scientists also analyze the expression of specific molecular markers unique to stem cells, such as Oct-4, Sox-2, Nanog, SSEA-3, and TRA-1-60. These markers help identify and characterize the cell population.