Divide and conquer

Seperating minute sub-populations of cells has taken cell biology forward in leaps and bounds. Here, Dr Ayad Eddaoudi and Joanna Sinclair tell us that flow cytometry is more than just a pure research tool - it can also enable monitoring of patients after gene therapy

THE ABILITY to separate even minute sub-populations of functioning cells with a high degree of accuracy has taken cell biology to the next level. The new generation of flow cytometers has made this possible - with their capacity to provide very fast and precise information about even the rarest cell sub-populations often present in only minute quantities.

The potential of gene therapy opens up the tantalising possibility of curing and treating many of the genetically inherited life-threatening childhood illnesses that we see every day at the Great Ormond Street Hospital. Our location alongside the hospital provides added impetus to the researchers who use our flow cytometry facilities. We are all driven - not just by the intellectual excitement of working towards a scientific breakthrough - but by close proximity to the everyday suffering, pain and distress of the hospital’s patients. 

The technique of cell sorting is based on flow cytometry which uses light excitation, light scattering, and emission of fluorochrome molecules to produce multi-parameter data on individual cells enabling their physical and biological properties to be determined.

The first flow cytometers were developed in the 1960s and allowed cells and other particles to be counted and sorted far more easily, revolutionising research in haematology and immunology. Cell purification was initially performed using technologies such as centrifugal elution, density gradient separation and complement-mediated lysis. Not only was this time consuming - it could only purify one of a very limited type of cells each time - it was slow and could damage the cells and lead to undesirable changes in cell function and cellular activation.

Recent technological advances in flow cytometry have enabled cells not only to be identified and counted but to be sorted at high speed with far greater precision. At the facility here, the sorter we use (Beckman Coulter MoFlo XDP Cell Sorter) has the capacity to sort four different types of cells at once, at a rate of up to 70,000 cells per second. At this speed we can easily collect a few thousands of very rare cells in just one hour. By being able to separate very rare populations - without pre purification of the cells - it is possible to avoid cell damage and contamination.

We currently deliver a purity of more than 98% - and may sometimes achieve 100% depending on sample preparation and the viability of sorted cells. Cell sorters use flow cytometry to collect data on each cell and then to siphon off cells with specified characteristics, enabling large numbers of specific cell subtypes to be collected quickly. Purity is essential for the ultra-sensitive genetic analysis required with gene therapy and other genetic research. In addition, the separated cells remain fully functioning - opening the door for further research and medical applications.

Ten years ago, the treatment of the severe combined immuno-deficiency disorder SCID-X1 became the first genetic disorder to be corrected successfully by gene therapy. Scientists at the Institute helped to lead the research. To ensure the effectiveness in patients of their gene therapy treatment, they need to be monitored long term. This involves cell sorting on a regular basis to confirm that the immune system of these patients continues to function.

Estimates of between one in 50,000 to 100,000 children are born with this inherited disorder. They have no immunity to infection because of a profound reduction or absence of T lymphocyte function. In some types of SCID, B lymphocytes and natural killer cells may also be affected. The condition is more commonly known as ‘bubble baby syndrome’ because without treatment the children must be imprisoned in sterile conditions to shield them from potentially fatal infections.

The most common form of SCID is X-linked and accounts for 40-50% of cases. SCID-X1 is caused by a mutation in a gene on the X chromosome. It affects only males because they have only one copy of the gene. As females have two copies they will be able to make the protein as long as they carry one functioning copy of the gene.

Conventional therapy for SCID was haematopoietic stem cell transplantation (HSCT) with survival rates of more than 90% where children are able to receive transplants from HLA-matched donors.  However, success rates are far higher when the donor is a sibling. With less well-matched donors, even a parent, the prognosis is less optimistic. Children must often continue to be given immunoglobulin replacement therapy and antibiotic prophylaxis. In addition before HSCT can take place, patients must undergo a course of chemotherapy, with the long-term complications of infertility and growth retardation.

Gene therapy was therefore seen as a more viable alternative by researchers.  It involves harvesting bone marrow cells expressing the CD34 stem cell markers. The stem cells are cultured in a cytokine enriched medium and then exposed to a retrovirus vector containing the correct common gamma chain gene sequence. The vector enters the cells and its RNA genome carrying the common gamma chain gene is reverse transcribed to DNA so that it integrates into the host genome. The genetically modified stem cells are then administered to the infant by peripheral infusion. It usually takes four - six weeks before newly emerging immune cells are detectable and three to six months for a functional immune system to develop.

