Cell therapy

Cell therapy describes the process of introducing new cells into a tissue in order to treat a disease. Cell therapies often focus on the treatment of hereditary diseases, with or without the addition of gene therapy. Cell therapy is a sub-type of Regenerative Medicine.

Cell therapy, and other regenerative medicines such as tissue engineering, comprise a separate therapeutic platform technology to that of the current three pillars of healthcare: pharma, biologics and medical devices. It is certainly a disruptive technology; however, it is not new. The cell therapy initative has its origins rooted in blood transfusion, bone marrow and organ transplantation, tissue banking and reproductive in vitro fertilization. Modern cell-based therapies have progressed from the first recorded human–human blood transfusion by James Blundell (Guy’s Hospital, London, UK) through to the advanced cellular therapies of today. This 200 year journey, based initially on clinical trial and error and more recently on laboratory science, has culminated in the necessary critical mass and unique challenges to justify being a distinct industry in its own right. Thus, today cell therapy is the fourth and final therapeutic pillar of global healthcare.

There are many potential forms of cell therapy, reviewed in more detail throughout this article:
 * The transplantation of stem cells or progenitor cells that are autologous (from the patient) or allogeneic (from another donor).


 * The transplantation of mature, functional cells (Cell Replacement Therapy).
 * The application of modified human cells that are used to produce a needed substance (cell-based gene therapy).

These forms of cell therapy are not currently known to exist, but may be possible in future depending on both research outcomes and ethical concerns:
 * The xenotransplantation of non-human cells that are used to produce a needed substance. For example, treating diabetic patients by introducing insulin-producing pig cells directly into their muscle.
 * The transplantation of transdifferentiated cells derived from the patient's own differentiated cells. For example, the use of insulin-producing beta cells transdifferentiated from isolated hepatocytes as a treatment for diabetes.

History
Cellular (cell) therapy can be defined as the use of cells to treat disease. Its origins can be traced to the early 1800's, when Dr Charles-Edward Brown-Séquard (1817-1894) injected animal testicle extracts to stop the effects of aging, followed by Paul Niehans (1882-1971), who practiced cell therapy using calf embryo cells in Switzerland.

Modern cellular therapy obtained its scientific legitimacy mainly from the field of bone marrow transplantation. The first breakthrough was provided by Prof Jean Dausset who in 1952 performed the experiments that lead to the identification of the first of many HLA antigens on the surface of cells. The understanding that HLA antigens are the body’s identification and when it does not recognize the patterns of an HLA antigen an immunological response is initiated, opened the field of allogeneic bone marrow transplantation. Dr Dausset's discovery and continuous work granted him the Nobel Prize in Physiology or Medicine in 1980. In the late 1950s his discoveries led to the first successful transplant of cells between identical twin patients. The transplant was performed by Dr E. Donnall Thomas, who went on to receive the Nobel Prize in physiology or medicine in 1990.

The consequence of such discoveries was the achievement of additional milestones in the area of cell transplantation and in 1968, in Minnesota, the first successful non-twin (allogeneic) transplantation took placed followed by the first unrelated bone marrow transplant in 1973, when a boy with a genetic immunodeficiency disorder received multiple marrow transplants from a donor identified as a match through a blood bank in Denmark.

While cellular compatibility issues were being deciphered, the cell therapy area continued to bloom. Stem cells from cord blood were successfully transplanted in 1988, in Paris, when it was used for the first time to regenerate blood and immune cells on a 6-year-old boy suffering from Fanconi anemia, a blood disorder, and in 1997 a successful cord blood transplant was performed on an adult suffering from chronic myelogenous leukemia using cord blood cells that were expanded ex vivo, which means outside of a living organism.

