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III. How do you culture stem cells in the laboratory?

How are stem cells grown in the laboratory?

Growing cells in the laboratory is known as “cell culture.” Stem cells can proliferate in laboratory environments in a culture dish that contains a nutrient broth known as culture medium (which is optimized for growing different types of stem cells). Most stem cells attach, divide, and spread over the surface of the dish.

The culture dish becomes crowded as the cells divide, so they need to be re-plated in the process of subculturing, which is repeated periodically many times over many months. Each cycle of subculturing is referred to as a “passage.” The original cells can yield millions of stem cells. At any stage in the process, batches of cells can be frozen and shipped to other laboratories for further culture and experimentation.

How do you “reprogram” regular cells to make iPSCs?

Differentiated cells, such as skin cells, can be reprogrammed back into a pluripotent state. Reprogramming is achieved over several weeks by forced expression of genes that are known to be master regulators of pluripotency. At the end of this process, these master regulators will remodel the expression of an entire network of genes. Features of differentiated cells will be replaced by those associated with the pluripotent state, essentially reversing the developmental process.

How are stem cells stimulated to differentiate?

As long as the pluripotent stem cells are grown in culture under appropriate conditions, they can remain undifferentiated. To generate cultures of specific types of differentiated cells, scientists may change the chemical composition of the culture medium, alter the surface of the culture dish, or modify the cells by forcing the expression of specific genes. Through years of experimentation, scientists have established some basic protocols, or “recipes,” for the differentiation of pluripotent stem cells into some specific cell types (see Figure 1 below).

Figure 1. Directed differentiation of mouse embryonic stem cells.Figure 1. Directed differentiation of mouse embryonic stem cells.

 

What laboratory tests are used to identify stem cells?

At various points during the process of generating stem cell lines, scientists test the cells to see whether they exhibit the fundamental properties that make them stem cells. These tests may include:

  • Verifying expression of multiple genes that have been shown to be important for the function of stem cells.
  • Checking the rate of proliferation.
  • Checking the integrity of the genome by examining the chromosomes of selected cells.
  • Demonstrating the differentiation potential of the cells by removing signals that maintain the cells in their undifferentiated state, which will cause pluripotent stem cells to spontaneously differentiate, or by adding signals that induce adult stem cells to differentiate into appropriate cell phenotypes.