Non-autologous Cells Research Articles

Nose-to-Brain: The Next Step for Stem Cell and Biomaterial Therapy in Neurological Disorders.

Neurological disorders are a leading cause of morbidity worldwide, giving rise to a growing need to develop treatments to revert their symptoms. This review highlights the great potential of recent advances in cell therapy for the treatment of neurological disorders. Through the administration of pluripotent or stem cells, this novel therapy may promote neuroprotection, neuroplasticity, and neuroregeneration in lesion areas. The review also addresses the administration of these therapeutic molecules by the intranasal route, a promising, non-conventional route that allows for direct access to the central nervous system without crossing the blood–brain barrier, avoiding potential adverse reactions and enabling the administration of large quantities of therapeutic molecules to the brain. Finally, we focus on the need to use biomaterials, which play an important role as nutrient carriers, scaffolds, and immune modulators in the administration of non-autologous cells. Little research has been conducted into the integration of biomaterials alongside intranasally administered cell therapy, a highly promising approach for the treatment of neurological disorders.

A T-cell independent universal cellular therapy strategy through antigen depletion.

Rationale: T cell therapeutic strategy using CD19-targeting chimeric antigen receptor (CAR) is a revolutionary, novel, and successful treatment for B-cell malignancies. However, the dependency on T-cell mediated cytotoxicity restricts CAR-T therapy as a patient-specific individualized therapy with severe side effects, such as cytokine release syndrome (CRS). Whether a non-T-cell based universal cellular therapy can substitute CAR-T therapy is largely unknown.Methods: Various artificial antigen-recognizing cells were prepared to determine whether non-T-cell-derived CD19-scFv bearing effector cells could cause target cell death. A universal strategy for CRS-free cellular therapeutics was proposed, utilizing artificial antigen-recognizing cells (AARC), which can be manufactured universally and routinely as “off-the-shelf” mesenchymal stromal cells (MSCs) or other types of non-autologous cells expressing anergic CARs.Results: We demonstrated that T-lymphocytic and non-lymphocytic cells could cause CD19 internalization and subsequent depletion when armed with a CD19-recognizing moiety. This CD19 antigen depletion could efficiently induce T-cell independent apoptosis in target cancer cells whose survival depends on CD19 expression, suggesting that CD19 antigen depletion constitutes a crucial tumor destroying mechanism for CD19-CAR-T, especially for its long-term efficacy.Conclusion: Our results uncovered an unrecognized CAR-T cytotoxicity and antigen loss mechanism and provided new insights into a shift from unique patient-specific autologous therapeutics to universal and standardized allogeneic treatment.

Requirements for Proper Immunosuppressive Regimens to Limit Translational Failure of Cardiac Cell Therapy in Preclinical Large Animal Models

Various cell-based therapies are currently investigated in an attempt to tackle the high morbidity and mortality associated with heart failure. The need for these therapies to move towards the clinic is pressing. Therefore, preclinical large animal studies that use non-autologous cells are needed to evaluate their potential. However, non-autologous cells are highly immunogenic and trigger immune rejection responses resulting in potential loss of efficacy. To overcome this issue, adequate immunosuppressive regimens are of imminent importance but clear guidelines are currently lacking. In this review, we assess the immunological barriers regarding non-autologous cell transplantation and immune modulation with immunosuppressive drugs. In addition, we provide recommendations with respect to immunosuppressive regimens in preclinical cardiac cell-replacement studies.

The role of nonautologous and autologous adipose-derived mesenchymal stem cell in acute pyelonephritis.

