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| Dr Aparna Khanna |
Nanoparticles are promising tools for various applications in biomedical research due to their small size and ability to penetrate cells. Stem cell research is a rapidly evolving area, despite being fraught with innumerable challenges and hurdles that are yet to be overcome to realise its ultimate potential. There has been a recent spate of activities with respect to the clinical application of foetal, cord and adult stem cells. Though the appears promising, there are severe limitations to stem cell based therapies. One major obstacle has been monitoring the bio-distribution and homing of implanted or injected stem cells in the human body. Hence, an urgent need exists to develop non-invasive and sensitive imaging techniques, which will prove valuable for optimising cell therapy.
The article outlines the therapeutic potential of stem cells, several hurdles faced by clinicians for optimising cell therapy and how some of the challenges can be overcome by using sensitive non-invasive imaging technology.
Stem cells: Sources, types and clinical application
Stem cells have two specialised properties that make them unique and unlike any other cell of the human body. First is their indefinite proliferative ability; which simply means that you can have an unlimited and inexhaustible supply of these cells. The second feature of these highly prolific cells, is their inherent capacity to differentiate upon receiving appropriate stimuli, into various cell types of the human body. The most versatile of the stem cells reported are the human embryonic stem cells (hESC). The source of the hESC are surplus embryos (five-day blastocyst), which can be obtained from in vitro fertilisation clinics, with proper informed consent and following national necessary ethical standards. Subsequently, a hESC line is established from the surplus embryos, brought to the laboratory and warrants a procedure that requires skilled technical expertise and takes about 8-10 months. So, from a five day blastocyst, a hESC line is established, which is said to be virtually immortal and at your disposal, possessing the potential to differentiate into neurons, oligodendrocytes, cardiac cells, pancreatic islet cells, hepatocytes; theoretically, to almost all the 210 cell types found in the human body. Such is the tremendous potential of stem cells and its use in regenerative medicine, that damaged or diseases cells can be replaced with the cell types derived from stem cells. The first hESC line established in the world was in 1998, by Dr James Thomson and co-workers at the University of Wisconsin1.
The other commonly reported sources of stem cells are bone marrow, umbilical cord blood, umbilical cord and adipose tissue/lipoaspirates. These sources are termed as “multipotent”, unlike the hESC which are termed as “pluripotent”, because of the restricted differentiation potential associated with adult stem cells. Adult stem cells are not as versatile as embryonic stem cells. More recently, another source of stem cells were discovered through a break-through research. These are the induced pluripotent stem cells (iPSC)2. Here, normal skin cells can be transformed (reprogrammed) into pluripotent stem cells using genetic manipulation. This implies that if this concept works in a clinical setting, the need for embryos would be eventually removed, and patient-specific stem cells could be made, thus enabling customised therapy. For their pioneering research on reprogramming, this year, the 2012 Noble prize in Physiology and Medicine was fittingly won by Sir John Gurdon, University of Oxford and Dr Shinya Yamanka, Kyoto University, Japan.
The goal of stem cell research is to obtain functional cells which can replace damaged cells in a variety of degenerative or debilitating disorders like Parkinson’s disease, type I diabetes, spinal cord injuries, liver diseases, to name a few. Fig 1 depicts a schematic representation of the use of stem cells in regenerative medicine.
Stem cell therapies: Where are we?
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| Figure 1 |
The therapeutic potential of stem cells is immense. Two modes of stem cell transplantation exists- patient specific (autologous), and not patient specific, i.e. from one individual to another (allogenic). One of the earliest examples of stem cell transplantation has been bone marrow transplantation, following which was the cord blood stem cell transplantation. Here, the haematopoietic stem cells (HSC) from an HLA matched individual is injected intravenously, and is widely used in haematological disorders or malignancies like acute and chronic myeloid and lymphoid leukaemia, myelodysplasias, bone marrow failure syndromes, haemoglobinopathies and immune deficiencies. Since the first cord blood transplant was performed in 1988, stem cells derived from umbilical blood have been used in more than 30,000 transplants worldwide to treat a wide range of blood diseases, genetic and metabolic disorders (US Cord Blood Banking Industry Report, 2012).
