2.Induced pluripotent stem cells in research and therapy
Hoon-Koon TEOH ; Soon-Keng CHEONG
The Malaysian Journal of Pathology 2012;34(1):1-13
Induced pluripotent stem cells (iPSC) are derived from human somatic cells through ectopic expression
of transcription factors. This landmark discovery has been considered as a major development towards patient-specifi c iPSC for various biomedical applications. Unlimited self renewal capacity, pluripotency and ease of accessibility to donor tissues contribute to the versatility of iPSC. The
therapeutic potential of iPSC in regenerative medicine, cell-based therapy, disease modelling and
drug discovery is indeed very promising. Continuous progress in iPSC technology provides clearer
understanding of disease pathogenesis and ultimately new optimism in developing treatment or cure for human diseases.
3.Leukaemic stem cells: Drug resistance, metastasis and therapeutic implications
Rebecca SY Wong ; Soon-Keng Cheong
The Malaysian Journal of Pathology 2012;34(2):77-88
Although there have been many new developments in the treatment of leukaemia with the use
of new anti-leukaemic agents and stem cell transplantation, drug resistance and treatment failure
remain a great challenge for the attending physician. Several studies have suggested that leukaemic
stem cells (LSCs) play a pivotal role in chemoresistance and metastasis and the mechanisms by
which these cells do so have also been elucidated. There is increasing evidence to show that there
exists a large pool of therapeutic targets in LSCs and that the eradication of these cells is feasible
with some promising results. This article gives an overview of different types of cancer stem cells
(CSCs) derived from various types of leukaemia, the mechanisms by which LSCs contribute to drug
resistance and metastasis and some recent advances in targeted therapy against LSCs.
4.Cellular mechanisms of emerging applications of mesenchymal stem cells
Pooi-Ling Mok ; Chooi-Fun Leong ; Soon-Keng Cheong
The Malaysian Journal of Pathology 2013;35(1):17-32
Mesenchymal stem cells (MSC) are multipotent, self-renewing cells that can be found mainly in the
bone marrow, and other post-natal organs and tissues. The ease of isolation and expansion, together
with the immunomodulatory properties and their capability to migrate to sites of infl ammation and
tumours make them a suitable candidate for therapeutic use in the clinical settings. We review here
the cellular mechanisms underlying the emerging applications of MSC in various fi elds.
5.Observation of dendritic cell morphology under light, phase-contrast or confocal laser scanning microscopy.
Tan, Yuen-Fen ; Leong, Chooi-Fun ; Cheong, Soon-Keng
The Malaysian Journal of Pathology 2010;32(2):97-102
Dendritic cells (DCs) are professional antigen presenting cells of the immune system. They can be generated in vitro from peripheral blood monocytes supplemented with GM-CSF, IL-4 and TNF alpha. During induction, DCs will increase in size and acquire multiple cytoplasmic projections when compared to their precursor cells such as monocytes or haematopoietic stem cells which are usually round or spherical. Morphology of DCs can be visualized by conventional light microscopy after staining or phase-contrast inverted microscopy or confocal laser scanning microscopy. In this report, we described the morphological appearances of DCs captured using the above-mentioned techniques. We found that confocal laser scanning microscopy yielded DCs images with greater details but the operating cost for such a technique is high. On the other hand, the images obtained through light microscopy after appropriate staining or phase contrast microscopy were acceptable for identification purpose. Besides, these equipments are readily available in most laboratories and the cost of operation is affordable. Nevertheless, morphological identification is just one of the methods to characterise DCs. Other methods such as phenotypic expression markers and mixed leukocyte reactions are additional tools used in the characterisation of DCs.
Dendritic Cells/*cytology
;
Microscopy, Confocal
;
Microscopy, Phase-Contrast
6.Experimental production of clinical-grade dendritic cell vaccine for acute myeloid leukemia.
Yuen-Fen Tan ; Geok-Choo Sim ; Aziz Habsah ; Chooi-Fun Leong ; Soon-Keng Cheong
The Malaysian journal of pathology 2008;30(2):73-9
Dendritic cells (DC) are professional antigen presenting cells of the immune system. Through the use of DC vaccines (DC after exposure to tumour antigens), cryopreserved in single-use aliquots, an attractive and novel immunotherapeutic strategy is available as an option for treatment. In this paper we describe an in vitro attempt to scale-up production of clinical-grade DC vaccines from leukemic cells. Blast cells of two relapsed AML patients were harvested for DC generation in serum-free culture medium containing clinical-grade cytokines GM-CSF, IL-4 and TNF-alpha. Cells from patient 1 were cultured in a bag and those from patient 2 were cultured in a flask. The numbers of seeding cells were 2.24 x 10(8) and 0.8 x 10(8), respectively. DC yields were 10 x 10(6) and 29.8 x 10(6) cells, giving a conversion rate of 4.7% and 37%, respectively. These DC vaccines were then cryopreserved in approximately one million cells per vial with 20% fresh frozen group AB plasma and 10% DMSO. At 12 months and 21 months post cryopreservation, these DC vaccines were thawed, and their sterility, viability, phenotype and functionality were studied. DC vaccines remained sterile up to 21 months of storage. Viability of the cryopreserved DC in the culture bag and flask was found to be 50% and 70% at 12 months post cryopreservation respectively; and 48% and 67% at 21 months post cryopreservation respectively. These DC vaccines exhibited mature DC surface phenotypic markers of CD83, CD86 and HLA-DR, and negative for haemopoietic markers. Mixed lymphocyte reaction (MLR) study showed functional DC vaccines. These experiments demonstrated that it is possible to produce clinical-grade DC vaccines in vitro from blast cells of leukemic patients, which could be cryopreserved up to 21 months for use if repeated vaccinations are required in the course of therapy.
