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  1. Safwani WK, Makpol S, Sathapan S, Chua K
    Arch Med Sci, 2014 Jun 29;10(3):597-606.
    PMID: 25097593 DOI: 10.5114/aoms.2014.43753
    Adipose tissue is a source of multipotent adult stem cells. Most studies on human adipose-derived stem cells (ASC) have been on the early passages. Studies in extensive expansion have not been well established yet. In this study, we aim to investigate the effects of extensive expansion on the adipogenic differentiation capability of ASC.
  2. Safwani WK, Makpol S, Sathapan S, Chua KH
    Cell Tissue Bank, 2013 Jun;14(2):289-301.
    PMID: 22476937 DOI: 10.1007/s10561-012-9309-1
    Adipose tissue is a source of multipotent stem cells and it has the ability to differentiate into several types of cell lineages such as neuron cells, osteogenic and adipogenic cells. Most studies on human adipose-derived stem cells (ASCs) have been carried out at the early passages. For clinical usage, ASCs need to be expanded in vitro for a period of time to get sufficient cells for transplantation into patients. However, the impact of long-term culture on ASCs molecular characteristics has not been established yet. Several studies have also shown that osteogenic and adipogenic cells have the ability to switch pathways during in vitro culture as they share the same progenitor cells. This data is important to ensure their functionality and efficacy before being used clinically in the treatment of bone diseases. Therefore, we aim to investigate the effect of long-term culture on the adipogenic, stemness and osteogenic genes expression during osteogenic induction of ASCs. In this study, the molecular characteristics of ASCs during osteogenic induction in long-term culture was analysed by observing their morphological changes during induction, analysis of cell mineralization using Alizarin Red staining and gene expression changes using quantitative RT-PCR. Morphologically, cell mineralization at P20 was less compared to P5, P10 and P15. Adipogenesis was not observed as negative lipid droplets formation was recorded during induction. The quantitative PCR data showed that adipogenic genes expression e.g. LPL and AP2 decreased but PPAR-γ was increased after osteogenic induction in long-term culture. Most stemness genes decreased at P5 and P10 but showed no significant changes at P15 and P20. While most osteogenic genes increased after osteogenic induction at all passages. When compared among passages after induction, Runx showed a significant increased at P20 while BSP, OSP and ALP decreased at later passage (P15 and P20). During long-term culture, ASCs were only able to differentiate into immature osteogenic cells.
  3. Safwani WK, Makpol S, Sathapan S, Chua KH
    Appl Biochem Biotechnol, 2012 Apr;166(8):2101-13.
    PMID: 22391697 DOI: 10.1007/s12010-012-9637-4
    Human adipose-derived stem cells (ASCs) have generated a great deal of excitement in regenerative medicine. However, their safety and efficacy issue remain a major concern especially after long-term in vitro expansion. The aim of this study was to investigate the fundamental changes of ASCs in long-term culture by studying the morphological feature, growth kinetic, surface marker expressions, expression level of the senescence-associated genes, cell cycle distribution and ß-galactosidase activity. Human ASCs were harvested from lipoaspirate obtained from 6 patients. All the parameters mentioned above were measured at P5, P10, P15 and P20. Data were subjected to one-way analysis of variance with a Tukey post hoc test to determine significance difference (P < 0.05). The data showed that growth of ASCs reduced in long-term culture and the ß-galactosidase activity was significantly increased at later passage (P20). The morphology of ASCs in long-term culture showed the manifestation of senescent feature at P15 and P20. Significant alteration in the senescence-associated genes expression levels was observed in MMP1, p21, Rb and Cyclin D1 at P15 and P20. Significant increase in CD45 and HLA DR DQ DP surface marker was observed at P20. While cell cycle analysis showed significant decrease in percentage of ASCs at S and G2/M phase at later passage (P15). Our data showed ASCs cultured beyond P10 favours the senescence pathway and its clinical usage in cell-based therapy may be limited.
  4. Wan Safwani WK, Makpol S, Sathapan S, Chua KH
    Biotechnol Appl Biochem, 2011 Jul-Aug;58(4):261-70.
