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  1. Yong KW, Safwani WKZW, Xu F, Zhang X, Choi JR, Abas WABW, et al.
    J Tissue Eng Regen Med, 2017 08;11(8):2217-2226.
    PMID: 26756982 DOI: 10.1002/term.2120
    Cryopreservation represents an efficient way to preserve human mesenchymal stem cells (hMSCs) at early culture/passage, and allows pooling of cells to achieve sufficient cells required for off-the-shelf use in clinical applications, e.g. cell-based therapies and regenerative medicine. To fully apply cryopreserved hMSCs in a clinical setting, it is necessary to evaluate their biosafety, e.g. chromosomal abnormality and tumourigenic potential. To date, many studies have demonstrated that cryopreserved hMSCs display no chromosomal abnormalities. However, the tumourigenic potential of cryopreserved hMSCs has not yet been evaluated. In the present study, we cryopreserved human adipose-derived mesenchymal stem cells (hASCs) for 3 months, using a slow freezing method with various cryoprotective agents (CPAs), followed by assessment of the tumourigenic potential of the cryopreserved hASCs after thawing and subculture. We found that long-term cryopreserved hASCs maintained normal levels of the tumour suppressor markers p53, p21, p16 and pRb, hTERT, telomerase activity and telomere length. Further, we did not observe significant DNA damage or signs of p53 mutation in cryopreserved hASCs. Our findings suggest that long-term cryopreserved hASCs are at low risk of tumourigenesis. These findings aid in establishing the biosafety profile of cryopreserved hASCs, and thus establishing low hazardous risk perception with the use of long-term cryopreserved hASCs for future clinical applications. Copyright © 2016 John Wiley & Sons, Ltd.
  2. Kakavand M, Yazdanpanah G, Ahmadiani A, Niknejad H
    J Tissue Eng Regen Med, 2017 06;11(6):1701-1709.
    PMID: 26190586 DOI: 10.1002/term.2064
    Amniotic membrane (AM), a placenta-derived natural biomaterial, has several characteristics which make it a potential substitute for blood vessels. However, there are no reports on the effects of the AM on blood components. The aim of this study was to evaluate the blood compatibility of the epithelial and mesenchymal surfaces of the amnion for potential use in vascular tissue engineering. The activation of intrinsic and extrinsic pathways of the clotting system, haemolysis and platelet adhesion were studied and the results were compared with heparin-coated expanded polytetrafluoroethylene (ePTFE) as a standard synthetic vascular graft. Prothrombin time (PT), activated partial thromboplastin time (aPTT), clotting time (CT) and haemolysis (%) tests showed that both the epithelial and mesenchymal sides of the AM are haemocompatible. Platelet aggregation and P-selectin production from the platelets showed that the epithelial surface of the AM has less induction of platelet activation than ePTFE. The results of scanning electron microscopy (SEM) demonstrated that platelets in contact with ePTFE had a higher rate of adhesion than with the epithelial and mesenchymal surfaces of the AM. Moreover, the morphological distribution of the platelets showed that the majority of platelets were round, while a large number of cells on ePTFE were dendritic. These results suggest that the AM which contains epithelial and mesenchymal stem cells has appropriate haemocompatibility to be employed in vascular tissue engineering, especially as a vein substitute. Copyright © 2015 John Wiley & Sons, Ltd.
  3. Syva SH, Ampon K, Lasimbang H, Fatimah SS
    J Tissue Eng Regen Med, 2017 02;11(2):311-320.
    PMID: 26073746 DOI: 10.1002/term.2043
    Human amnion mesenchymal stem cells (HAMCs) show great differentiation and proliferation potential and also other remarkable features that could serve as an outstanding alternative source of stem cells in regenerative medicine. Recent reports have demonstrated various kinds of effective artificial niche that mimic the microenvironment of different types of stem cell to maintain and control their fate and function. The components of the stem cell microenvironment consist mainly of soluble and insoluble factors responsible for regulating stem cell differentiation and self-renewal. Extensive studies have been made on regulating HAMCs differentiation into specific phenotypes; however, the understanding of relevant factors in directing stem cell fate decisions in HAMCs remain underexplored. In this review, we have therefore identified soluble and insoluble factors, including mechanical stimuli and cues from the other supporting cells that are involved in directing HAMCs fate decisions. In order to strengthen the significance of understanding on the relevant factors involved in stem cell fate decisions, recent technologies developed to specifically mimic the microenvironments of specific cell lineages are also reviewed. Copyright © 2015 John Wiley & Sons, Ltd.
  4. Abu Kasim NH, Govindasamy V, Gnanasegaran N, Musa S, Pradeep PJ, Srijaya TC, et al.
    J Tissue Eng Regen Med, 2015 Dec;9(12):E252-66.
