CAR-T-Cell-Based Cancer Immunotherapies: Potentials, Limitations, and Future Prospects.

Cancer encompasses various elements occurring at the cellular and genetic levels, necessitating an immunotherapy capable of efficiently addressing both aspects. T cells can combat cancer cells by specifically recognizing antigens on them. This innate capability of T cells has been used to develop cellular immunotherapies, but most of them can only target antigens through major histocompatibility complexes (MHCs). New gene-editing techniques such as clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (CRISPR-cas9) can precisely edit the DNA sequences. CRISPR-cas9 has made it possible to generate genetically engineered chimeric antigen receptors (CARs) that can overcome the problems associated with old immunotherapies. In chimeric antigen receptor T (CAR-T) cell therapy, the patient's T cells are isolated and genetically modified to exhibit synthetic CAR(s). CAR-T cell treatment has shown remarkably positive clinical outcomes in cancers of various types. Nevertheless, there are various challenges that reduce CAR-T effectiveness in solid tumors. It is required to address these challenges in order to make CAR-T cell therapy a better and safer option. Combining CAR-T treatment with other immunotherapies that target multiple antigens has shown positive outcomes. Moreover, recently generated Boolean logic-gated advanced CARs along with artificial intelligence has expanded its potential to treat solid tumors in addition to blood cancers. This review aims to describe the structure, types, and various methods used to develop CAR-T cells. The clinical applications of CAR-T cells in hematological malignancies and solid tumours have been described in detail. In addition, this discussion has addressed the limitations associated with CAR-T cells, explored potential strategies to mitigate CAR-T-related toxicities, and delved into future perspectives.

Minimum criteria for defining induced mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are a promising cell type for cell-based therapies. The therapeutic potential of MSCs has been verified in preclinical and clinical studies, however; low cell number in adult tissues, restricted expansion and differentiation capacity, and donor-related heterogeneity limit their use. To address these issues, there has been considerable interest in induced pluripotent stem cells (iPSCs) derived MSCs (induced mesenchymal stem cells [iMSCs]). Investigators obtain iMSCs from iPSCs of different origins, with variable methods of generation and expansion. Results of current studies have suggested iMSCs as a unique alternative source of MSCs. However, iMSCs are defined using the same criteria (proposed previously for primary MSCs by the International Society for Cellular Therapy [ISCT]) without realizing the distinct nature of iMSCs as compared to primary MSCs. To rationally define iMSCs, additional characterization is proposed along with ISCT's minimum criteria for defining primary MSCs. Minimum criteria for defining iMSCs should include (1) spindle-shaped morphology, (2) plastic adherent growth, (3) positive expression of CD29, CD44, CD73, CD90, CD105, along with negative expression of hematopoietic markers (CD45, CD34, CD14 or CD11b, CD79α or CD19, HLA-DR), (4) lack of expression of iPSCs induction factors, (5) trilineage differentiation potential, (6) lack of ability to form teratoma, and (7) release of MSC relevant paracrine factors. Defining the minimum criteria for iMSCs will be of great interest in the field and will provide a uniform description and identification of iMSCs to expedite progress in the field. Furthermore, due to increased interest in the clinical use of iMSCs, the above-mentioned additional characterization before the clinical application is important to avoid unwanted complications for recipients.

Insight into generation of induced mesenchymal stem cells from induced pluripotent cells.

Mesenchymal stem cells (MSCs) have the potential for use in cell-based regenerative therapies. Currently, hundreds of clinical trials are using MSCs for the treatment of various diseases. However, MSCs are low in number in adult tissues; they show heterogeneity depending upon the cell source and exhibit limited proliferative potential and early senescence in in vitro cultures. These factors negatively impact the regenerative potential of MSCs and therefore restrict their use for clinical applications. As a result, novel methods to generate induced MSCs (iMSCs) from induced pluripotent stem cells have been explored. The development and optimization of protocols for generation of iMSCs from induced pluripotent stem cells is necessary to evaluate their regenerative potential in vivo and in vitro. In addition, it is important to compare iMSCs with primary MSCs (isolated from adult tissues) in terms of their safety and efficacy. Careful investigation of the properties of iMSCs in vitro and their long term behavior in animals is important for their translation from bench to bedside.

Human neonatal stem cell-derived skin substitute improves healing of severe burn wounds in a rat model.

Conventional approaches can repair minor skin injuries; however, severe burn injuries require innovative approaches for efficient and better wound repair. Recent studies indicate that stem cell-based regenerative therapies can restore severe damaged skin both structurally and functionally. The current study aims to evaluate the wound healing potential of skin substitute derived from human neonatal stem cells (hNSCs) using a severe burn injury rat model. Amniotic epithelial cells (AECs) and mesenchymal stem cells (MSCs) were isolated from placenta (a source of neonatal stem cells) by explant culture method. After characterization, AECs and umbilical cord-MSCs were differentiated into keratinocyte and fibroblasts, respectively. Morphological changes, and expression of corresponding keratinocyte and fibroblast specific markers were used to verify differentiation into respective lineage. A skin substitute was developed by mixing hNSCs-derived skin cells (hNSCs-SCs) in plasma for transplantation in a rat model of severe burn injury. Results indicated that placenta-derived AECs and MSCs were efficiently differentiated into skin cells, that is, keratinocytes and fibroblasts, respectively, as indicated by morphological changes, immunostaining, and polymerase chain reaction analysis. Further, transplantation of hNSCs-SCs seeded in plasma significantly improved basic skin architecture, re-epithelization rate, and wound healing concurrent with reduced apoptosis. In conclusion, neonatal stem cell-derived skin substitute efficiently improved severe burn wounds in a rat model of burn injury. Unique properties of placenta-derived stem cells make them superlative candidates for the development of "off-the-shelf" artificial skin substitutes for future use.

