Journal of Regenerative MedicineISSN: 2325-9620

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Commentary, J Regen Med Vol: 2 Issue: 1

Use of Immortalized Differentiated Cells for Regenerative Medicine

John A Loudon*
1Wetherill Park Medical Centre, Australia
Corresponding author : John A Loudon, PhD
Wetherill Park Medical Centre, Suite 101, Stockland Mall, Polding St., Wetherill Park, Sydney, NSW, 2164, Australia,
Tel: +61-2-9756-3636; Fax: +61-2-9756-3637
Received: January 27, 2013 Accepted: March 16, 2013 Published: March 21, 2013
Citation: Loudon JA (2013) Use of Immortalized Differentiated Cells for Regenerative Medicine J Regen Med 2:1. doi:10.4172/2325-9620.1000105


Use of Immortalized Differentiated Cells for Regenerative Medicine

Stem cells are touted to offer a potential avenue for curing many conditions such as diabetes, cardiovascular disease and Alzheimer’s disease. The comments raised were of particular significance in regards hurdles for the use of stem cells in regenerative medicine. These include in vivo issues such as cellular survival, senescence, proliferative capacity and differentiation into a functional tissue.

Keywords: Autologous cell harvesting; Cell-based therapies; Cell immortalization; Cell reprogramming; Cell senescence; Contact inhibition; Differentiation; Ex vivo genetics; Human telomerase; MicroRNAs; Oncogene; Pluripotency; Prosurvival factors; Stem cell; Tissue microenvironment; Tissue regeneration; Tumorigenicity; Tumor suppressor


Autologous cell harvesting; Cell-based therapies; Cell immortalization; Cell reprogramming; Cell senescence; Contact inhibition; Differentiation; Ex vivo genetics; Human telomerase; MicroRNAs; Oncogene; Pluripotency; Prosurvival factors; Stem cell; Tissue microenvironment; Tissue regeneration; Tumorigenicity; Tumor suppressor