Another scientific leap occurred in 1998, when James Thompson from the University of Wisconsin – Madison developed the first embryonic stem cell lines. The capacity to generate different cell types from a common cell ancestor under control conditions and the increase understanding on how to grow cells ex-vivo, boosted the basic research and development of potential cell products in the field of regenerative medicine. Initially the studies were aimed at replacing cells that were damaged by disease with cells grown ex-vivo, however the field evolved and cells are instead used as secretors of factors that can ameliorate or change the course of disease. Due to the ethical controversy associated with the use of embryonic cells for research purposes, the field suffered a temporary setback, however in 2006, Shinya Yamanaka's team at Kyoto University, Japan first generated induced pluripotent stem cells (iPSCs) from mouse fibroblasts and in November 2007, James Thomson at University of Wisconsin–Madison and Shinya Yamanaka and colleagues, reported the production of iPSCs from adult human cells. This scientific discovery serves as a catalyst for new research in the area of regenerative medicine and diagnostics based on cellular therapies. During the last 20 years stem cells and adult cells derived from different tissues such as blood, bone marrow, muscle, brain, hair, skin, etc., have been characterized, grown in culture and tested in animals and in the clinic for a diverse number of illnesses.

Autologous Cell Therapy
There are two general classes of cell therapy approaches to treat patients. First, cells may be harvested from a patient and treated or expanded and introduced back into the same patient. This patient-specific, or autologous, method has historically been favored due to the lack of required immunologic matching. A second approach involves the harvesting of cells from one, or a few, universal donors followed by large scale expansion and banking of multiple doses (see Allogeneic Cell Therapy below).

Allogeneic Cell Therapy
Allogeneic cell therapy involves harvesting donor cells from one, or a few, universal donors. These cells will be expanded in a manufacturing facility and cryopreserved for later manipulation or as therapeutic doses. This allogeneic approach utilizes cell types that do not elicit immune responses upon implantation and therefore have the potential to treat hundreds of patients from a single manufactured lot of cells. The allogeneic methodology fits the pharmaceutical model of drug manufacturing because the product can be readily available for “off the shelf” distribution.

Currently, there are numerous clinical trials involving allogeneic-derived adult stem cells. Increasingly, mesenchymal stem cells are being proposed as agents for cell-based therapies, due to their plasticity, established isolation procedures, and capacity for ex vivo expansion. Manufacturing models are in development for bone marrow mesenchymal stem cells (such as Osiris’ Prochymal platform) and multipotent adult progenitor cells (MSCs, such as Athersys’ MultiStem platform). Other cell types included in this model are human embryonic stem cells (hESCs, such as Geron’s hESC-based platform of differentiated cells, described further in Section 2.1), hematopoietic stem cells (HSCs), as well as engineered cells used in tissue grafts. Therapeutic targets for such allogeneic therapies include graft versus host disease, myocardial infarction, central nervous system disorders, stroke, diabetes, osteoarthritis, and other diseases.

Challenges to the allogeneic approach primarily reside in the manufacture of the cellular product. Large numbers of cells (greater than 1011-12) must be produced per lot to satisfy the larger dosing requirement (which may exceed 106 cells/kg/dose). The cells must be efficiently expanded in culture while retaining their proper cell characterization profile and efficacy. In addition, the cellular product must be amenable to cryopreservation and subsequent revival in order for the “off the shelf” production model to be successful. The successful manufacture of these products must also rely on a stringent quality control policy to demonstrate lot-to-lot consistency and safety.

Embryonic Stem Cells
Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass of the blastocyst. They have the ability to renew themselves and to differentiate into a variety of different cell types that are found in the body. Unlike somatic or ‘adult’ stem cells, hESCs proliferate indefinitely. This, together with their ability to differentiate into most adult cell types, has resulted in the preferred use of these cells for research and therapeutic applications, as they represent a potentially indefinite source of therapeutic cells. Any cell therapy derived from hESCs would be allogeneic by nature. Some current studies involve the potential therapeutic application of hESCs for spinal cord injury, age-related macular degeneration (AMD), cardiovascular diseases, and diabetes. Among the start up cell therapy companies, Geron and Advanced Cell Technologies have pioneered clinical trials using cells differentiated from hESCs.

Regulation of derivation of hESCs
Since its inception, stem cell research has remained controversial. At the heart of the debate lies research involving human embryonic stem cells (hESC). hESC research is not conducted globally due to a complete ban imposed by the governments of some countries like Italy and Austria. In places where hESC research is permitted, the regulations on hESC derivation from embryos are imposed by local governments and hence there is no global consensus on these regulations ).