    PMID: 21838801 DOI: 10.1002/bab.38
    One of the advantages of human adipose-derived stem cells (ASCs) in regenerative medicine is that they can be harvested in abundance. However, the stemness biomarkers, which marked the safety and efficacy of ASCs in accordance with the good manufacturing practice guidelines, is not yet well established. This study was designed to investigate the effect of long-term culture on the stemness properties of ASCs using quantitative real-time polymerase chain reaction and flow cytometry. Results showed the growth rate of ASCs was at its peak when they reached P10 (population doubling; PD = 26) but started to decrease when they were expanded to P15 (PD = 36) and P20 (PD = 46). The ASCs can be culture expanded with minimal alteration in the stemness genes and cluster of differentiation (CD) markers expression up to P10. Expression level of Sox2, Nestin, and Nanog3 was significantly decreased at later passage. CD31, CD45, CD117, and human leukocyte antigen DR, DQ, and DP were lowly expressed at P5 and P10 but their expressions increased significantly at P15 or P20. The differentiation ability of ASCs (adipogenesis, osteogenesis, and neurogenesis) also decreased in long-term culture. Our findings suggested that P10 (PD = 26) should be the "cutoff point" for clinical usage because ASCs at passage 15 onward showed significant changes in the stemness genes, CD markers expression, and differentiation capability.
  5. Wan Safwani WK, Makpol S, Sathapan S, Chua KH
    PMID: 22221649 DOI: 10.1186/1477-5751-11-3
    Adipose tissue is a source of multipotent adult stem cells and it has the ability to differentiate into several types of cell lineages such as neuron cells, osteogenic cells and adipogenic cells. Several reports have shown adipose-derived stem cells (ASCs) have the ability to undergo cardiomyogenesis. Studies have shown 5-azacytidine can successfully drive stem cells such as bone marrow derived stem cells to differentiate into cardiomyogenic cells. Therefore, in this study, we investigated the effect 5-azacytidine on the cardiogenic ability of ASCs.
  6. Yong KW, Choi JR, Wan Safwani WK
    Adv Exp Med Biol, 2016;951:99-110.
    PMID: 27837557
    Human mesenchymal stem cells (hMSCs), a type of adult stem cells that hold great potential in clinical applications (e.g., regenerative medicine and cell-based therapy) due to their ability to differentiate into multiple types of specialized cells and secrete soluble factors which can initiate tissue repair and regulate immune response. hMSCs need to be expanded in vitro or cryopreserved to obtain sufficient cell numbers required for clinical applications. However, long-term in vitro culture-expanded hMSCs may raise some biosafety concerns (e.g., chromosomal abnormality and malignant transformation) and compromised functional properties, limiting their use in clinical applications. To avoid those adverse effects, it is essential to cryopreserve hMSCs at early passage and pool them for off-the-shelf use in clinical applications. However, the existing cryopreservation methods for hMSCs have some notable limitations. To address these limitations, several approaches have to be taken in order to produce healthy and efficacious cryopreserved hMSCs for clinical trials, which remains challenging to date. Therefore, a noteworthy amount of resources has been utilized in research in optimization of the cryopreservation methods, development of freezing devices, and formulation of cryopreservation media to ensure that hMSCs maintain their therapeutic characteristics without raising biosafety concerns following cryopreservation. Biobanking of hMSCs would be a crucial strategy to facilitate clinical applications in the future.
  7. Yong KW, Wan Safwani WK, Xu F, Wan Abas WA, Choi JR, Pingguan-Murphy B
    Biopreserv Biobank, 2015 Aug;13(4):231-9.
    PMID: 26280501 DOI: 10.1089/bio.2014.0104
    Mesenchymal stem cells (MSCs) hold many advantages over embryonic stem cells (ESCs) and other somatic cells in clinical applications. MSCs are multipotent cells with strong immunosuppressive properties. They can be harvested from various locations in the human body (e.g., bone marrow and adipose tissues). Cryopreservation represents an efficient method for the preservation and pooling of MSCs, to obtain the cell counts required for clinical applications, such as cell-based therapies and regenerative medicine. Upon cryopreservation, it is important to preserve MSCs functional properties including immunomodulatory properties and multilineage differentiation ability. Further, a biosafety evaluation of cryopreserved MSCs is essential prior to their clinical applications. However, the existing cryopreservation methods for MSCs are associated with notable limitations, leading to a need for new or improved methods to be established for a more efficient application of cryopreserved MSCs in stem cell-based therapies. We review the important parameters for cryopreservation of MSCs and the existing cryopreservation methods for MSCs. Further, we also discuss the challenges to be addressed in order to preserve MSCs effectively for clinical applications.
  8. Chua KH, Raduan F, Wan Safwani WK, Manzor NF, Pingguan-Murphy B, Sathapan S
    Cell Prolif, 2013 Jun;46(3):300-11.