    PMID: 23229816 DOI: 10.1002/term.1663
    The discovery of mesenchymal stem cells (MSCs) from a myriad of tissues has triggered the initiative of establishing tailor-made stem cells for disease-specific therapy. Nevertheless, lack of understanding on the inherent differential propensities of these cells may restrict their clinical outcome. Therefore, a comprehensive study was done to compare the proliferation, differentiation, expression of cell surface markers and gene profiling of stem cells isolated from different sources, viz. bone marrow, Wharton's jelly, adipose tissue and dental pulp. We found that although all MSCs were phenotypically similar to each other, Wharton's jelly (WJ) MSCs and dental pulp stem cells (DPSCs) were highly proliferative as compared to bone marrow (BM) MSCs and adipose tissue (AD) MSCs. Moreover, indistinguishable cell surface characteristics and differentiation capacity were confirmed to be similar among all cell types. Based on gene expression profiling, we postulate that BM-MSCs constitutively expressed genes related to inflammation and immunodulation, whereas genes implicated in tissue development were highly expressed in AD-MSCs. Furthermore, the transcriptome profiling of WJ-MSCs and DPSCs revealed an inherent bias towards the neuro-ectoderm lineage. Based on our findings, we believe that there is no unique master mesenchymal stem cell that is appropriate to treat all target diseases. More precisely, MSCs from different sources exhibit distinct and unique gene expression signatures that make them competent to give rise to specific lineages rather than others. Therefore, stem cells should be subjected to rigorous characterization and utmost vigilance needs to be adopted in order to choose the best cellular source for a particular disease.
  5. Zaman WS, Makpol S, Sathapan S, Chua KH
    J Tissue Eng Regen Med, 2014 Jan;8(1):67-76.
    PMID: 22552847 DOI: 10.1002/term.1501
    In the field of cell-based therapy and regenerative medicine, clinical application is the ultimate goal. However, one major concern is: does in vitro manipulation during culture expansion increases tumourigenicity risk on the prepared cells? Therefore, the aim of this study was to investigate the effect of long-term in vitro expansion on human adipose-derived stem cells (ASCs). The ASCs were harvested from lipo-aspirate samples and cultured until passage 20 (P20), using standard culture procedures. ASCs at P5, P10, P15 and P20 were analysed for morphological changes, DNA damage (Comet assay), tumour suppressor gene expression level (quantitative PCR), p53 mutation, telomerase activity, telomere length determination and in vivo tumourigenicity test. Our data showed that ASCs lost their fibroblastic feature in long-term culture. The population doubling time of ASCs increased with long-term culture especially at P15 and P20. There was an increase in DNA damage at later passages (P15 and P20). No significant changes were observed in both p53 and p21 genes expression throughout the long-term culture. There was also no p53 mutation detected and no significant changes were recorded in the relative telomerase activity (RTA) and mean telomere length (TRF) in ASCs at all passages. In vivo implantation of ASCs at P15 and P20 into the nude mice did not result in tumour formation after 4 months. The data showed that ASCs have low risk of tumourigenicity up to P20, with a total population doubling of 42 times. This indicates that adipose tissue should be a safe source of stem cells for cell-based therapy.
  6. Kardia E, Ch'ng ES, Yahaya BH
    J Tissue Eng Regen Med, 2018 Feb;12(2):e995-e1007.
    PMID: 28105760 DOI: 10.1002/term.2421
    Aerosol-based cell therapy has emerged as a novel and promising therapeutic strategy for treating lung diseases. The goal of this study was to determine the safety and efficacy of aerosol-based airway epithelial cell (AEC) delivery in the setting of acute lung injury induced by tracheal brushing in rabbit. Twenty-four hours following injury, exogenous rabbit AECs were labelled with bromodeoxyuridine and aerosolized using the MicroSprayer® Aerosolizer into the injured airway. Histopathological assessments of the injury in the trachea and lungs were quantitatively scored (1 and 5 days after cell delivery). The aerosol-based AEC delivery appeared to be a safe procedure, as cellular rejection and complications in the liver and spleen were not detected. Airway injury initiated by tracheal brushing resulted in disruption of the tracheal epithelium as well as morphological damage in the lungs that is consistent with acute lung injury. Lung injury scores were reduced following 5 days after AEC delivery (AEC-treated, 0.25  ±  0.06 vs. untreated, 0.53  ±  0.05, P  
  7. Das AK, Pal R
    J Tissue Eng Regen Med, 2010 Aug;4(6):413-21.
