Prospects For Cell-based Therapy Research Articles

Human Genomic Safe Harbors and the Suicide Gene-Based Safeguard System for iPSC-Based Cell Therapy.

The use of human induced pluripotent stem cells (hiPSCs) and recent advances in cell engineering have opened new prospects for cell‐based therapy. However, there are concerns that must be addressed prior to their broad clinical applications and a major concern is tumorigenicity. Suicide gene approaches could eliminate wayward tumor‐initiating cells even after cell transplantation, but their efficacy remains controversial. Another concern is the safety of genome editing. Our knowledge of human genomic safe harbors (GSHs) is still insufficient, making it difficult to predict the influence of gene integration on nearby genes. Here, we showed the topological architecture of human GSH candidates, AAVS1, CCR5, human ROSA26, and an extragenic GSH locus on chromosome 1 (Chr1‐eGSH). Chr1‐eGSH permitted robust transgene expression, but a 2 Mb‐distant gene within the same topologically associated domain showed aberrant expression. Although knockin iPSCs carrying the suicide gene, herpes simplex virus thymidine kinase (HSV‐TK), were sufficiently sensitive to ganciclovir in vitro, the resulting teratomas showed varying degrees of resistance to the drug in vivo. Our findings suggest that the Chr1‐eGSH is not suitable for therapeutic gene integration and highlight that topological analysis could facilitate exploration of human GSHs for regenerative medicine applications. Our data indicate that the HSV‐TK/ganciclovir suicide gene approach alone may be not an adequate safeguard against the risk of teratoma, and suggest that the combination of several distinct approaches could reduce the risks associated with cell therapy. stem cells translational medicine 2019;8:627&638

Human adipose-derived stem cells display myogenic potential and perturbed function in hypoxic conditions

Here, we enriched a human cell population from adipose tissue that exhibited both mesenchymal plasticity, self-renewal capacity, and a cell-surface marker profile indistinguishable from that of bone marrow-derived mesenchymal stem cells. In addition to adipogenic and osteogenic differentiation, these adipose-derived stem cells displayed skeletal myogenic potential when co-cultured with mouse skeletal myocytes in reduced serum conditions. Physical incorporation of stem cells into multinucleated skeletal myotubes was determined by genetic lineage tracing, whereas human-specific antibody staining was employed to demonstrate functional contribution of the stem cells to a myogenic lineage. To investigate the effects of hypoxia, cells were maintained and differentiated at 2% O 2. In contrast with reports on bone marrow-derived stem cells, both osteogenic and adipogenic differentiation were significantly attenuated. In summary, the relative accessibility of adipose-derived mesenchymal stem cells from human donors provides opportunity for molecular investigation of mechanistic dysfunction in disease settings and may introduce new prospects for cell-based therapy.

Prospects for cell-based therapy

O TREAT disease with cells is not a new concept. In the 17 th century, before the nature of cell structure and function were known, blood transfusion experiments were performed between animals using feather quill needles by Christopher Wren and his friends. For blood transfusions to be of value, rather than a “Russian roulette” for sudden death, a means of preventing clot formation and an understanding of red blood cell groups were necessary then the veil of ignorance was removed and blood transfusions became life-saving and opened the door to modern major surgery. In the 1950s advances in immunology spearheaded by Peter Medawar and his colleagues revealed an immune system vital to life that could be manipulated by cell injection of animals in utero and allow acceptance of skin grafts from the cell donors— called “acquired immunolog

Altered traffic to the lysosome in an ex vivo lacrimal acinar cell model for chronic muscarinic receptor stimulation

