Growing the potential of stem cells

Stem cells have become one of the most promising areas of biomedical research, yet there are many problems to overcome - could flow cytometry get the technology to its full potential?  

The market for stem cell research was fairly small in 2005 but three years later expanded to $87 million. Robyn Young, CEO of consulting firm PearlDriver Technologies, predicts that stem cell therapeutics could become an $8.5 billion business within a decade. 

Stem cells can be broadly classified as either embryonic stem cells or adult stem cells.  Embryonic stem cells, which are derived from the inner cell mass of the blastocyst, are pluripotent, meaning they have the capacity to differentiate into all cells of a living organism. Mouse embryonic stem cells were first isolated in the 1980s, and human embryonic stem cells (hESCs) were successfully isolated and cultured a little over 10 years ago1-4. Much research has focused on differentiating both mouse and human embryonic stem cells into various somatic cell types relevant to disease, such as hepatocytes, cardiomyocytes, neurons, hematopoietic progenitors and pancreatic beta islets5.  

In contrast, adult stem cells exist in many tissues of adult organisms and have limited differentiation potential. Examples include hematopoietic stem cells that give rise to blood cells, neural stem cells that give rise to cells of the nervous system and mesenchymal stem cells that can give rise to fat, bone and cartilage. Clinical trials using many adult stem cells are ongoing, and there is a strong push from many companies to bring hESC-derived cells to the clinic as many animal transplantation models have shown encouraging results6,7. Furthermore, pluripotent stem cells can also be created by reprogramming somatic cells to induced pluripotent stem cells (iPSCs)8. This discovery has changed the landscape of stem cell biology, opening up exciting new areas of research, including the generation of autogenic cell types for cell therapy and for generating in vitro disease models from diseased individuals9. 
 
With all of these advancements, many obstacles must still be overcome in order to realise the full potential of stem cells for both in vitro and in vivo applications. One issue is that differentiation methods for both adult and pluripotent stem cells can result in heterogeneous and variable cell populations. This heterogeneity can hamper downstream studies requiring consistent, defined or pure cell populations, such as micro arrays, transplantation and cell-based assays. In addition, an understanding of the molecular and biochemical events that occur during stem cell self-renewal and differentiation is required. Finally, cells used in stem cell therapy need to be well characterised and purified from potential contaminants that can lead to tumour formation.

Flow cytometry is one technology that is being employed to address these problems by allowing the identification, analysis and isolation of cells based on their light scattering properties, including size and granularity, and their protein expression patterns. The latter is made possible by utilising multiple antibodies conjugated with fluorescent dye or proteins. This multiparametric approach enables researchers to analyse both homogeneous and heterogeneous cell populations, at a single cell level. Tens to hundreds of thousands of cells can be analysed from each experimental sample, enabling researchers to accurately discern cell fate and function by examining self-renewal and differentiation states, as well as cellular processes, such as the cell cycle, apoptotic pathways and cell signalling. In addition, specialised flow cytometers enable cell sorting of live cells, which is a powerful tool for enriching or purifying cells of interest from heterogeneous populations of cells. 

Figure 1:  Characterisation of the hESC line, H9 using the BD StemFlow Human and Mouse Pluripotent Stem Cell Analysis Kit.  The majority of the cell population expresses the pluripotent stem cell markers Oct3/4 and SSEA-4.  Only a small percentage of these cells are differentiating, which are marked by the expression of SSEA-1.  Stained cells were run on a BD LSR II flow cytomter.

Many companies have responded to the needs of stem cell researchers by producing fluorochrome-conjugated antibodies to a variety of stem cell markers, as well as kits and panels that facilitate technology adoption. Figure 1 illustrates characterisation of the hESC line, H9 using the BD StemFlow Human and Mouse Pluripotent Stem Cell Analysis Kit (RUO), which was designed to streamline multi-colour flow cytometry experiments of pluripotent stem cells. In addition to these reagents, stem cell biologists are making use of the numerous, flow cytometry-validated antibodies to cell surface markers that were developed over decades in the study of hematopoiesis10. While a majority of these antibodies were first validated on terminally differentiated cells of hematopoietic and endothelial origin, they are proving to be useful reagents for identifying cell-surface antigen signatures of both pluripotent and adult stem cells and their differentiated progeny.