    PMID: 20084623 DOI: 10.1002/term.258
    Pluripotent stem cells possess the unique property of differentiating into all other cell types of the human body. Further, the discovery of induced pluripotent stem cells (iPSCs) in 2006 has opened up new avenues in clinical medicine. In simple language, iPSCs are nothing but somatic cells reprogrammed genetically to exhibit pluripotent characteristics. This process utilizes retroviruses/lentiviruses/adenovirus/plasmids to incorporate candidate genes into somatic cells isolated from any part of the human body. It is also possible to develop disease-specific iPSCs which are most likely to revolutionize research in respect to the pathophysiology of most debilitating diseases, as these can be mimicked ex vivo in the laboratory. These models can also be used to study the safety and efficacy of known drugs or potential drug candidates for a particular diseased condition, limiting the need for animal studies and considerably reducing the time and money required to develop new drugs. Recently, functional neurons, cardiomyocytes, pancreatic islet cells, hepatocytes and retinal cells have been derived from human iPSCs, thus re-confirming the pluripotency and differentiation capacity of these cells. These findings further open up the possibility of using iPSCs in cell replacement therapy for various degenerative disorders. In this review we highlight the development of iPSCs by different methods, their biological characteristics and their prospective applications in regenerative medicine and drug screening. We further discuss some practical limitations pertaining to this technology and how they can be averted for the betterment of human life.
  8. Gnanasegaran N, Govindasamy V, Kathirvaloo P, Musa S, Abu Kasim NH
    J Tissue Eng Regen Med, 2018 Feb;12(2):e881-e893.
    PMID: 28079995 DOI: 10.1002/term.2401
    Parkinson's disease (PD) is characterized by tremors and cognitive issues, and is due to the death of dopaminergic (DA-ergic) neurons in brain circuits that are responsible for producing neurotransmitter dopamine (DA). Currently, cell replacement therapies are underway to improve upon existing therapeutic approaches such as drug treatments and electrical stimulation. Among the widely available sources, dental pulp stem cells (DPSCs) from deciduous teeth have gained popularity because of their neural crest origin and inherent propensity toward neuronal lineage. Despite the various pre-clinical studies conducted, an important factor yet to be elucidated is the influence of growth phases in a typical trans-differentiation process. This study selected DPSCs at three distinct time points with variable growth phase proportions (G0/G1, S and G2/M) for in vitro trans-differentiation into DA-ergic-like cells. Using commercially available PCR arrays, we identified distinct gene profiles pertaining to cell cycles in these phases. The differentiation outcomes were assessed in terms of morphology and gene and protein expression, as well as with functional assays. It was noted that DPSCs with the highest G0/G1 phase were comparatively the best, representing at least a 2-fold up regulation (p 
  9. Sebastian AA, Kannan TP, Norazmi MN, Nurul AA
    J Tissue Eng Regen Med, 2018 Aug;12(8):1856-1866.
    PMID: 29774992 DOI: 10.1002/term.2706
    Stem cells derived from human exfoliated deciduous teeth (SHED) represent a promising cell source for bone tissue regeneration. This study evaluated the effects of interleukin-17A (IL-17A) on the osteogenic differentiation of SHED. SHED were cultured in complete alpha minimum essential medium supplemented with osteoinducing reagents and treated with recombinant IL-17A. The cells were quantitatively analysed for proliferative activity by MTS assay, cell markers expression, and apoptotic activity by flow cytometry. For osteogenic differentiation, alkaline phosphatase (ALP) activity was quantified; mineralization assays were carried out using von Kossa and Alizarin red, and expression of osteogenic markers were analysed by real-time polymerase chain reaction and Western blot. The results showed that treatment with IL-17A increased proliferative activity in a dose-dependent manner, but reduced the expression of stem cell markers (c-Myc and Nanog) as the days progressed. IL-17A induced osteogenic differentiation in SHED as evidenced by high ALP activity, increased matrix mineralization, and upregulation of the mRNA expression of the osteogenic markers ALP, alpha 1 type 1 collagen (Col1A1), runt-related transcription factor 2 (RUNX2), osteopontin (OPN), osteocalcin (OCN), and osteoprotegerin (OPG) but downregulation of receptor activator of nuclear factor κB ligand (RANKL) as well as altering the OPG/RANKL ratio. Findings from our study indicate that IL-17A enhances proliferation and osteogenic differentiation of SHED by regulating OPG/RANKL mechanism thus suggests therapeutic potential of IL-17A in bone regeneration.
  10. Lee SY, George JH, Nagel DA, Ye H, Kueberuwa G, Seymour LW
    J Tissue Eng Regen Med, 2019 Mar;13(3):369-384.