See also: Stem cell research policy

While there are no consistent global regulations on deriving hESC, an international effort has been made by the International Society for Stem Cell Research (ISSCR) to unify such guidelines. The ISSCR guidelines state that informed consent should be obtained from fully autonomous individuals and that donor privacy must be maintained at all times. Also, unwarranted inducements should be avoided so as to maintain the voluntary nature of the donation. The ISSCR also requires regulatory reviews to be conducted before any embryos, gametes or somatic cells are obtained.

Scientists who wish to derive hESC from human embryos are required by the ISSCR to submit proposals to a stem cell research oversight (SCRO) body for approval and give sound scientific reasoning for the need to conduct such derivations. Furthermore, researchers are required to justify the need for the number of embryos to be used. Researchers who aim to use nuclear transfer (NT) derived embryos need to provide justification for the use of such embryos and for the number of trials to be made.

The ISSCR guidelines state that only qualified researchers who have experience is maintaining and culturing of existing hESCs together with experience in deriving pluripotent non-hESCs can attempt the derivation process. In addition, researchers are required to have a thorough, documented plan for characterisation, storage, banking and distribution of the newly created cell lines. International efforts known as the International Stem Cell Banking Initiative (ISCBI) have also been made to harmonize standards in cell banking, characterization, storage distribution. The initiative was implemented by the International Stem Cell Forum.

Use of hESCs in research
Current research focuses on differentiating hESC into a variety of cell types for eventual use as cell replacement therapies (CRTs). Some of the cell types that have or are currently being developed include cardiomyocytes (CM), neurons, hepatocytes, bone marrow cells, islet cells and endothelial cells. However, the derivation of such cell types from hESCs is not without obstacles and hence current research is focused on overcoming these barriers. For example, studies are underway to differentiate hESC in to tissue specific CMs and to eradicate their immature properties that distinguish them from adult CMs.

Besides in the future becoming an important alternative to organ transplants, hESC are also being used in field of toxicology and as cellular screens to uncover new chemical entities (NCEs) that can be developed as small molecule drugs. Studies have shown that cardiomyocytes derived from hESC are validated in vitro models to test drug responses and predict toxicity profiles. hESC derived cardiomyocytes have been shown to respond to pharmacological stimuli and hence can be used to assess cardiotoxicity like Torsades de Pointes.

hESC-derived hepatocytes are also useful models that could be used in the preclinical stages of drug discovery. However, the development of hepatocytes from hESC has proven to be challenging and this hinders the ability to test drug metabolism. Therefore, current research is focusing on establishing fully functional hESC-derived hepatocytes with stable phase I and II enzyme activity.

Researchers have also differentiated hESC into dopamine-producing cells with the hope that these neurons could be used in the treatment of Parkinson’s disease. hESCs have also been differentiated to natural killer (NK) cells and bone tissue. Studies involving hESC are also underway to provide an alternative treatment for diabetes. For example, D’Amour et al were able to differentiate hESC into insulin producing cells.

Use of hESCs in therapy
While an abundance of research on hESC is being carried out in laboratories, only three clinical trials have resulted thus far. Phase I trials using hESC derived Oligodendrocytes (GRNOPC1) developed by Geron are currently underway in human subjects to treat spinal cord injuries, and Advanced Cell Technologies (ACT) are launching Phase I/II trials in human subjects using hESC derived retinal epithelial pigment (RPE) cells in the treatment of Stargadt’s Macular Dystrophy (SMD) and age related macular degeneration:

Geron has been developing several hESC-derived products to treat spinal cord injury, cardiac and other chronic degenerative diseases. Currently, one of their products for spinal cord injury, GRNOPC1, is in a phase I clinical trial. GRNOPC1 are oligodendrocyte progenitor cells that were differentiated from hESCs. In this stem cell process, various growth factors are applied to induce hESCs into oligodendrocyte precursors. Oligodendrocytes play an important role in repairing the myelin insulation around the nerve cells so that the nerve cells may transmit signals. Upon injection of GRNOPC1 into the spinal cord injury site, restoration of motor and load-bearing functions were significantly improved in rat models. In 2009, the Food and Drug Administration (FDA) granted Geron clearance for a phase I clinical trial: a multi-center assessment aimed at evaluating the safety and tolerability of GRNOPC1 in patients with complete ASIA (American Spinal Injury Association) Impairment Scale grade A thoracic spinal cord injuries. Studies were also conducted to further assess the safety and effectiveness of GRNOPC1 in patients affected in the thoracic and cervical regions. However, due to cystic development at the injury site, the clinical trial was put on hold. New candidate markers and release specifications were subsequently established for GRNOPC1 and the phase I clinical trial resumed in 2010.

Advanced Cell Technology (ACT) is the second company in the United States to begin clinical trials on their hESC-based therapy targeting dry acute macular degeneration (AMD). Retinal pigment epithelial (RPE) cells in dry AMD degenerate and result in the breakdown of the epithelia in the patient’s macula. ACT aims to provide treatment to the disease via transplantation of hESC-derived RPE cells. Using a differentiation system free of growth factors and independent of exposure to other cell types, zoonoses-free RPE cells were produced for transplantation. Significant improvements to visual acuity were observed without any adverse effects in rat models. Like Geron, ACT will adopt a multi-center trial to determine the safety and tolerability of the RPE cells transplanted into patients with dry AMD. In following trials, ACT aims to demonstrate that these RPE cells will be able to impede the progression of the disease and improve visual acuity in patients. Given approved phase I/II clinical trial clearance, ACT will also use the hESC-derived RPE cells in a trial for Stargardt’s Disease simultaneously. A similar proposal to treat AMD with hESC-derived RPE cells is being developed under The London Project, which is expected to begin towards the end of 2011.

These clinical trials represent potential treatments for diseases that have no other treatments.

Cell Therapies derived from Adult Stem Cells
Adult stem cells are multipotent cells committed to specific lineages. They can replenish dying cells and damaged tissues by multiplying through cell division and differentiating into a subset of cell types specific to its lineage. As such, they hold vast regenerative and therapeutic potential. Furthermore, their use in cell therapy is less controversial as they can be harvested from various sources in humans whereas the use of human embryonic stem cells (hESCs) often entails destruction of human embryos. Thus, recent efforts are focused on efficient expansion and differentiation of adult stem cells for clinical purposes. Notably, there have been significant advancements in clinical applications of neural, mesenchymal and hematopoietic stem cells.

Neural Stem Cell Therapy
There have been extensive developments in the use of NSCs for treatment of neurological disorders such as lysosomal storage diseases, stroke and cancer. StemCells Inc. has proposed a clinical trial to use human central nervous system-stem cells (HuCNS-SCs) in the treatment of Batten disease and the NSC therapy for stroke patient developed by ReNeuron has reached the stage of a first-in-man clinical trial. Importantly, NSCs can also be used as possible drug delivery agents to brain tumors. An investigation by Aboody et al. described the ability of NSCs to track tumor cells and deliver cytosine deaminase, which converts a non-toxic pro-drug into a chemotherapeutic agent. This cancer treatment offers the advantage of selectively targeting and killing the cancer cells. The clinical trial for such a treatment is underway and currently recruiting participants with recurrent brain tumors. As early evidence with a mouse model suggests, NSCs could also potentially be used in the treatment of neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease.

Mesenchymal Stem Cell Therapy
MSCs are immunomodulatory, multipotent and fast proliferating and these unique capabilities mean they can be used for a wide range of treatments including immune-modulatory therapy, bone and cartilage regeneration, myocardium regeneration and the treatment of Hurler syndrome, a skeletal and neurological disorder. MSCs therapy for the treatment of graft-versus-host-disease (GVHD) and Crohn’s disease has been developed by Osiris Therapeutics and the clinical trials are largely successful and pending completion of phase III.