Cell transplantation has emerged as a potential therapy for Parkinson’s disease and has been under rigorous experimentation, both in animal models and human patients. In two well-defined double blind trials, carried out in the US, in the 1990s, the possibility of using stem cells from aborted foetuses (foetal embryonic transplantation) was investigated3,4 and provided a cell therapy paradigm for neuronal repair in the human brain. This was one of the first published reports and the concept that stem cells could help in neuronal repair emerged. However, some of the limitations included; donor dependent variation, scarcity of foetal tissue, number of cells required and ethical concerns. Additionally, a fraction of patients suffered from dyskinesia (jerky movements) after transplantation, either due to the stem cells or the procedure (sterotactic surgery). Further, in a recent report foetal stem cells were injected into a patient’s brain, who suffered from a rare genetic disorder, ataxia telangiectasia, and it triggered tumours5. Hence, a word of caution is to be aware of the adverse reaction and associated problems.
Adult stem cells, the mesenchymal stem/stromal cells (MSC) are another population of stem cells found in bone marrow and in a number of other tissues, and have used for stem cell therapy. The plethora of diseases that can be cured using MSCs are graft versus host disease (GVHD), Crohn’s disease multiple sclerosis (MS), motor neuron disease (MND) and ALS, Parkinson’s disease (PD), diabetes mellitus (DM), chronic obstructive pulmonary disorder (COPD), acute myocardial Infarction (MI), dilated cardiomyopathy (DCM), osteogenesis imperfecta, osteodysplasia and liver failure to mention a few.
The details of the ongoing/completed clinical trials can be obtained from www.clinicaltrial.gov, a registry of the US National Institute of Health (NIH). ClinicalTrials.gov currently lists 4196 ongoing clinical trials using stem cells, with approximately 90 per cent of the studies using adult stem cells.
Similarly, in India all clinical trials being carried out using human participants have to be registered at the Clinical Registry-India, a site maintained by the Indian Council of Medical Research (ICMR). According to the registry, currently there are 29 ongoing studies registered using stem cells. The details are given at www.ctri.nic.in.
Despite, the promising clinical data, a number of unanswered questions with respect to the bio-distribution of stem cells in the human body, homing or movement of the injected stem cells into desired organs and their long term effects need to be addressed. These issues can be overcome by developing sensitive non-invasive imaging modalities, which will prove valuable in optimising cell based therapies. The treating physician should be able to address questions with respect to number of cells to be injected, viability of cells after injection and the migration pattern to the targeted organ.
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| Figure 2 |
Nanotechnology and biomedical applications
Nanoparticles, as the word suggests, are particles with size in the “nanó” range that is 1-100 nm. The advantages of these very small sized particles are that they can traverse through blood vessels, can be administered either intravenously, oral route or inhalation. They can also be targeted to reach specific organs or tissues in the human body. For biomedical applications, it becomes essential to ensure that the nanoparticles synthesised possess characteristics such as biocompatiblilty, indicating safety and non-toxicity; otherwise they will be rejected by the body. Further, they have to be retained in vivo for reasonable long periods. Various methods have been employed to synthesise nanoparticles, which involves basic and inorganic chemistry. With the advancements in synthetic chemistry, nanomaterials can be modified to required size, shapes and properties. Due to their small size and high surface energy, the bare nanoparticles tend to aggregate. Hence, they need to be coated with suitable agents so as to increase their stability and solubility. A number of approaches are used to functionalise nanoparticles, involving the use of materials such as proteins, polysaccharides and synthetic polymers.
Once the nanoparticles are synthesised, detailed characterisation should be performed to understand whether the properties of the nanoparticles with respect to its size, coating are retained. Detailed characterisation entails sophisticated instrumentation like UV-vis spectroscopy, fourier-transformed infra red spectroscopy (FT-IR), X-Ray diffraction (XRD), transmission electron microscopy (TEM), etc. The ultimate challenge is to design multiplex systems for drug delivery, imaging and therapeutics, using nanotechnology. Fig 2 shows a schematic structure of a functionalised super paramagnetic iron oxide nanoparticle (SPIO) with a tagged fluorophore to allow in vivo imaging.
In vivo imaging technology for tracking stem cells
Various imaging modalities available for cell tracking are computed tomography (CT), positron emission tomography (PET), magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT) and optical imaging. Each of these methods have their pros and cons. Some of the major hurdles are insensitive contrast agents, short half life, dilution of signal because of cell division, and genetic modification of stem cells, posing regulatory concerns for human stem cell clinical trials. Hence, it is recommended that a multimodality approach that would ensure sensitivity and reproducibility should be used for in vivo tracking. Such dual optical/MRI have been described using visible-wavelength fluorophores and Gd3+ chelators conjugated to high-molecular-weight scaffolds such as dextran.