We compared the therapeutic effects of autologous and nonautologous adipose-derived mesenchymal stem cell (ADMSC), in ameliorating the renal function in a rabbit model of acute pyelonephritis. The difference of perirenal and neck subcutaneous ADMSCs were also evaluated. Twenty female rabbits were apportioned to 5 groups. In group I (n=4), the rabbits were injected direct inoculation of Escherichia coli (E. coli) into the right kidney. In group II (n=4), autologous ADMSCs obtained from nape adipose tissue were injected into the subcapsular space 1week after E. coli injection, while nonautologous ADMSCs of the same origin (from male rabbits) were applied in group III (n=4). In group IV (n=4), autologous perirenal ADMSCs were applied with the same method, while perirenal nonautologous ADMSCs from male rabbits were used in group V (n=4). Technetium-99m-DMSA renal scan was performed 1, 2 and 4months post-injection in all groups. Kidneys were excised for the evaluation of histopathological changes in the same time points. PCR examination for detection of Y-chromosome (in group III and V) and fluorescent evaluation (in group II and IV) were also performed to determine the fate of injected cells. Injection of autologous ADMSCs resulted in more satisfactory outcomes in reduction of interstitial fibrosis, tubular, and glomerular atrophy as compared to nonautologous groups. However, histopathological ameliorations were significantly better in group IV in which autologous perirenal ADMSC was applied. Remarkably, two months after the injection, Technetium-99m-DMSA renal scan showed that right kidney reached to near normal cortical function (48 and 45%) in group IV and V, respectively as compared to groups II (41%) and III (37%). Autologous ADMSCs may have better results in cell therapy as compared to nonautologous cells. However, more satisfactory outcomes may be obtained when the cell source is selected from the surrounding adipose tissue.

Applications of Cell Microencapsulation.

The goal of this chapter is to provide an overview of the different purposes for which the cell microencapsulation technology can be used. These include immunoisolation of non-autologous cells used for cell therapy; immobilization of cells for localized (targeted) delivery of therapeutic products to ablate, repair, or regenerate tissue; simultaneous delivery of multiple therapeutic agents in cell therapy; spatial compartmentalization of cells in complex tissue engineering; expansion of cells in culture; and production of different probiotics and metabolites for industrial applications. For each of these applications, specific examples are provided to illustrate how the microencapsulation technology can be utilized to achieve the purpose. However, successful use of the cell microencapsulation technology for whatever purpose will ultimately depend upon careful consideration for the choice of the encapsulating polymers, the method of fabrication (cross-linking) of the microbeads, which affects the permselectivity, the biocompatibility and the mechanical strength of the microbeads as well as environmental parameters such as temperature, humidity, osmotic pressure, and storage solutions.The various applications discussed in this chapter are illustrated in the different chapters of this book and where appropriate relevant images of the microencapsulation products are provided. It is hoped that this outline of the different applications of cell microencapsulation would provide a good platform for tissue engineers, scientists, and clinicians to design novel tissue constructs and products for therapeutic and industrial applications.

Fat for fostering: Regenerating injured heart using local adipose tissue

Fat for fostering: Regenerating injured heart using local adipose tissue

Microencapsulation of cells in alginate through an electrohydrodynamic process

The encapsulation of living cells within a semi-permeable matrix is an attractive process for transplanting nonautologous cells by limiting the interaction with the host immune system. The electrohydrodynamic process is a low-cost and high-throughput system to encapsulate cells by means of a static potential. We evaluated the use of this system for cell entrapment by assessing and then manufacturing capsules that had the best dimensions. The effect of different cell densities on the beads was determined to set up the basic parameters of the encapsulation system. The cell viability inside the beads and as a function of release time was observed for their biological response.

Mechanism and methods to induce pluripotency

Pluripotent stem cells are able to self-renew indefinitely and differentiate into all types of cells in the body. They can thus be an inexhaustible source for future cell transplantation therapy to treat degenerative diseases which currently have no cure. However, non-autologous cells will cause immune rejection. Induced pluripotent stem cell (iPSC) technology can convert somatic cells to the pluripotent state, and therefore offers a solution to this problem. Since the first generation of iPSCs, there has been an explosion of relevant research, from which we have learned much about the genetic networks and epigenetic landscape of pluripotency, as well as how to manipulate genes, epigenetics, and microRNAs to obtain iPSCs. In this review, we focus on the mechanism of cellular reprogramming and current methods to induce pluripotency. We also highlight new problems emerging from iPSCs. Better understanding of the fundamental mechanisms underlying pluripotenty and refining the methodology of iPSC generation will have a significant impact on future development of regenerative medicine.

