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Guest Commentary: Combinatorial screening and the future of therapeutics
December 2017
by Dr. Shahzad Ali of Plasticell  |  Email the author
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The purpose of regenerative medicine is to replace or regenerate diseased cells, tissues or entire organs to restore or establish normal function. This may be achieved by a variety of means, including the use of cell and gene therapy, tissue engineering or regenerative drug therapy.
 
The pioneering regenerative medicine therapy was the allogeneic transplantation of bone marrow hematopoietic stem cells (HSCs), which can be used to both replace blood stem cells as well as reconstitute the full blood lineage spectrum. Decades later, this remains a gold standard in care for many hematological disorders.
 
For all the developments that have occurred in this field, however, a number of limitations remain. One is the need to match human leukocyte antigen (HLA) groups between donors and recipients in order to avoid the prospect of transplant rejection, a problem which is remedied somewhat with the use of umbilical cord blood, a more primitive form of blood cell than bone marrow and thus less requiring of exact donor-recipient HLA matching.
 
Another issue is the discrepancy between supply and demand. This is due to a rapidly aging population, the cell volumes required per transplantation, a shortage of bone marrow donors and some uncertainty over whether existing stocks of frozen umbilical cord blood cells can meet current and future demand.
 
A highly promising solution to this problem is to use ex-vivo expansion to boost HSC numbers prior to transplantation. However, existing methods of HSC expansion remain relatively inefficient and expensive due to reliance on expensive cytokines.
 
Challenges and advances
 
The discovery of human pluripotent stem cells (hPSCs) such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) has created another viable source of cells to meet future demand, due to their ability to expand almost indefinitely in in-vitro culture. Furthermore, their ability to differentiate into any somatic cell (this quality is known as pluripotency) has opened up the possibility of developing cellular therapies for a host of diseases for which there are no current cures, such as Parkinson’s disease, age-related macular degeneration and diabetes.
 
This approach also provides a defined and scalable method for the generation of rare progenitor cell types that can be further used for discovery of regenerative drugs with a capacity to stimulate tissue regeneration in vivo. However, as valid safety concerns remain over direct transplantation of hPSCs, these cells require in-vitro expansion and differentiation to direct them to their target cell or lineage of interest in order to fulfill their therapeutic potential. Unfortunately, current strategies to do so remain inefficient and costly.
 
The latest exciting development in the regenerative medicine space has been the emergence of cellular gene therapies, targeting a host of (mainly oncological) disorders. Whilst these have mainly been limited to blood cancers so far, the major players in the industry remain optimistic that these therapies will eventually be able to treat solid tumors in the future.
 
The two most promising therapeutic strategies in this area are engineered T cells and the gene editing of diseased cells (using either of or a combination of traditional viral vectors and novel gene-editing tools such as CRISPR/Cas9) in patients suffering from genetic disorders.
 
The outstanding efficacy of one such therapy, CTL019 (developed in collaboration between Novartis and the University of Pennsylvania), has resulted in a pioneering FDA approval for the treatment of B cell precursor ALL. However, one cause for concern in the case of autologous therapies is that diseased tissue from the patient subjected to the manufacturing process often expand inefficiently compared to the cell line or tissue used to develop the process. In terms of gene editing, the excitement surrounding the recent approval of Strimvelis, GSK’s gene therapy product for the treatment of ADA-SCID, has led the way for a host of therapeutic programs looking at rare genetic diseases for which there are no current cures. Being mainly autologous therapies does mean, however, that manufacturing processes for such technologies remain expensive, reflected in higher costs being passed on to healthcare systems. Furthermore, the use of autologous tissue also raises similar prospects of process variability, as discussed previously.
 
How does cell-based combinatorial screening work?
 
To exploit the capabilities of such cells for use in regenerative medicine and drug discovery we must be able to direct their differentiation (in the case of stem cells) and promote their expansion in vitro in a manner that is efficient, reproducible and cost-effective. Cell-based combinatorial screening technology was developed in order to directly confront this challenge, leading to rapid and efficient identification of optimized protocols for cell expansion and cell differentiation, as well as the provision of human cells for use in drug discovery. This technology is capable of testing thousands of combinations of cell culture variables simultaneously by miniaturizing and multiplexing large numbers of stepwise cell culture experiments, increasing throughput by orders of magnitude.
 
The combinatorial screening process begins with the design of an experimental matrix. In the case of stem cell differentiation, for example, this may be a series of consecutive culture steps, reflecting development in the embryo or adult from stem cells to progenitors and finally to functional mature cell types. Each of these steps contains a range of discrete media conditions consisting of known agonists and inhibitors for a particular developmental pathway.
 
Some of these conditions may contain small-molecule agents that can target similar pathways to cytokines but in a manner that is substantially more cost-effective. Each media condition is in turn labelled with a specific fluorescent tag. The cells to be tested are then either seeded onto microcarrier beads (in the case of adherent cells) or encapsulated within hydrogel beads (in the case of suspension cells). These beads are then equally distributed amongst the culture conditions of the first step.
 
Following a defined time period, beads are pooled together, washed and randomly distributed into the next stage of conditions, fluorescently tagged in a way similar to that described above. The beads proceed sequentially through the matrix until the combinatorial screening process is complete.
 
At the end of the process, cells on/in beads are immunostained with an antibody marking a particular phenotype (alternatively cells can be GFP-labelled in the case of transcription factors difficult to access using antibodies). Beads can then be sorted based on whether they are positive or negative for a certain marker of interest. Cells on positive beads can then be dissociated from their parent beads and analyzed using flow cytometry. Flow cytometry analysis of the tags associated with each cell gives access to a unique cell culture history for each positive bead through the combinatorial screening matrix.
 
By grouping together positives that have followed similar pathways or families of pathways, we can choose a list of cost-effective combinatorial cell culture protocols capable of significantly increasing the efficiency of cell-based processes such as expansion, differentiation and lentiviral transduction. Ideally, analysis and grouping should be performed using a bioinformatics tool in order to significantly reduce the manual computation effort required in choosing small numbers of protocols from the vast data space of the initial matrix.
 
A recent development in combinatorial screening has been the ability to multiplex cell lines. By differentially tagging cell lines, we can identify protocols or families of protocols maximally efficient for a particular cell line or even protocols that work across multiple cell lines. This is a direct route towards addressing the challenge of reproducibility of protocols across cell lines or donor tissue, as described earlier.
 
Conclusions
 
Regenerative medicine offers a highly promising new therapeutic paradigm for fighting a host of severe unmet clinical needs, many of which are likely to create an unsustainable burden on healthcare systems as future lifestyle and demographic changes inevitably occur. Whilst these therapies have drawn much attention due to their impressive efficacies in clinical trials, they have drawn equal attention due to their expensive costs and questions over reproducibility. Cell-based combinatorial culture offers an opportunity to increase the efficiency and reproducibility of cell bioprocesses, whilst reducing associated costs substantially. Despite the challenges mentioned, through the combined efforts of academia, traditional pharmaceutical and newer biotechnology companies, there is much reason for optimism for the future of regenerative medicine.

Shahzad Ali, Ph.D., is a senior research scientist at Plasticell Ltd. Part of his role at the company has been to lead a project aimed at identifying natural compounds to induce the formation of brown fat tissue, which is known to reduce the risk of metabolic diseases such as obesity and type 2 diabetes. In parallel, Ali also has worked on a number of projects differentiating induced pluripotent and human embryonic stem cells into various blood lineages such as platelet-producing megakaryocytes and red blood cells and he has been working with Ariadne, Plasticell’s bioinformatics software, for the selection of protocols for the differentiation of stem cells.

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