    PMID: 23672290 DOI: 10.1111/cpr.12029
    OBJECTIVES: This study investigated effects of reduced serum condition and vascular endothelial growth factor (VEGF) on angiogenic potential of adipose stromal cells (ASCs) in vitro.

    MATERIALS AND METHODS: Adipose stromal cells were cultured in three different types of medium: (i) F12/DMEM (FD) supplemented with 10% FBS from passage 0 (P0) to P6; (ii) FD supplemented with 2% FBS at P6; and (iii) FD supplemented with 2% FBS plus 50 ng/ml of VEGF at P6. Morphological changes and growth rate of ASCs were recorded. Changes in stemness, angiogenic and endogenic genes' expressions were analysed using Real-Time PCR.

    RESULTS: Adipose stromal cells changed from fibroblast-like shape when cultured in 10% FBS medium to polygonal when cultured in 2% FBS plus VEGF-supplemented medium. Their growth rate was lower in 2% FBS medium, but increased with addition of VEGF. Real-Time PCR showed that ASCs maintained most of their stemness and angiogenic genes' expression in 10% FBS at P1, P5 and P6, but this increased significantly in 2% FBS at P6. Endogenic genes expression such as PECAM-1, VE chaderin and VEGFR-2 decreased after serial passage in 10% FBS, but increased significantly at P6 in 2% FBS. Addition of VEGF did not cause any significant change in gene expression level.

    CONCLUSION: Adipose stromal cells had greater angiogenic potential when cultured in reduced serum conditions. VEGF did not enhance their angiogenic potential in 2% FBS-supplemented medium.

  9. Yong KW, Li Y, Huang G, Lu TJ, Safwani WK, Pingguan-Murphy B, et al.
    Am J Physiol Heart Circ Physiol, 2015 Aug 15;309(4):H532-42.
    PMID: 26092987 DOI: 10.1152/ajpheart.00299.2015
    Cardiac myofibroblast differentiation, as one of the most important cellular responses to heart injury, plays a critical role in cardiac remodeling and failure. While biochemical cues for this have been extensively investigated, the role of mechanical cues, e.g., extracellular matrix stiffness and mechanical strain, has also been found to mediate cardiac myofibroblast differentiation. Cardiac fibroblasts in vivo are typically subjected to a specific spatiotemporally changed mechanical microenvironment. When exposed to abnormal mechanical conditions (e.g., increased extracellular matrix stiffness or strain), cardiac fibroblasts can undergo myofibroblast differentiation. To date, the impact of mechanical cues on cardiac myofibroblast differentiation has been studied both in vitro and in vivo. Most of the related in vitro research into this has been mainly undertaken in two-dimensional cell culture systems, although a few three-dimensional studies that exist revealed an important role of dimensionality. However, despite remarkable advances, the comprehensive mechanisms for mechanoregulation of cardiac myofibroblast differentiation remain elusive. In this review, we introduce important parameters for evaluating cardiac myofibroblast differentiation and then discuss the development of both in vitro (two and three dimensional) and in vivo studies on mechanoregulation of cardiac myofibroblast differentiation. An understanding of the development of cardiac myofibroblast differentiation in response to changing mechanical microenvironment will underlie potential targets for future therapy of cardiac fibrosis and failure.
  10. Choi JR, Pingguan-Murphy B, Wan Abas WA, Noor Azmi MA, Omar SZ, Chua KH, et al.
    Biochem Biophys Res Commun, 2014 May 30;448(2):218-24.
    PMID: 24785372 DOI: 10.1016/j.bbrc.2014.04.096
    Adipose-derived stem cells (ASCs) have been found adapted to a specific niche with low oxygen tension (hypoxia) in the body. As an important component of this niche, oxygen tension has been known to play a critical role in the maintenance of stem cell characteristics. However, the effect of O2 tension on their functional properties has not been well determined. In this study, we investigated the effects of O2 tension on ASCs stemness, differentiation and proliferation ability. Human ASCs were cultured under normoxia (21% O2) and hypoxia (2% O2). We found that hypoxia increased ASC stemness marker expression and proliferation rate without altering their morphology and surface markers. Low oxygen tension further enhances the chondrogenic differentiation ability, but reduces both adipogenic and osteogenic differentiation potential. These results might be correlated with the increased expression of HIF-1α under hypoxia. Taken together, we suggest that growing ASCs under 2% O2 tension may be important in expanding ASCs effectively while maintaining their functional properties for clinical therapy, particularly for the treatment of cartilage defects.