Tissue engineering of human bladder

There are a number of conditions of the bladder that can lead to loss of function. Many of these require reconstructive procedures. However, current techniques may lead to a number of complications. Replacement of bladder tissues with functionally equivalent ones created in the laboratory could improve the outcome of reconstructive surgery. A review of the literature was conducted using PubMed to identify studies that provide evidence that tissue engineering techniques may be useful in the development of alternatives to current methods of bladder reconstruction. A number of animal studies and several clinical experiences show that it is possible to reconstruct the bladder using tissues and neo-organs produced in the laboratory. Materials that could be used to create functionally equivalent urologic tissues in the laboratory, especially non-autologous cells that have the potential to reject have many technical limitations. Current research suggests that the use of biomaterial-based, bladder-shaped scaffolds seeded with autologous urothelial and smooth muscle cells is currently the best option for bladder tissue engineering. Further research to develop novel biomaterials and cell sources, as well as information gained from developmental biology, signal transduction studies and studies of the wound healing response would be beneficial.

Microencapsulating and Banking Living Cells for Cell‐Based Medicine

A major challenge to the eventual success of the emerging cell-based medicine such as tissue engineering, regenerative medicine, and cell transplantation is the limited availability of the desired cell sources. This challenge can be addressed by cell microencapsulation to overcome the undesired immune response (i.e., to achieve immunoisolation) so that non-autologous cells can be used to treat human diseases, and by cell/tissue preservation to bank living cells for wide distribution to end users so that they are readily available when needed in the future. This review summarizes the status quo of research in both cell microencapsulation and banking the microencapsulated cells. It is concluded with a brief outlook of future research directions in this important field.

Oct4 and Klf4 Reprogram Dermal Papilla Cells into Induced Pluripotent Stem Cells

Direct reprogramming of somatic cells into induced pluripotent stem (iPS) cells by only four transcription factors (Oct4, Sox2, Klf4, and c-Myc) has great potential for tissue-specific regenerative therapies, eliminating the ethical issues surrounding the use of embryonic stem cells and the rejection problems of using non-autologous cells. The reprogramming efficiency generally is very low, however, and the problems surrounding the introduction of viral genetic material are only partially investigated. Recent efforts to reduce the number of virally expressed transcription factors succeeded at reprogramming neural stem cells into iPS cells by overexpressing Oct4 alone. However, the relative inaccessibility and difficulty of obtaining neural cells in humans remains to be resolved. Here we report that dermal papilla (DP) cells, which are specialized skin fibroblasts thought to instruct hair follicle stem cells, endogenously express high levels of Sox2 and c-Myc, and that these cells can be reprogrammed into iPS cells with only Oct4 and Klf4. Moreover, we show that DP cells are reprogrammed more efficiently than skin and embryonic fibroblasts. iPS cells derived from DP cells expressed pluripotency genes and differentiated into cells from all germ layers in vitro and widely contributed to chimeric mice in vivo, including the germline. Our work establishes DP cells as an easily accessible source to generate iPS cells with efficiency and with less genetic material. This opens up the possibility of streamlined generation of skin-derived, patient-specific pluripotent stem cells and of ultimately replacing the remaining two factors with small molecules for safe generation of transplantable cells.

A Cell-Based Approach for Diabetes Treatment Using Engineered Non-β Cells

Implantation of insulin-secreting cells has the potential to provide tight glycemic regulation in diabetic subjects. Implantation of cadaveric human islets in immunosuppressed human patients is currently applied at a very small scale. To overcome the limitations of tissue availability and recipient immunosuppression, encapsulation of nonautologous cells and use of potentially autologous nonislet cells, the latter engineered for insulin secretion, are being pursued. This article reports on recent findings with the implantation of tissue constructs containing enteroendocrine cells stably expressing recombinant insulin in diabetic mice. The concept of a dual recombinant hepatic and enteroendocrine cell system, which may better approximate the secretory response of islets, is discussed. Mouse GLUTag-INS cells engineered to secrete human insulin were developed and incorporated in tissue constructs as reported previously. Constructs were implanted intraperitoneally in diabetic mice, and blood glucose levels, animal weights, and plasma insulin levels were measured at various time points. GLUTag-INS-containing tissue constructs secreted insulin preimplantation and postexplantation, and human insulin was detected in the plasma of diabetic mice. However, normoglycemia was not restored. A variety of cell types and of encapsulation methods to enhance immune acceptance of insulin-secreting grafts are being pursued. Recombinant enteroendocrine cells show promise, but it is likely that they need to be combined with recombinant hepatic cells to achieve glycemic normalization.