Researchers have demonstrated the use of MSCs for the treatment of osteogenesis imperfecta (OI). Horwitz et al. transplanted bone marrow (BM) cells from human leukocyte antigen (HLA)-identical siblings to patients suffering from OI. Results show that MSCs can develop into normal osteoblasts, leading to fast bone development and reduced fracture frequencies. A more recent clinical trial showed that allogeneic fetal MSCs transplanted in utero in patients with severe OI can engraft and differentiate into bone in a human fetus. The Children’s Hospital of Philadelphia in the United States is currently recruiting study subjects for their phase I study to assess the safety and feasibility of repeated infusions of MSCs in children with OI.

Although not conclusively proven to be effective, application of autologous BM-derived MSCs into patients with osteoarthritis has been reported recently. The usefulness of MSCs in cartilage regeneration has also been demonstrated with animal models and results look promising. Medipost Co. Ltd is currently recruiting participants for a study on the efficiency and safety of using umbilical cord blood (UCB) derived MSCs for treatment of articular cartilage defect in old patients.

Besides bone and cartilage regeneration, cardiomyocyte regeneration with autologous BM MSCs has also been reported recently. Introduction of BM MSCs following myocardial infarction (MI) resulted in significant reduction of damaged regions and improvement in heart function. Clinical trials for treatment of acute MI with Prochymal by Osiris Therapeutics are underway. Also, a clinical trial revealed huge improvements in nerve conduction velocities in Hurler’s Syndrome patients infused with BM MSCs from HLA-identical siblings.

Hematopoietic Stem Cell Therapy
HSCs possess the ability to self-renew and differentiate into all types of blood cells, especially those involved in the human immune system. Thus, they can be used to treat blood and immune disorders. Since human bone marrow (BM) grafting was first published in 1957, there have been significant advancements in HSCs therapy. Following that, syngeneic marrow infusion and allogeneic marrow grafting were performed successfully. HSCs therapy can also render its cure by reconstituting damaged blood-forming cells and restoring the immune system after high-dose chemotherapy to eliminate disease.

There are three types of HSCT: syngeneic, autologous, and allogeneic transplants. Syngeneic transplantations occur between identical twins. Autologous transplantations use the HSCs obtained directly from the patient and hence do not cause any complications of tissue incompatibility; whereas allogeneic transplantations involve the use of donor HSCs, either genetically related or unrelated to the recipient. To lower the risks of transplant, which include graft rejection and GVHD, allogeneic HSCT must satisfy compatibility at the HLA loci (i.e. genetic matching to reduce the immunogenicity of the transplant). Mismatch of HLA loci would result in treatment-related mortality and higher risk of acute GVHD.

In addition to BM derived HSCs, the use of alternative sources such as umbilical cord blood (UCB) and peripheral blood stem cells (PBSCs) has been increasing. In comparison with BM derived HSCs recipients, PBSCs recipients afflicted with myeloid malignancies reported a faster engraftment and better overall survival. However, this was at the expense of increased rate of GVHD. Also, the use of UCB requires less stringent HLA loci matching, although the time of engraftment is longer and graft failure rate is higher.

Mechanisms of Action
Cell therapy is targeted at many clinical indications in multiple organs and by several modes of cell delivery. Accordingly, the specific mechanisms of action involved in the therapies are wide ranging. However, there are two main principles by which cells facilitate therapeutic action:

1. Stem cell or progenitor cell engraftment, differentiation, and long term replacement of damaged tissue. In this paradigm multipotent or unipotent cells differentiate into a specific cell type in the lab or after reaching the site of injury (via local or systemic administration). These cells then integrate into the site of injury, replacing damaged tissue, and thus facilitate improved function of the organ or tissue. An example of this is the use of cells to replace cardiomyocytes after myocardial infarction.

2. Cells that have the capacity to release soluble factors such as cytokines, chemokines, and growth factors which act in a paracrine or endocrine manner. These factors facilitate self-healing of the organ or region. The delivered cells (via local or systemic administration) remain viable for a relatively short period (days-weeks) and then die. This includes cells that naturally secrete the relevant therapeutic factors, or which undergo epigenetic changes or genetic engineering that causes the cells to release large quantities of a specific molecule. Examples of this include cells that secrete factors which facilitate angiogenesis, anti-inflammation, and anti-apoptosis. This mode of action is proposed by companies such as Pluristem and Pervasis that use adherent stromal cells or mature endothelial cells to treat peripheral artery disease and arteriovenous access complications.