Magnetic iron oxide nanoparticles are primarily used in medical resonance imaging (MRI), serving as excellent contrast agents. Magnetic nanoparticles can be classified into a few groups like superparamagnetic iron oxide particles (SPIO) or ultrasmall SPIOs (USPIO) and function by creating local field inhomogeneities that cause decreased signal on T2- and T2*-weighted images in a MRI scanner. Besides, they help to distinguish between soft tissues; the iron content is biodegradable and biocompatible and excess iron will be recycled by the cells using biochemical pathways for iron metabolism. Moreover, as the magnetic properties of the cells are retained after coating, these properties can be exploited to deliver the cells at appropriate sites in the human body.
Several clinical studies utilising magnetic nanoparticles to label cells have been conducted. Some of the clinically approved iron oxide particles are Feridex/Endorem (dextran coated SPIO), Resovist (SPIO particles that are coated with carboxydextran).The first report involving magnetically labeled stem cells was from a patient with brain trauma who received an autologous transplant of Feridex-labeled neural stem cells (NSCs) into the damaged temporal lobe6. NSCs were isolated from exposed neural tissue (brain injury region) from the patient. Cells were cultured to select for neural progenitor cells and labeled with Feridex prior to stereotactical transplantation. The patient was then imaged with a 3.0-T MR scanner weekly for 10 weeks after transplantation. Hypointense signals at the injection sites were only observed after transplantation and the signal persisted for seven weeks.
Although there are a few encouraging results on stem cell tracking, some of the commercially available magnetic iron oxide particles have certain limitations, and the search is on for an ideal biocompatible nanoparticles for use in stem cell tracking in humans.
Research is underway at the School of Science, NMIMS, in collaboration with IIT, Mumbai, where we are in the process of designing novel nanoparticles (SPIO) to be used for long term stem cell tracking. We have been able to synthesise homogenous magnetic iron oxide particles, sized 7-9 nm (see inset) and have performed in-depth characterisation of the generated SPIOs. Further, the detailed cytotoxicity and stem cell-nanoparticle interaction, in vitro are in progress. The ultimate aim of the research is to select the most promising magnetic nanoparticles generated during the in vitro studies, for preclinical studies, to enable tracking of the stem cells in vivo.
References:
1. Thomson, JA; Itskovitz-Eldor, J; Shapiro, SS; Waknitz, MA; Swiergiel, JJ; Marshall, VS; Jones, JM. Embryonic stem cell lines derived from human blastocysts. Science 1998, 282 (5391), 1145–1147.
2. Takahashi, K; Tanabe, K; Ohnuki, M; Narita, M; Ichisaka, T; Tomoda, K; Yamanaka, S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 2007 131(5), 861-72.
3. Freed, CR; Greene, PE; Breeze, RE; Tsai, WY; DuMouchel, W; Kao, R; Dillon, S; Winfield, H; Culver, S; Trojanowski, JQ; Eidelberg, D; Fahn, S. Transplantation of embryonic dopamine neurons for severe Parkinson’s disease. N Engl J Med. 2001, 344, 710-19.
4. Olanow, CW; Goetz, CG; Kordower, JH; Stoessl, AJ; Sossi, V; Brin, MF; Shannon, KM; Nauert, GM; Perl, DP; Godbold, J; Freeman, TB. A double-blind controlled trial of bilateral fetal nigral transplantation in Parkinson’s disease. Ann.Neuro. 2003, 54, 403-414.
5. Amariglio, N; Hirshberg, A; Scheithauer, BW; Cohen, Y; Loewenthal, R; Trakhtenbrot, L; Paz, N; Koren-Michowitz, M; Waldman, D; Leider-Trejo, L; Toren, A; Constantini, S; Rechavi, G. Donor-derived brain tumor following neural stem cell transplantation in an ataxia telangiectasia patient. PLoS Med. 2009 Feb 17;6 (2):e1000029.
6. Zhu J, Zhou L, XingWu F. Tracking neural stem cells in patients with brain trauma. N Engl JMed 2006, 355:2376–2378.
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