Local Activation or Implantation of Cardiac Progenitor Cells Rescues Scarred Infarcted Myocardium Improving Cardiac Function

Ischemic heart disease is characterized chronically by a healed infarct, foci of myocardial scarring, cavitary dilation, and impaired ventricular performance. These alterations can only be reversed by replacement of scarred tissue with functionally competent myocardium. We tested whether cardiac progenitor cells (CPCs) implanted in proximity of healed infarcts or resident CPCs stimulated locally by hepatocyte growth factor and insulin-like growth factor-1 invade the scarred myocardium and generate myocytes and coronary vessels improving the hemodynamics of the infarcted heart. Hepatocyte growth factor is a powerful chemoattractant of CPCs, and insulin-like growth factor-1 promotes their proliferation and survival. Injection of CPCs or growth factors led to the replacement of approximately 42% of the scar with newly formed myocardium, attenuated ventricular dilation and prevented the chronic decline in function of the infarcted heart. Cardiac repair was mediated by the ability of CPCs to synthesize matrix metalloproteinases that degraded collagen proteins, forming tunnels within the fibrotic tissue during their migration across the scarred myocardium. New myocytes had a 2n karyotype and possessed 2 sex chromosomes, excluding cell fusion. Clinically, CPCs represent an ideal candidate cell for cardiac repair in patients with chronic heart failure. CPCs may be isolated from myocardial biopsies and, following their expansion in vitro, administered back to the same patients avoiding the adverse effects associated with the use of nonautologous cells. Alternatively, growth factors may be delivered locally to stimulate resident CPCs and promote myocardial regeneration. These forms of treatments could be repeated over time to reduce progressively tissue scarring and expand the working myocardium.

Immuno-Isolation in Cancer Gene Therapy

The implantation of genetically-modified non-autologous cells in immuno-protected microcapsules is an alternative to ex vivo gene therapy. Such cells delivering a recombinant therapeutic product are isolated from the host's immune system by being encapsulated within permselective microcapsules. This approach has been successful in pre-clinical animal studies involving delivery of hormone or enzymes to treat dwarfism, lysosomal storage disease, or hemophilia B. Recently, this platform technology has shown promise in the treatment for more complex diseases such as cancer. One of the earliest strategy was to augment the chemotherapeutic effect of a prodrug by implanting encapsulated cells that can metabolise prodrugs into cytotoxic products in close proximity to the cancer cells. More recent approaches include enhancing tumor cell death through immunotherapy, or suppressing tumor cell proliferation through anti-angiogenesis. These can be achieved by delivering single molecules of cytokines or angiostatin, respectively, by implanting microencapsulated cells engineered to secrete these recombinant products. Recent refinements of these approaches include genetic fusion of cytokines or angiostatin to additional functional groups with tumor targeting or tumor cell killing properties, thus enhancing the potency of the recombinant products. Furthermore, a COMBO strategy of implanting microencapsulated cells to deliver multiple products targeted to diverse pathways in tumor suppression also showed much promise. This review will summarise the application of microencapsulation of genetically-modified cells to cancer treatment in animal models, the efficacy of such approaches, and how these studies have led to better understanding of the biology of cancer treatment. The flexibility of this modular system involving molecular engineering, cellular genetic modification, and polymer chemistry provides potentially a huge range of application modalities, and a tremendous multi-disciplinary challenge for the future.