To the Editor

I was interested to read the recent article by McNiece on stem cells and regenerative medicine [1]. Stem cells are touted to offer a potential avenue for curing many conditions such as diabetes, cardiovascular disease and Alzheimer’s disease. The comments raised were of particular significance in regards hurdles for the use of stem cells in regenerative medicine. These include in vivo issues such as cellular survival, senescence, proliferative capacity and differentiation into a functional tissue. Immune reactions too need to be taken into account. Limited integration of infused mesenchymal stem cells is discussed in regards cell-based therapies and any transient benefits probably arise from soluble mediators released (paracrine activity) rather than engraftment. Further, multicellular components are needed to replace damaged tissues and as a drawback, terminally differentiated cells need to be used multiple times to replace multitudinous cell varieties. It is proposed that mesenchymal stem cells (MSCs) initially are transfused to repair the tissue microenvironment followed by a second cell product to replace the functional mature cells of the tissue. Clinical trials need to go ahead in a stepwise fashion for any cellular treatments and adhere to the framework of the FDA. Hematopoietic stem-cell transplantation appears as a relative success story yet has been the result of years of optimizing studies.
As an alternative to MSCs another relatively recent approach outlines reprogramming or conversion of somatic cells into pluripotent cells (induced pluripotent cells – iPS) which can then undergo multiple cell lineage differentiation [2]. Use of reprogramming factors such as Sox2, Oct4, Klf4 and c-Myc have been introduced into porcine fibroblast cells [3] and these cells express pluripotent markers and in vitro differentiation potential and normal karyotype – that is, do not appear tumorigenic. Nonetheless, there is a real risk with iPS for creating a tumorigenic capacity [4]. Introduction of reprogramming factors Oct4, Sox2, Klf4 and c-Myc into nontumorigenic mammary epithelial cells transformed the bulk into tumor-like cells which possess cancer stem cell properties and form tumors of multiple lineages in vivo. Notably, introduction of p16 cyclin – dependent kinase inhibitor blocked the cancer stem cells and produced cellular senescence.
My own proposition has sought to address at least some of these apparent downsides of stem cell therapy through use of already terminally differentiated cells that are immortalized and hence tailored to the purpose required [5]. By immortalizing mature cells one aims to remove the variables of senescence and differentiation and provide for a more durable and predictable engrafting cell type.
In this protocol, autologous adult differentiated cells are genetically modified with genes that confer immortality and cell survival as inferred from cancer genetics [5]. This method aims to introduce the patient’s own adult immortalized differentiated cells into the damaged site to replace lost cells, for example, fibroblasts to aid wound healing, or cardiomyocytes into ischemic-damaged heart tissue. Autologously harvested cells can be cultured in vitro and genetically modified ex vivo and re-transplanted – already differentiated - essentially as a ‘prepackaged’ commodity. As adult cells are routinely now stably immortalized in vitro by the American Type Culture Collection (ATCC) for important research purposes via human telomerase reverse transcriptase (hTERT) transfection [6] then conceivably this protocol can be carried across into regenerative strategies. Introduction of hTERT and loss of p16 tumor suppressor has been noted to lead to cellular immortalization - cellular senescence bypass - without production of a cancer-like phenotype [5]. This is crucial for the procedure as obviously alterations such as loss of contact inhibition of growth would signify undesirable tumorigenic changes. The stability of such immortalization procedures can be attested by the production of immortalized in vitro cell lines of benign tumors that do not show any propensity for malignant transformation [5].
As a measure of the feasibility of such a strategy, a recent study shows that immortalized Schwann cells can be created via hTERT transfection [7]. Expression of p53 and p16 was maintained along with release of neurotrophic factors thus providing a cell-based sustentacular environment for neurites. hTERT expression induces immortalization of cell types without significant phenotypic alteration - Schwann cell transfection with hTERT did not alter mRNA expression of senescence associated genes such as p53 and p16 -although some degree of alteration in the profile of neurotrophic factors occurred compared to non-transfected cells. How effective these cells might be in supporting nerve regeneration remains open to further examinations. Clearly, such cells might provide a resource for future ‘prepackaged’ cell-based neuronal regenerative medicine.
Immortalization of arachnoid cell lines has been achieved of late to overcome such hurdles as slow growth and early senescence by use of retroviral gene transfer of SV40 large T antigen either with or without hTERT [8]. These transfected cells show a high proliferative rate, contact inhibition at confluence and stable arachnoid-specific protein marker expression for over 160 passages in vitro. Such cells may well form a valuable bank for neurological repair of damaged central nervous system.