Cell Bioprocessing
Cell Therapy Bioprocessing is a discipline that bridges the fields of Cell Therapy and Bioprocessing (ie. biopharmaceutical manufacturing), and is a sub-field of Bioprocess Engineering. The goals of Cell Therapy Bioprocessing are to establish reproducible and robust manufacturing processes for the production of therapeutic cells. Commercially relevant bioprocesses will:

1) produce products that maintain all of the quality standards of biopharmaceutical drugs http://www.fda.gov/cber/gdlns/cmcsomcell.pdf

2) supply both clinical and commercial quantities of therapeutic cells throughout the various stages of development. The processes and production technologies must be scalable, and

3) control the Cost of Goods (CoGs) of the final drug product. This aspect is critical to building the foundation for a commercially viable industry.

Upstream Bioprocessing
Therapeutic cell manufacturing processes can be separated into upstream processes and downstream processes. The upstream process is defined as the entire process from early cell isolation and cultivation, to cell banking and culture expansion of the cells until final harvest (termination of the culture and collection of the live cell batch).

Downstream Bioprocessing
At the point where the cells will not be expanded any further the downstream process begins. This includes the final harvest, and subsequent process steps of concentration/volume reduction of the harvested cells, washing or clarification of the harvested cells, formulation of the cells into an appropriate solution for biopreservation and filling the formulated cells into their final container for cryopreservation and storage, or for delivery direct to clinic. When the cell drug is needed for a patient, it must be shipped under appropriate conditions to the clinical site, prepared for delivery to the patient and then administered by a medical doctor or trained healthcare professional. The final shipping and clinical manipulations are technically manufacturing steps, but may lay outside of what most bioprocessing facilities would consider to be downstream processing.

Manufacturing Challenges
The manufacturing processes for allogeneic or universal cell therapies, and autologous or patient-specific cell therapies share many of the same steps, but the two forms of commercial bioprocess will have very different challenges. Allogeneic processes must be scaled-up to an optimal lot size while maintaining the quality parameters of research or trial-sized processes. Autologous processes, conversely, must be scaled-out to where tens to hundreds of small-scale individual unit operations are be performed in parallel, as each patient constitutes their own “lot” of product. While both types of processes may be producing cells of similar identity, potency and purity, the technologies to achieve the different lot sizes are dramatically different.

Future Potential
While cell therapy describes the transplantation of stem/progenitor cells into an organism by various measures (usually intravenously), this characterization does not describe the mechanism of action by which the cells treat a disease. In cell therapy, the cells may act by secreting paracrine factors or by a receptor-ligand interaction with other cells if the immune system permits. Most work to date concentrates on this methodology.

Cell replacement therapy requires the (trans)differentiation of therapeutic stem/progenitor) cells into tissue cells of the target organ, e.g. stem cells becoming insulin-producing cells. While cell replacement therapy is the primary objective of cell therapy, it is only in clinical testing for the treatment of spinal cord injury and AMD thus far.  Tissue engineering so far has only been modestly successful as the understanding of, and ability to artificially engineer, complete organ structures is still in its early days. Rather, the future potential of cell therapy may lie in the field of modulating the immune system for the treatment of autoimmune diseases like inflammatory bowel disease or rheumatoid arthritis (RA) by the mechanisms described above.

While xenotransplantation is still in its clinical infancy, without current clinical trials and exhibiting a host of ethical and technical difficulties, another very promising field is cell-based gene therapy. This would be a sequential step towards a clinically successful gene therapy and the treatment of numerous diseases like inherited disorders or cancers. It involves the genetic manipulation of patient cells, and their subsequent re-transplantation to the patient. However, any manipulation of stem or progenitor cells in vitro (cultivation, expansion or gene transfer) is a substantial matter and therefore requires a strictly regulated environment as described in guidelines by the European Medicinal Agency (EMA) or the Food and Drug Administration (FDA) to protect the potential patient from medical malpractice and to ensure the highest degree of clinical safety.