    PMID: 30550638 DOI: 10.1002/term.2786
    Development of an optogenetically controllable human neural network model in three-dimensional (3D) cultures can provide an investigative system that is more physiologically relevant and better able to mimic aspects of human brain function. Light-sensitive neurons were generated by transducing channelrhodopsin-2 (ChR2) into human induced pluripotent stem cell (hiPSC) derived neural progenitor cells (Axol) using lentiviruses and cell-type specific promoters. A mixed population of human iPSC-derived cortical neurons, astrocytes and progenitor cells were obtained (Axol-ChR2) upon neural differentiation. Pan-neuronal promoter synapsin-1 (SYN1) and excitatory neuron-specific promoter calcium-calmodulin kinase II (CaMKII) were used to drive reporter gene expression in order to assess the differentiation status of the targeted cells. Expression of ChR2 and characterisation of subpopulations in differentiated Axol-ChR2 cells were evaluated using flow cytometry and immunofluorescent staining. These cells were transferred from 2D culture to 3D alginate hydrogel functionalised with arginine-glycine-aspartate (RGD) and small molecules (Y-27632). Improved RGD-alginate hydrogel was physically characterised and assessed for cell viability to serve as a generic 3D culture system for human pluripotent stem cells (hPSCs) and neuronal cells. Prior to cell encapsulation, neural network activities of Axol-ChR2 cells and primary neurons were investigated using calcium imaging. Results demonstrate that functional activities were successfully achieved through expression of ChR2- by both the CaMKII and SYN1 promoters. The RGD-alginate hydrogel system supports the growth of differentiated Axol-ChR2 cells whilst allowing detection of ChR2 expression upon light stimulation. This allows precise and non-invasive control of human neural networks in 3D.
  11. Gunawardena TNA, Rahman MT, Abdullah BJJ, Abu Kasim NH
    J Tissue Eng Regen Med, 2019 Apr;13(4):569-586.
    PMID: 30644175 DOI: 10.1002/term.2806
    Recent studies suggest that the main driving force behind the therapeutic activity observed in mesenchymal stem cells (MSCs) are the paracrine factors secreted by these cells. These biomolecules also trigger antiapoptotic events to prevent further degeneration of the diseased organ through paracrine signalling mechanisms. In comparison with the normal physiological conditions, an increased paracrine gradient is observed within the peripheral system of diseased organs that enhances the migration of tissue-specific MSCs towards the site of infection or injury to promote healing. Thus, upon administration of conditioned media derived from mesenchymal stem cell cultures (MSC-CM) could contribute in maintaining the increased paracrine factor gradient between the diseased organ and the stem cell niche in order to speed up the process of recovery. Based on the principle of the paracrine signalling mechanism, MSC-CM, also referred as the secretome of the MSCs, is a rich source of the paracrine factors and are being studied extensively for a wide range of regenerative therapies such as myocardial infarction, stroke, bone regeneration, hair growth, and wound healing. This article highlights the current technological applications and advances of MSC-CM with the aim to appraise its future potential as a regenerative therapeutic agent.
  12. Mh Busra F, Rajab NF, Tabata Y, Saim AB, B H Idrus R, Chowdhury SR
    J Tissue Eng Regen Med, 2019 May;13(5):874-891.
    PMID: 30811090 DOI: 10.1002/term.2842
    The full-thickness skin wound is a common skin complication affecting millions of people worldwide. Delayed treatment of this condition causes the loss of skin function and integrity that could lead to the development of chronic wounds or even death. This study was aimed to develop a rapid wound treatment modality using ovine tendon collagen type I (OTC-I) bio-scaffold with or without noncultured skin cells. Genipin (GNP) and carbodiimide (EDC) were used to cross-link OTC-I scaffold to improve the mechanical strength of the bio-scaffold. The physicochemical, biomechanical, biodegradation, biocompatibility, and immunogenicity properties of OTC-I scaffolds were investigated. The efficacy of this treatment approach was evaluated in an in vivo skin wound model. The results demonstrated that GNP cross-linked OTC-I scaffold (OTC-I_GNP) had better physicochemical and mechanical properties compared with EDC cross-linked OTC-I scaffold (OTC-I_EDC) and noncross-link OTC-I scaffold (OTC-I_NC). OTC-I_GNP and OTC-I_NC demonstrated no toxic effect on cells as it promoted higher cell attachment and proliferation of both primary human epidermal keratinocytes and human dermal fibroblasts compared with OTC-I_EDC. Both OTC-I_GNP and OTC-I_NC exhibited spontaneous formation of bilayer structure in vitro. Immunogenic evaluation of OTC-I scaffolds, in vitro and in vivo, revealed no sign of immune response. Finally, implantation of OTC-I_NC and OTC-I_GNP scaffolds with noncultured skin cells demonstrated enhanced healing with superior skin maturity and microstructure features, resembling native skin in contrast to other treatment (without noncultured skin cells) and control group. The findings of this study, therefore, suggested that both OTC-I scaffolds with noncultured skin cells could be promising for the rapid treatment of full-thickness skin wound.
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