My protocol for isolating novel genes involved in determining senescence by-pass from cancer that on transformation still maintain a benign phenotype has been outlined and is based on shuttle vector technology [5]. The aim here is to further look for factors that may be used for production of immortalized cells via genetic modification ex vivo. More recently, along this line of thought, MSCs have been immortalized through a combination of p53 knockdown and hTERT expression to improve their overall survival characteristics [9]. Notably, differentiation potential was maintained along with cell surface markers. Gene expression profiling of these modified MSCs identified immortalization associated genes related to up-regulation of cell cycle regulator and DNA repair thus allowing them to bypass cell crisis and complete mitosis [9]. This use of cancer-like ‘hits’ and the search for immortalization associated genes is reminiscent of my own strategic approach [5]. Doubtlessly, genetic modification and immortalization of stem cells may therefore prove a useful addition to the regeneration armamentarium.
It has been considered that cellular prosurvival factors are involved in regenerative pathways [5] and may be involved in senescence bypass. This cross-road in survival mechanism between regenerating tissues and cancer prosurvival paths ought to be exploited to isolate further factors that may be used for creating ex vivo survival-proficient differentiated cells that are durable to unfavorable environments oftentimes found in regenerating sites such as wounds or ischemic damaged tissues. Recently, MSCs have been proposed for the heart [10] to aid in contractility, promote endogenous cardiac regeneration, neovascularisation, and act as anti-inflammation, anti-apoptosis and against adverse remodelling [10]. Inefficient engraftment due to poor viability of the MSCs remains a key problem to date. In this study, microRNAs are seen as key determinants of engraftment survival and controlling microRNA expression may improve MSCs success. Preconditioning the MSCs with microRNAs to strengthen the cells to the rigors of the new environment they find themselves in is a tangible approach. As an example, over-expression of miR-21 – an oncomir and prosurvival factor [11] may promote survival of MSCs exposed to hypoxia. This supports the link to prosurvival mechanisms in cancer and how understanding of these may assist in regenerative strategies. Indeed prosurvival factors are involved in iPS generation, for example Poly(ADP-ribose) polymerase 1 [12] which itself is a prosurvival factor in cancer [11].
Further along the lines of this mechanistic connection between cancer and regeneration, cancer stem cells have been hypothesized to be of potential future use for tissue regeneration [13] although the risks of this must be borne in mind as already alluded to above [4]. Exploring this link or cross-roads between cancer and regeneration has been further looked at recently from another viewpoint [14]. Here the dominance of the local tissue microenvironment is shown to determine cancer cell fate and redirect these cells to a normal phenotype. The regenerating mammary gland microenvironment is able to redirect mammary gland carcinoma cells into contributing the cellular element of that regenerating tissue in vivo. In a sense therefore the cancer is being ‘reprogrammed’. This cross-road between cancer and normal tissue regeneration is by no means new as wound healing has been long considered a mimic of cancer signaling and prosurvival mechanisms [15]. My approach is to appreciate this connection and to manipulate already differentiated cells with cancer-like prosurvival factors and/or produce controlled cancer ‘hits’ to increase the resistance of the cells to the rigors of engraftment.
There may be downsides though to my concept [5]. Telomerase immortalization could certainly pave the way to other less desirable ‘hits’ taking place such as beta-catenin activation in pathways to carcinogenic transformation from immortalized cells [16]. As a further note of caution, repression of p16 in telomerase immortalized bone marrow endothelial cell lines led to a subset of cells that were highly atypical and proliferative and failed to undergo morphogenic differentiation and form vessel like structures in vitro [17]. Therefore p16 repression/hTERT introduction in this scenario can be considered as ‘hits’ towards a pathway of malignant transformation.
Perhaps in the end, having said all the above, ideally some form of spontaneous immortalization would be ideal [18]. In this case, a microvascular dermal endothelial cell line could be established that was based on optimized culture conditions without resorting to exogenous oncogenes or carcinogen action. The phenotypic properties of these cells appear as normal endothelium and it is concluded that these cells shall provide a valuable resource for studying angiogenesis. I would also add that they can be used for wound healing and other repair needs.
Matters of concern surround several notable practical points with my currently proposed concept. These involve aspects of manufacture of personalized ‘banks’ of immortalized adult somatic cells and the all-important attendant costs. An initial approach to these points of practical concern can be made through appreciating what efforts have been achieved to date in terms of commercializing immortalized human cells and their quality assurance (QA). As intimated above, the ATCC already manufacture immortalized human cell lines for research [6]. In fact, a whole range of human ‘prepackaged’ immortalized adult somatic cells are currently available [6]. Further, and importantly, a good initial ‘measuring stick’ in regard costing for this methodology can be gleaned from reviewing the ATCC catalogue. As an example from the ATCC, hTERT immortalized dermal microvascular endothelial cells are available with extended lifespan and express a panel of endothelial cell surface proteins. They produce tubules in culture and are karyotypically and phenotypically similar to the primary parent cells. Cultures of these are available from ATCC at $1,200 American on ‘non-profit’ use basis. Clinical scaled quantities would have to be approximated from animal models yet further expansion can be readily achieved in cell culture at an added but relatively low cost. Other readily available cell lines that are immortalized and have undergone QA include fibroblast, hepatocyte, pancreatic duct and retinal amongst others. Costs are similar across the board for all of these cell types.