Evidence suggests that lacrimal and salivary epithelial cells constitutively expose potentially pathogenic autoantigens, but that active regulatory networks normally suppress pathological autoimmune responses [Mircheff, A.K., 2003. Sjögren's syndrome as failed local immunohomeostasis: prospects for cell-based therapy. Ocular Surface 1, 160–179; Mircheff, A.K., Hamm-Alvarez, S.F., Kaslow, H.R., Schechter, J.E., Trousdale, M.D., 2004. Epithelial cells in lymphocyte regulation in the lacrimal gland. In: Zierhut, M. (Ed.), Immunology of the Ocular Surface and the Lacrimal Glands. Swets & Zeitlinger, Amsterdam; in press]. Events that potentially disrupt the regulatory networks include increased exposure of constitutive autoantigens and induced exposure of previously cryptic autoantigen epitopes. Chronic muscarinic receptor (MAChR) stimulation in an ex vivo rabbit lacrimal acinar cell model induces functional and biochemical alterations reminiscent of the functional quiescence associated with Sjögren's syndrome [Qian, L., Wang, Y., Xie, J., Rose, C.M., Yang, T., Nakamura, T., Sandberg, M., Zeng, H., Schechter, J.E., Chow, R.H., Hamm-Alvarez, S.F., Mircheff, A.K., 2003. Biochemical changes contributing to functional quiescence in lacrimal gland acinar cells after chronic ex vivo exposure to a muscarinic agonist. Scand. J. Immunol. 58, 550–565]. Chronic MAChR stimulation also elicits changes in the compartmental distribution of β-hexosaminidase, a product that normally is dually targeted into the lysosomal pathway and the regulated apical secretory pathway. Here, we use subcellular fractionation analyses to further explore the nature of the stimulation-induced traffic changes and to identify effectors that might mediate this change. Overnight stimulation of primary cultured rabbit lacrimal gland acinar cells with 10 μ m carbachol (CCh) significantly decreased the abundance of mature cathepsin B in the pre-lysosome and lysosome; decreased the abundance of preprocathepsin B in fractions containing the TGN and late endosome; increased the abundance of procathepsin B in fractions containing the basal-lateral membrane; and increased the accumulation of endocytosed [ 125I]-EGF in the recycling endosome. Alterations in distribution or abundance of traffic effectors included: increased abundances of rab5A and rab6 in the TGN; decreased overall abundance of γ-adaptin; remarkably increased relative abundance of membrane phase-associated actin; redistribution of cytoplasmic dynein from biosynthetic and proximal endocytic compartments to the lysosome; and redistribution of p150 Glued from the lysosome to biosynthetic or proximal endocytic compartments. We conclude that chronic MAChR stimulation blocks traffic from the early endosome and the TGN to the lysosome, causing lysosomal proteins to reflux to the TGN, endosomes, and basal-lateral membrane. These traffic alterations may be mediated through action on one or more of the effectors noted.

Sjogrens Syndrome as Failed Local Immunohomeostasis: Prospects for Cell-Based Therapy

Sjogrens syndrome has been estimated to affect between 0.2% and 2% or more of the population. It is an autoimmune disease with the hallmark histopathology of focal, periductal, and perivascular CD4+ cell infiltration of the lacrimal and salivary glands. The immunohistopathology is typically associated with severe lacrimal and salivary dysfunctions, which contribute to debilitating ocular surface and oral symptoms. The quality of life of patients with Sjogrens syndrome often is degraded further by serious, multisystemic manifestations, and they are subject to a forty-fold increased risk of developing B cell lymphomas. In normal lacrimal glands, secretory epithelial cells, autoimmune effector lymphocytes, and regulatory lymphocytes can be seen as collaborating to maintain a local immunohomeostasis. The epithelium contributes by secreting immunomodulatory paracrine factors and also by continuously exposing autoantigens, which thereby become available for uptake by professional antigen presenting cells (APCs). Local or systemic perturbations may initiate autoimmune pathophysiology by impairing the replacement of normally-turning-over regulatory cells, by altering epithelial production of immunomodulatory paracrine factors, by inducing intact epithelial cells to begin secreting previously cryptic epitopes (epitopes that previously were not available to bind to major histocompatibility complex (MHC) molecules and so could not be recognized by T cell antigen receptors), and by inducing epithelial cells to begin expressing MHC Class II molecules and presenting formerly cryptic epitopes directly to CD4+ cells. This process has been modeled ex vivo with mixed cell reactions comprised of isolated epithelial cells and autologous lymphocytes. This development has occurred as studies of anterior chamber-associated immune deviation (ACAID) and other immunoregulatory phenomena have elucidated the origins and functions of several different kinds of regulatory lymphocytes and shown that regulatory lymphocytes can be generated ex vivo. It now is possible to envision strategies for exploiting each possible mode of epithelial autoantigen exposure to produce therapeutic regulatory cells that might be capable of re-establishing normal immunohomeostasis. Consideration of the hypothetical therapies identifies a number of basic questions that warrant investigation.