  11. Chua KH, Zaman Wan Safwani WK, Hamid AA, Shuhup SK, Mohd Haflah NH, Mohd Yahaya NH
    Cytotherapy, 2014 May;16(5):599-611.
    PMID: 24290076 DOI: 10.1016/j.jcyt.2013.08.013
    The use of retropatellar fat pad-derived mesenchymal stromal cells (RFMSCs) for cell-based therapy, particularly for cartilage repair, has been reported by several investigators in recent years. However, the effects of the donor's age and medical condition on the characteristics of RFMSCs have not been well established. The aim of this study was to determine whether age and medical condition can reduce the multipotential of stem cells isolated from the retropatellar fat pad.
  12. Rozila I, Azari P, Munirah S, Wan Safwani WK, Gan SN, Nur Azurah AG, et al.
    J Biomed Mater Res A, 2016 Feb;104(2):377-87.
    PMID: 26414782 DOI: 10.1002/jbm.a.35573
    The osteogenic potential of human adipose-derived stem cells (HADSCs) co-cultured with human osteoblasts (HOBs) using selected HADSCs/HOBs ratios of 1:1, 2:1, and 1:2, respectively, is evaluated. The HADSCs/HOBs were seeded on electrospun three-dimensional poly[(R)-3-hydroxybutyric acid] (PHB) blended with bovine-derived hydroxyapatite (BHA). Monocultures of HADSCs and HOBs were used as control groups. The effects of PHB-BHA scaffold on cell proliferation and cell morphology were assessed by AlamarBlue assay and field emission scanning electron microscopy. Cell differentiation, cell mineralization, and osteogenic-related gene expression of co-culture HADSCs/HOBs were examined by alkaline phosphatase (ALP) assay, alizarin Red S assay, and quantitative real time PCR, respectively. The results showed that co-culture of HADSCs/HOBs, 1:1 grown into PHB-BHA promoted better cell adhesion, displayed a significant higher cell proliferation, higher production of ALP, extracellular mineralization and osteogenic-related gene expression of run-related transcription factor, bone sialoprotein, osteopontin, and osteocalcin compared to other co-culture groups. This result also suggests that the use of electrospun PHB-BHA in a co-culture HADSCs/HOBs system may serve as promising approach to facilitate osteogenic differentiation activity of HADSCs through direct cell-to-cell contact with HOBs.
  13. Wan Safwani WK, Wong CW, Yong KW, Choi JR, Mat Adenan NA, Omar SZ, et al.
    Cytotechnology, 2016 Oct;68(5):1859-72.
    PMID: 26728363 DOI: 10.1007/s10616-015-9939-9
    The need to have a better and safer culture condition for expansion of human mesenchymal stem cells (MSCs) is crucial particularly to prevent infection and immune rejection. This is normally associated with the use of animal-based serum in the culture media for cell expansion. The aim of this study is to investigate alternative culture conditions which may provide better and safer environment for cell growth. In the present study, human adipose-derived stem cells (ASCs) at passage 3 were subjected to treatment in 4 conditions: (1) 21 % O2 with fetal bovine serum (FBS), (2) 21 % O2 without FBS, (3) 2 % O2 with FBS and (4) 2 % O2 without FBS followed by subsequent analysis of their phenotype, viability and functionality. We observed that ASCs cultured in all conditions present no significant phenotypic changes. It was found that ASCs cultured in 2 % O2 without serum showed an increase in viability and growth to a certain extent when compared to those cultured in 21 % O2 without serum. However, ASCs cultured in 2 % O2 without serum displayed a relatively low adipogenic and osteogenic potential. On the other hand, interestingly, there was a positive enhancement in chondrogenic differentiation of ASCs cultured in 21 % O2 without serum. Our findings suggest that different culture conditions may be suitable for different indications. In summary, ASCs cultured in serum-free condition can still survive, proliferate and undergo subsequent adipogenic, osteogenic and chondrogenic differentiation. Therefore, FBS is feasible to be excluded for culture of ASCs, which avoids clinical complications.
  14. Choi JR, Pingguan-Murphy B, Wan Abas WA, Yong KW, Poon CT, Noor Azmi MA, et al.
    PLoS One, 2015;10(1):e0115034.
