Stem Cells
randomly, stepwise through multiple differentia- tion media using a split-pool method, systemati- cally sampling all possible combinations of media in a predetermined matrix (Figure 2). The tagging system allows the cell culture history (ie differenti- ation protocol) of beads bearing cells of the desired lineage to be deduced. Bespoke bioinfor- matics software, which uses criteria such as hier- archical clustering and probability analysis is used to analyse the positive protocols and select the optimal ones for further validation. The system has been successfully used to discover novel dif- ferentiation protocols for many different starting stem cell types and differentiated progeny, eg hepatocytes, neurons and osteoblasts from hES, mES and hMSCs. Since large numbers of condi- tions can be tested in each screen it is possible to efficiently discover optimised protocols that have advantages over more traditional cell culture methods, eg are serum-free, use only small mole- cules or exclude other variable and expensive products. For example, a screen of 10,000 proto- cols identified serum-free, feeder cell-free proto- cols for the generation of megakaryocytes (platelet precursor cells) from hES cells. In several of these protocols growth factors were replaced with small bioactive molecules.
Several groups have taken the approach of using automated robotic cell culture systems to screen multiple growth and differentiation conditions in multiwell format. These are typically coupled with an automated screening readout such as high con- tent analysis platforms that enable simultaneous assessment of multiple cellular features in an auto- mated and quantitative way. In particular, focus has been on the screening of small molecule libraries for their effect on self-renewal and stem cell differentiation21-23. The use of small molecules in place of standard growth factors and cytokines is preferable in terms of increasing reproducibility and cost-effectiveness. In one example, Studer’s group performed an automated screen of more than 2,900 compounds for their effects on hES cell fate. Following compound treatment, cells were assessed by automated immunostaining and high content analysis. Four compounds were identified that support short-term self-renewal of hES cells in the absence of factors normally required, while 10 compounds were identified that resulted in early differentiation, inducing commitment to different lineages, ie trophectoderm, mesendoderm and neurectoderm24. In another example, more than 5,000 small molecules were screened for their effect on pancreatic differentiation of hES cells using high-content analysis of pdx-1 expression as
Drug Discovery World Winter 2011/12
a readout. One compound in particular was found to promote efficient generation and expansion of pancreatic progenitor cells23.
2. Lineage selection
Although the addition of optimal combinations of soluble factors can direct the differentiation of stem cells to a particular lineage, enriching for a chosen cell type, purity can vary extensively and never reaches 100%. For many applications there is therefore the need to purify populations of a specific cell type from the heterogeneous mix gen- erated even during directed stem cell differentia- tion. Lineage marking and lineage selection strategies allow for the identification and selec- tion of specified cell populations and the elimina- tion of cells which are not of interest, generating highly purified populations of cells. The targeted introduction of a reporter gene such as green flu- orescent protein (GFP) to one allele of a lineage restricted gene makes it possible to monitor appearance of that lineage during stem cell differ- entiation and to purify a specific population of GFP-expressing differentiated cells by fluores- cence-activated cell sorting (FACS). Similarly, tar- geted insertion of a drug resistance gene (eg neomycin) enables the purification of a popula- tion of lineage restricted cells by positive selection with drug treatment. Such a strategy was used by Pfizer in the generation of neurons from mES cells for a high throughput drug screen (cf Stem Cell Applications)7. With recent advances in the effi- ciency of genetic manipulation in hES cells, in particular for homologous recombination, such strategies are increasingly being applied in hES cell differentiation. For example, hES cells have been generated that contain a neomycin resistance gene under the control of the lung alveolar type II (ATII) specific gene, SPC. Exposure of these cells to G418 during differentiation, resulted in gener- ation of more than 99% pure populations of ATII cells which were morphologically and functional- ly normal. Without G418 selection only 12% of cells were of an ATII phenotype25. In another example, an eGFP gene was targeted to the Nkx2.5 allele in hES cells and eGFP expression used to track cardiac differentiation and to purify committed cardiac progenitors and cardiomy- ocytes26. Negative selection strategies can also be applied through the targeting of suicide genes such as thymidine kinase under the control of lin- eage specific promoters allowing unwanted cells to be eliminated from cultures27. This would be particularly valuable in the development of cell therapies from ES cells where transplant of con-
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