Other aspects of my presently proposed concept can be considered as well. Since the RNA component of hTERT is generally expressed in all cells the only limiting feature is the protein itself and introducing this into cells via a readily available construct produced in kit format reconstitutes telomerase activity for primary cells. Licensing for production of such ‘self-made’ hTERT cell lines with ATCC hTERT recombinant material is a straightforward process. Efficiency of hTERT transfection is good as well despite the fact that primary cells are not always easy to stably transfect. Various protocols are recommended such as via lipofectamine, electroporation or with standard transfection approaches or by viral vectors.
Quality control as mentioned above is important especially in regards the protocols used to confirm that the cells have become immortalized and are stable. The ATCC has QA activities stringently set-up for this process. Establishing immortalization parameters such as extended proliferative capacity, stable genotype, phenotypic markers of the tissue of interest and continued expression of hTERT are routine and available across the board for many cell types. Costs for this are included in the ATCC expenses and may not therefore be implied to be overly burdensome.
Engineering the addition of prosurvival factors to strengthen the cells to rigors of their intended new environment can be achieved in cell culture and tested under hypoxic conditions ex vivo - for example, with selected prosurvival microRNAs. Co-transfection with hTERT may follow standard protocols and selection may be based on growth characteristics in culture. Costing can be anticipated to be relatively favorable.
Overall speaking, the costs would be aimed to be significantly under $5000 American per patient per procedure to establish ex vivo personalized bank of tailored adult immortalized somatic cells for whatever regenerative purpose undertaken. This is still a significant financial outlay particularly for Government-based health systems such as the NHS in Great Britain or Medicare in Australia. Thirdparty insurance payers in America would also have to come to some determination over what they will allow in their policies. Nevertheless, the era of personalized medicine is virtually upon us and my current regenerative approach represents another avenue of personalized care. In the end, it is the balance of costs that would appear important – in other words, can one afford not to consider this particular strategy? After all, other approaches to cell based therapeutics such as with stem cells have a potentially significant costing. This is particularly relevant since with stem cells optimal conditions are needed to isolate, expand and differentiate in vitro, ex vivo and in vivo – and are far from being streamlined to the point of manufacture [19]. This is especially true for embryonal stem cells. Biomarkers too are needed to distinguish between progenitor stem cells and the differentiated progeny. With each particular challenge and step comes a certain cost –not to mention issues of ethics especially with embryonal stem cell manipulation.
There are other mechanistic obstacles with my proposed strategy which include time required to establish and QA suitable personalized cell lines. Obviously, such time frames need to be taken into account and several months may be required to establish the necessary celltherapy based product tailored for the need intended. If a patient presents with an acute problem such as an injury then the needed autologous ex vivo generated stable and QA tested immortalized cell line would not be readily available. Of course, for more chronic conditions such as heart failure or liver failure or wound breakdown then cell lines can be derived, The obvious answer is to prepare in advance cell lines to ‘cover one’s bases’ as it were in a preventive sense to anticipate an acute need. Costs and ethical justification would be important players to decide if this would at all ever prove satisfactory for the general public. Indeed this may only encourage an ‘elite medicine’ tailored for those who are willing to stock-pile cell lines for all occasions and pay for this in turn. So there is little doubt that hurdles are yet to be entirely overcome before my proposition can be given the ‘green light’ as a workable therapeutic modality.
In summary, various possible approaches to regenerative strategies can be visualized at this stage in the development of the field (Figure 1).
Figure 1: Outline of various potential approaches for achieving regeneration of damaged tissues from around the body. Included are: MSCs and genetically modified MSCs [1,5,9]; somatic cell reprogramming – iPS [2,3]; genetic immortalization of somatic differentiated cells [5,7,8]; spontaneous immortalization of differentiated cells [18].
In the final analysis, animal testing with FDA approval for human trials is needed for any new approach for regenerative medicine, such as outlined [5]. Ultimately, a combination of approaches may need to be used and no one method for a given clinical situation may be allsufficient – in these respects regenerative biologists need to keep very open minded. Perhaps as per McNiece’s comments [1] MSCs could initially be transfused to repair the tissue microenvironment, followed by a second cell product to replace the functional mature cells of the tissue. My concept of using differentiated ‘prepackaged’ or tailored immortalized cells to functionally repair here could be beneficial and fit into this type of format. This supports the use of immortalized differentiated cells with programmed enhanced survival features that may well be able to form an important contribution to the durable scaffold of cell-based therapies required to repair damaged tissues around the body.


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