    PMID: 25615717 DOI: 10.1371/journal.pone.0115034
    Adipose tissue-derived stromal cells (ASCs) natively reside in a relatively low-oxygen tension (i.e., hypoxic) microenvironment in human body. Low oxygen tension (i.e., in situ normoxia), has been known to enhance the growth and survival rate of ASCs, which, however, may lead to the risk of tumourigenesis. Here, we investigated the tumourigenic potential of ASCs under their physiological condition to ensure their safe use in regenerative therapy. Human ASCs isolated from subcutaneous fat were cultured in atmospheric O2 concentration (21% O2) or in situ normoxia (2% O2). We found that ASCs retained their surface markers, tri-lineage differentiation potential, and self-renewal properties under in situ normoxia without altering their morphology. In situ normoxia displayed a higher proliferation and viability of ASCs with less DNA damage as compared to atmospheric O2 concentration. Moreover, low oxygen tension significantly up-regulated VEGF and bFGF mRNA expression and protein secretion while reducing the expression level of tumour suppressor genes p16, p21, p53, and pRb. However, there were no significant differences in ASCs telomere length and their relative telomerase activity when cultured at different oxygen concentrations. Collectively, even with high proliferation and survival rate, ASCs have a low tendency of developing tumour under in situ normoxia. These results suggest 2% O2 as an ideal culture condition for expanding ASCs efficiently while maintaining their characteristics.
  15. Yong KW, Pingguan-Murphy B, Xu F, Abas WA, Choi JR, Omar SZ, et al.
    Sci Rep, 2015;5:9596.
    PMID: 25872464 DOI: 10.1038/srep09596
    Cryopreservation represents an effective technique to maintain the functional properties of human adipose-derived stem cells (ASCs) and allows pooling of cells via long-term storage for clinical applications, e.g., cell-based therapies. It is crucial to reduce freezing injury during the cryopreservation process by loading the ASCs with the optimum concentration of suitable cryoprotective agents (CPAs). In this study, human ASCs were preserved for 3 months in different combinations of CPAs, including 1) 0.25 M trehalose; 2) 5% dimethylsulfoxide (DMSO); 3) 10% DMSO; 4) 5% DMSO + 20% fetal bovine serum (FBS); 5) 10% DMSO + 20% FBS; 6) 10% DMSO + 90% FBS. Interestingly, even with a reduction of DMSO to 5% and without FBS, cryopreserved ASCs maintained high cell viability comparable with standard cryomedium (10% DMSO + 90% FBS), with normal cell phenotype and proliferation rate. Cryopreserved ASCs also maintained their differentiation capability (e.g., to adipocytes, osteocytes and chondrocytes) and showed an enhanced expression level of stemness markers (e.g., NANOG, OCT-4, SOX-2 and REX-1). Our findings suggest that 5% DMSO without FBS may be an ideal CPA for an efficient long-term cryopreservation of human ASCs. These results aid in establishing standardized xeno-free long-term cryopreservation of human ASCs for clinical applications.
  16. Yong KW, Li Y, Liu F, Bin Gao, Lu TJ, Wan Abas WA, et al.
    Sci Rep, 2016 10 05;6:33067.
    PMID: 27703175 DOI: 10.1038/srep33067
    Human mesenchymal stem cells (hMSCs) hold great promise in cardiac fibrosis therapy, due to their potential ability of inhibiting cardiac myofibroblast differentiation (a hallmark of cardiac fibrosis). However, the mechanism involved in their effects remains elusive. To explore this, it is necessary to develop an in vitro cardiac fibrosis model that incorporates pore size and native tissue-mimicking matrix stiffness, which may regulate cardiac myofibroblast differentiation. In the present study, collagen coated polyacrylamide hydrogel substrates were fabricated, in which the pore size was adjusted without altering the matrix stiffness. Stiffness is shown to regulate cardiac myofibroblast differentiation independently of pore size. Substrate at a stiffness of 30 kPa, which mimics the stiffness of native fibrotic cardiac tissue, was found to induce cardiac myofibroblast differentiation to create in vitro cardiac fibrosis model. Conditioned medium of hMSCs was applied to the model to determine its role and inhibitory mechanism on cardiac myofibroblast differentiation. It was found that hMSCs secrete hepatocyte growth factor (HGF) to inhibit cardiac myofibroblast differentiation via downregulation of angiotensin II type 1 receptor (AT1R) and upregulation of Smad7. These findings would aid in establishment of the therapeutic use of hMSCs in cardiac fibrosis therapy in future.
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