This method of using antibodies to define cell-surface antigen signatures is known as immunophenotyping and can be performed using flow cytometry or imaging techniques.  It can be used to screen both homogeneous and heterogeneous cell populations to identify prospective antibody combinations for future cell sorting and analysis applications.  Although a powerful approach, immunophenotyping is limited to the hundreds of available antibodies to cell-surface markers, which only cover a fraction of the cell-surface proteome. Moreover, general unbiased screens using the hundreds of available antibodies are hampered due to the costs associated with purchasing individual vials of each antibody. For this reason, most screens are biased approaches using a limited number of antibodies. To meet the need for large cost-effective immunophenotyping screens, BD Biosciences introduced the BD Lyoplate Cell Surface Marker Screening Panel (RUO), a microtiter plate-based assay system that incorporates 242 validated antibodies to known cell surface markers in lyophilised form. Figure 2 illustrates some of the positive and negative markers identified using this kit in a screen on the hESC line, H9. 

Figure 2:  Immunophenotyping of the hESC line, H9 using the BD Lyoplate Cell Surface Marker Screening Panel.  Cells stained positive CD9, CD30, CD49b, CD50 and CD81, and were largely negative for CD15, CD20 and CD26.  These data represent 8 of the 242 antibodies which identify the unique cell-surface antigen signature of this cell population of H9.

RNA analysis by deep sequencing or microarray methods can also been used to examine cell surface marker expression. Although this provides a large range of analytic targets, one cannot be certain that RNA expressed within cells is translated into proteins, or that antibodies to those proteins will become available. Moreover, some antibodies to cell surface antigens are specific for particular post-translational modifications, such as glycosylation, and these modifications (or lack thereof) cannot be gleaned from RNA sequences alone. Also, RNA analysis is best performed on homogeneous cell populations and cannot be used to identify prospective subpopulations as in immunophenotyping approaches.

Immunophenotyping methods have been used to scrutinise a number of adult stem cells, such as hematopoietic stem cells and mesenchymal stem cells, as well as human embryonic stem cells and their derivatives. Based on multiple studies, the International Society for Cell Therapy has suggested an immune signature that all MSC lines should possess to be considered a bona fide MSC, and research is ongoing to further refine this signature and correlate cell-surface marker expression to the fate, function and therapeutic efficacy of MSCs11-13. 

Immunophenotyping of hESC-derived cells is proving to be equally fruitful.  Pruszak et al., used a set of cell-surface markers to distinguish neural stem cells, neural crest-like and mesenchymal phenotypes from neuroblasts and neurons14. The objective was to isolate cell types suitable for forming neuronal grafts and to eliminate cells with the potential to form tumours in vivo. In earlier work, this group demonstrated a general flow cytometry-based method for separating neuronal cells from their stem cell precursors15.  Similarly, Sundberg et al., discovered that the cell surface marker, CD326, was a useful marker for distinguishing residual hESCs from neuronal precursors in neural induction cultures16.  Zhao et al., has recently reported the generation of hepatic progenitor cells from human embryonic stem cells17. By screening for antibodies that detected their cells of interest, this group was able to purify these progenitors using flow cytometry.

Overall, stem cell biologists are utilising flow cytometry in numerous ways, such as in the development of antibody panels for quality control assays and in the discovery of cell-surface signatures to purify cells for transplantations and in vitro assays. To define cell-surface signatures for a number of specific therapeutically relevant cell types derived from stem cells, the use immunophenotyping by researchers will expand. For example, it is possible that cell surface signatures could be discovered for the isolation of specific neural subtypes such as dopaminergic neurons or for cardiac cell subtypes, such as atria- or ventricular-specific cardiomyocytes. Additionally, cell surface signatures will continue to be discovered that identify potentially tumour-forming cells in a number of stem cell culture systems, so they can be effectively removed from cell populations prior to grafting. As the field of stem cell biology continues to grow, so will the potential need for clinical-grade cell sorters and reagents to enable these cell therapy applications. Overall, flow cytometric techniques align with information gaps in the field of stem cell biology and facilitate the growth of the field and enable clinical translation.