Enhancement of myoblast microencapsulation for gene therapy

One method of nonviral-based gene therapy is to implant microencapsulated nonautologous cells genetically engineered to secrete the desired gene products. Encapsulating the cells within a biocompatible permselective hydrogel, such as alginate-poly-L-lysine-alginate (APA), protects the foreign cells from the host immune system while allowing diffusion of nutrients and the therapeutic gene products. An important consideration is which kind of cells is the best candidate for long-term implantation. Our previous work has shown that proliferation and differentiation of encapsulated C2C12 myoblasts in vitro are significantly improved by inclusion of basic fibroblast growth factor (bFGF), insulin growth factor II (IGF-II), and collagen within the microcapsules ("enhanced" capsules). However, the effects of such inclusions on the functional status of the microcapsules in vivo are unknown. Here we found that comparing the standard with the enhanced APA microcapsules; there was no difference in the rates of diffusion of recombinant products of different sizes, that is, human factor IX (FIX, 65 kDa), murine IgG (150 kDa), and a lysosomal enzyme, beta-glucuronidase (300 kDa), thus providing a key requirement of such an immunoprotective device. Furthermore, the creatine phosphokinase activity and myosin heavy chain staining (markers for differentiation of the myoblasts) and the cell number per capsule in the enhanced microcapsules indicated a higher degree of differentiation and proliferation when compared to the standard microcapsules, thus demonstrating an improved microenvironment for the encapsulated cells. Efficacy was tested in a melanoma cancer tumor model by treating tumor induced by B16-F0/neu tumor cells in mice with myoblasts secreting angiostatin from either the standard or enhanced APA microcapsules. Mice treated with enhanced APA-microcapsules had an 80% reduction in tumor volume at day 21 compared to a 70% reduction in those treated with standard APA-microcapsules. In conclusion, enhancement of APA microcapsules with growth factors and collagen did not adversely affect their permeability property and therapeutic efficacy. However, the enhanced differentiation and viability of the encapsulated myoblasts in vivo should be advantageous for long-term delivery with this method of gene therapy.

A multiprong approach to cancer gene therapy by coencapsulated cells

Immune-isolation of nonautologous cells with microencapsulation protects these cells from graft rejection, thus allowing the same recombinant therapeutic cell line to be implanted in different recipients. This approach was successful in treating HER2/neu-expressing tumors in mice by delivering an interleukin-2 fusion protein (sFvIL-2), or angiostatin. However, treatment with interleukin-2 led to profuse inflammation, while angiostatin delivery did not result in long-term tumor suppression, in part due to endothelial cell-independent neovascularization (vascular mimicry). We hypothesize that coencapsulating the two producer cells in the same microcapsules may enhance the efficacy and ameliorate the above side effects. Hence, B16-F0/neu tumor-bearing mice were implanted with sFvIL-2- and angiostatin-secreting cells coencapsulated in the same alginate-poly-L-lysine-alginate microcapsules. However, this protocol only produced an incremental but not synergistic improvement, as measured with greater tumor suppression and improved survival. Compared to the single sFvIL-2 treatment, the coencapsulation protocol showed improved efficacy associated with: mobilization of sFvIL-2 from the spleen; a higher level of cytokine delivery systemically and to the tumors; increased tumor and tumor-associated endothelial cell apoptosis; and a reduced host inflammatory response. However, compared to the single angiostatin treatment, the efficacy was reduced, primarily due to a "bystander" effect in which the angiostatin-secreting cells suffered similar transgene silencing as the coencapsulated cytokine-secreting cells. Nevertheless, the level of "vascular mimicry" of the single angiostatin treatment was significantly reduced. Hence, while there was no synergy in efficacy, an incremental improvement and some reduction in undesirable side effects of inflammation and vascular mimicry were achieved over the single treatments.

Immunogenicity of human embryonic stem cells: can we achieve tolerance?

Human embryonic stem cells are unique in their capacity to propagate without losing pluripotency, and at the same time may readily differentiate to various cell types of the three embryonic germ layers. It is widely accepted today that differentiated human embryonic stem cells may in the future enable repair of vital tissues of the body. Detailed differentiation protocols need to be developed and safety issues associated with cellular therapeutics must be examined. One of the greatest hurdles facing transplantation is the development of immune rejection processes towards non-autologous cells. Profiling of histocompatibility antigens expressed on the cells reveals that they might be subjected to immune response. Here we describe the routes of immune recognition that can identify these antigens and the proposed ways for overcoming the rejection of human embryonic stem cell derivatives.