Patient blood samples are collected three months after treatment and stained with fluorescently labelled antibodies to isolate specific cell subtypes. This process is repeated regularly to monitor the regeneration of the immune system and to identify the gene insertion site within these new immune cells.

To do this successfully, we need to use a cell sorter able to separate out the cell lineage to a minimum number of 50,000 recovered cells. Each of these cell samples needs to be extremely pure to enable further DNA analysis to be carried out. This stage is vital because it must identify where the correction gene has inserted into the DNA. It is important to monitor that the integration is occurring at a random site within and between cell types. In addition to cell sorting, flow cytometric data is also obtained on the cell number and development of immune cell types during the post treatment period.

Adenosine deaminase (ADA) deficiency is responsible for around 15% of SCID. In ADA-SCID, the substrates for ADA accumulate in cells and become toxic. It is an extremely rare condition but results in the immune system being severely compromised or completely lacking.

The first attempts at gene therapy in the 1990s used patient T cells and these

Figure1: Cells expressing a gene related to sight and GFP tagged are sorted. Region 1 (R1, plot A) targets live cell population, region R2 (plot B) targets single cells and R4 (plot C, R3, plot D) gives GFP positive cells. R4 (and R3) contains GFP positive cells. MoFlo XDP cell sorter was set to sort cells that satisfy these criteria: Live (R1), single (R2) and GFP positive cells (R4). These cells are only about 40% of pre-sorted cells.

were transduced using an ADA-expressing retroviral vector and then reinfused into the patient. The resulting T cell populations were still in evidence 10 years later. Similar results have also been achieved using bone marrow stem cells, and cord stem cells, as target cells rather than lymphocytes.

However, patients have had to remain on enzyme (PEG-ADA) replacement therapy, making it difficult to clearly establish the effectiveness of the genetically modified T cell populations. Recent trials attempted to enhance the advantage of gene modified CD34 cells by withholding PEG-ADA. This has included a mild pre-conditioning chemotherapy regime to make space for the corrected cells. Using the latest cell sorting technology and anti-ADA antibody staining we have subsequently been able to isolate ADA expressing T cells as part of the ongoing monitoring of patients and therefore verify that cellular immunity has started to recover.

Flow cytometry is being used in research examining the potential use of cell

Figure 2: Sorted cells are rerun to check for purity. R4 (plot C and R3 in plot D): sorted cells are 98% pure. The 2% GFP negative have probably lost GFP after cell sorting because of cell death

transplantation for the treatment of retinal degeneration, which is the leading cause of blindness in the Western world. This is an inherited disorder and for which there is no current cure. 

A defect in a single gene is responsible for photoreceptor loss in around one in 3000 people.  In terms of potential for repair of the central nervous system by cell transplantation, retinal damage has the greatest potential. As photoreceptor degeneration initially leaves the inner retinal circuitry intact, new photoreceptors need only make single short synaptic connections.

It has been demonstrated that cell transplantation is technically feasible but only if the donor cells are at a certain stage of development.2 They must be post-mitotic photoreceptor precursor cells - ones that have left the cell cycle and have started to mature into the adult photoreceptor.

The exit of these cells from the cell cycle correlates with the expression of a number of genes. It is possible to tag the expression of one of these genes with the reporter - green fluorescent protein (GFP). By combining this with cell sorting, it becomes possible for the scientist to single out just those precise cells from a mixed donor population; and then to transplant  200-800 of these cells per eye into a sightless model, enabling some restoration of visual function. 

Around 120 sight genes have now been identified and the focus of research is on early onset severe retinal degeneration, particularly Leber’s congenital amaurosis, because it is believed this will offer the best chance of success

Cell sorting has the potential to help scientists in the treatment of immunological disorders, degenerative diseases and even help provide treatments for HIV and cancer. As more genes responsible for cellular malfunctions in disease processes are identified through genetic mapping, the requirement of high-speed precision sorting of cells has the potential to play an even greater role in aiding research.

The flow cytometry facility at the Institute of Child Health has the very latest in high-speed cell sorting and analysis technology and is open to both internal and external researchers. Scientists come to the laboratory to talk through their experiments and then make a formal application to use the facility. The precision we offer enables them to work with almost 100% purity at all stages, including being able to monitor the effectiveness of the potential treatments under investigation.