In Vitro Differentiation Potential of Human Placenta Derived Cells into Skin Cells.

Skin autografting is the most viable and aesthetic technique for treatment of extensive burns; however, this practice has potential limitations. Harvesting cells from neonatal sources (such as placental tissue) is a simple, inexpensive, and noninvasive procedure. In the current study authors sought to evaluate in vitro potential of human placenta derived stem cells to develop into skin-like cells. After extensive washing, amniotic membrane and umbilical cord tissue were separated to harvest amniotic epithelial cells (AECs) and umbilical cord mesenchymal stem cells (UC-MSCs), respectively. Both types of cells were characterized for the expression of embryonic lineage markers and their growth characteristics were determined. AECs and UC-MSCs were induced to differentiate into keratinocytes-like and dermal fibroblasts-like cells, respectively. After induction, morphological changes were detected by microscopy. The differentiation potential was further assessed using immunostaining and RT-PCR analyses. AECs were positive for cytokeratins and E-Cadherin while UC-MSCs were positive for fibroblast specific makers. AECs differentiated into keratinocytes-like cells showed positive expression of keratinocyte specific cytokeratins, involucrin, and loricrin. UC-MSCs differentiated into dermal fibroblast-like cells indicated expression of collagen type 3, desmin, FGF-7, fibroblast activation protein alpha, procollagen-1, and vimentin. In conclusion, placenta is a potential source of cells to develop into skin-like cells.

Mesenchymal stem cells conditioned with glucose depletion augments their ability to repair-infarcted myocardium.

Mesenchymal stem cells (MSCs) are an attractive candidate for autologous cell therapy, but their ability to repair damaged myocardium is severely compromised with advanced age. Development of viable autologous cell therapy for treatment of heart failure in the elderly requires the need to address MSC ageing. In this study, MSCs from young (2 months) and aged (24 months) C57BL/6 mice were characterized for gene expression of IGF-1, FGF-2, VEGF, SIRT-1, AKT, p16(INK4a) , p21 and p53 along with measurements of population doubling (PD), superoxide dismutase (SOD) activity and apoptosis. Aged MSCs displayed senescent features compared with cells isolated from young animals and therefore were pre-conditioned with glucose depletion to enhance age affected function. Pre-conditioning of aged MSCs led to an increase in expression of IGF-1, AKT and SIRT-1 concomitant with enhanced viability, proliferation and delayed senescence. To determine the myocardial repair capability of pre-conditioned aged MSCs, myocardial infarction (MI) was induced in 24 months old C57BL/6 wild type mice and GFP expressing untreated and pre-conditioned aged MSCs were transplanted. Hearts transplanted with pre-conditioned aged MSCs showed increased expression of paracrine factors, such as IGF-1, FGF-2, VEGF and SDF-1α. This was associated with significantly improved cardiac performance as measured by dp/dt(max), dp/dt(min), LVEDP and LVDP, declined left ventricle (LV) fibrosis and apoptosis as measured by Masson's Trichrome and TUNEL assays, respectively, after 30 days of transplantation. In conclusion, pre-conditioning of aged MSCs with glucose depletion can enhance proliferation, delay senescence and restore the ability of aged cells to repair senescent infarcted myocardium.

Bone marrow derived mesenchymal stem cells from aged mice have reduced wound healing, angiogenesis, proliferation and anti-apoptosis capabilities.

Decline in the function of stem cells with age, such as other cells of the body, results in an imbalance between loss and renewal. Increasing age of the donor thus diminishes the effectiveness of MSCs (mesenchymal stem cells) transplantation in age-related diseases. The clinical use of stem cell therapies needs autologous stem cell transplantation; it is essential therefore to study the repair ability and survivability of cells before transplantation. Bone marrow derived MSCs possess multi-lineage differentiation potential, but aging adversely affects their therapeutic efficacy. MSCs from young (2-3 months) and aged (23-24 months) GFP (green fluorescent protein)-expressing C57BL/6 mice were isolated and their regenerative potential was assessed in vitro. Real-time RT-PCR (reverse transcriptase-PCR) showed significantly higher expression of Sirt1 in MSCs isolated from young than older animals. Down-regulation of VEGF (vascular endothelial growth factor), SDF-1 (stromal-cell-derived factor 1), AKT (also known as protein kinase B) and up-regulation of p53, p21, Bax and p16 occurred in aged cells. Tube formation, wound healing and proliferative abilities of the young MSCs were better than the aged MSCs. The results suggest that age-related increased expression of apoptotic and senescent genes, with concomitant decrease in Sirt1 gene expression, inhibits to some extent stem cell functioning.