Encapsulated transgene cells attenuate hypertension, cardiac hypertrophy and enhance renal function in Goldblatt hypertensive rats.

The success of any gene-therapy approach depends on the survival of the genetically engineered cells that are implanted in the patient to deliver the therapeutic product. Immunoisolation of nonautologous cells within a microcapsule is a unique approach for gene therapy. We employed an immunoisolation device that protects nonautologous cells from destruction, to implant human atrial natriuretic peptide (hANP)-producing Chinese hamster ovary (CHO) cells in two-kidney, one-clip (2K1C) hypertensive rats. CHO cells transfected with the plasmid CMV-cANP were encapsulated in biocompatible polycaprolactone (PCL) capsules, and then the PCL capsules were implanted into 2K1C hypertensive rats intraperitoneally. The implantation of encapsulated hANP-producing cells caused a significant delay of blood pressure (BP) increase 2 weeks post-implantation and the effect lasted for more than 5 months. The implantation of encapsulated hANP-producing cells also caused significant increases in renal blood flow (RBF), glomerular filtration rate (GFR), sodium output, urine excretion, and urinary cGMP levels. These beneficial effects were reflected morphologically by an attenuation of the glomerular sclerotic lesions, reduction in cardiomyocyte size, tubular injury and renal arterial thickening. Immunoreactive hANP can be detected in the blood of 2K1C rats after implantation of the PCL capsules containing hANP-producing cells. This study demonstrates the usefulness of encapsulated ANP gene transfected cells as a new tool for ANP gene delivery in studying renovascular hypertension and cardiovascular diseases. Thus, our results may have important implications for clinical use of transgene cells as therapeutic agents in the treatment of cardiovascular diseases.

Prospects for Delivery of Recombinant Angiostatin by Cell-Encapsulation Therapy

Implantation of encapsulated nonautologous cells that have been genetically modified to secrete proteins with tumor suppressor properties represents an alternative nonviral strategy to cancer gene therapy. We report an approach to raise the yield of recombinant proteins from encapsulated cells substantially. We hypothesized that by optimizing the encapsulation procedure, the production efficacy from the encapsulated cells could be increased. HEK 293 EBNA cells were genetically engineered to produce angiostatin. Encapsulation was performed by varying bead size, cellular density, homogeneity, and ion composition of the gel. The morphology and viability of the cells and the release of angiostatin were studied. Computer software was developed for three-dimensional imaging and quantification of cell viability. Angiostatin production was assessed at 3, 6, and 11 weeks using enzyme-linked immunosorbent assay (ELISA). Inhomogeneous gels facilitated cell growth and viability. The most efficient inhomogeneous microcapsules were generated by reducing the size and cellular density of the beads. The viability and the production of angiostatin were 3 to 5 times higher than in the homogeneous capsules. Significant amounts of viable cells were present in both homogeneous and inhomogeneous beads after 6 months of culture. The stability of the alginate matrix was greatly enhanced by gelling in the presence of barium. In conclusion, the viability and production efficacy of recombinant angiostatin from alginate-encapsulated cells can be increased considerably by optimizing the encapsulation procedure. The development of such optimized microcapsules brings cell-encapsulation therapy further towards clinical use in cancer therapy.

Survival of different cell lines in alginate-agarose microcapsules

Cell microencapsulation has emerged as a promising therapeutic strategy to treat a wide range of diseases. The optimisation of this technology depends on several critical issues such as the careful selection of the cell line, the controlled manufacture of microcapsules and the suitable adaptation of the construct design to the selected cell line. In this work, we studied the behavior of hybridoma cells once enclosed in solid and liquefied core alginate-agarose beads. Results show that hybridoma cells presented a better growing pattern and improved their viability and antibody production within liquefied beads. However, when these beads were evaluated with a compression resistance study, they were found to be mechanically more fragile than solid ones. To address this problem, we entrapped non-autologous cells (BHK fibroblast and C2C12 myoblast) in solid alginate-agarose beads and observed that they showed an improved growing profile and prolonged their viability up to 70 days in comparison to the 15 days seen for the hybridoma cells.