Vaccines
;
month
;
Grade
;
Clinical
;
Lower case ecks
7.Subtractive Hybridization Identifies Stem Cell-Associated Genes in an Acute Myeloid Leukemia with Poor Prognosis
Ngiow Shin Foong ; Maha Abdullah ; Jasmine Lim ; Cheong Soon-Keng ; Seow Heng-Fong
Malaysian Journal of Medicine and Health Sciences 2016;12(1):19-31
Introduction: Current prognostic markers have improved survival prediction, however, it has not
advanced treatment strategies. Gene expression profiling may identify biological markers suitable as
therapeutic targets. Leukaemia stem cell is associated with adverse outcome, however, its biological
characteristics are still being investigated. We observed higher in vitro cell viability in acute myeloid
leukaemia (AML) samples with poor prognosis, which may be stem cell related. Objective: The
objective of this study was to profile highly expressed genes in an AML sample of poor prognosis/high
viability and compare with a sample of good prognosis/low viability. Method: Subtractive hybridization
was performed on two AML samples with high blast counts (>80%), a poor prognosis, PP (disease free
survival, DFS<12 months) and a good prognosis, GP (DFS>12 months) sample. The PP sample had
higher CD34+ counts (73% vs 46%) and higher cell viability than the GP sample. cDNA libraries were
subsequently cloned and sequenced. Results: cDNA subtracted from the PP samples was identified
as genes active during fetal/embryonic development (LCOR, CNOT1, ORMDL1), HOX- related genes
(HOXA3, PBX3, SF3B1), hematopoiesis (SELL, IL-3RA) and aerobic glycolysis/hypoxia (PGK1,
HIGD1A) -associated genes. Majority of GP clones isolated contained genes involved in oxidative
phosphorylation, OXPHOS (COXs, ATPs, MTND4 and MTRNR2), protein synthesis (including
ribosomal proteins, initiating and elongation factors), chromatin remodeling (H2AFZ, PTMA), cell
motility (MALAT1, CALM2, TMSB4X), and mitochondria (HSPA9, MPO) genes. Conclusion: Thus,
the PP sample exhibited stem cell-like features while the GP sample showed cells at a high level of cell
activity. These genes are potential prognostic markers and targets for therapy.
Leukemia, Myeloid, Acute
8.Differential Expression Patterns of Leukaemia Associated Genes in Leukaemia Cell Lines Compared to Healthy Controls
Ang Pei-Shen ; Rajesh Ramasamy ; Noor Hamidah Hussin ; Cheong Soon-Keng ; Seow Heng-Fong ; Maha Abdullah
Malaysian Journal of Medicine and Health Sciences 2016;12(1):32-45
Introduction: The phenotype and genotype of cancer cells portray hallmarks of cancer which may
have clinical value. Cancer cell lines are ideal models to study and confirm these characteristics. We
previously established two subtracted cDNA libraries with differentially expressed genes from an
acute myeloid leukaemia patient with poor prognosis (PP) and good prognosis (GP). Objective: To
compare gene expression of the leukaemia associated genes with selected biological characteristics
in leukaemia cell lines and normal controls. Methodology: Expression of 28 PP genes associated
with early fetal/embryonic development, HOX-related genes, hematopoiesis and aerobic glycolysis/
hypoxia genes and 36 GP genes involved in oxidative phosphorylation, protein synthesis, chromatin
remodelling and cell motility were examined in B-lymphoid (BV173, Reh and RS4;11) and myeloid
(HL-60, K562) leukaemia cell lines after 72h in culture as well as peripheral blood mononuclear cells
from healthy controls (N=5) using semi-quantitative polymerase chain reaction (PCR) method. Cell
cycle profiles were analysed on flow cytometry while MTT cytotoxicity assay was used to determine
drug resistance to epirubicin. Results: Genes expressed significantly higher in B-lymphoid leukaemia
cell lines compared to healthy controls were mostly of the GP library i.e. oxidative phosphorylation
(3/10), protein synthesis (4/11), chromatin remodelling (3/3) and actin cytoskeleton genes (1/5). Only
two genes with significant difference were from the PP library. Cancer associated genes, HSPA9 and
PSPH (GP library) and BCAP31 (PP library) were significantly higher in the B-lymphoid leukemia cell
lines. No significant difference was observed between myeloid cell lines and healthy controls. This
may also be due heterogeneity of cell lines studied. PBMC from healthy controls were not in cell cycle.
G2/M profiles and growth curves showed B-lymphoid cells just reaching plateau after 72 hour culture
while myeloid cells were declining. IC50 values from cytotoxicity assay revealed myeloid cell lines had
an average 13-fold higher drug resistance to epirubicin compared to B-lymphoid cell lines. Only CCL1,
was expressed at least two-fold higher in myeloid compared to B-lymphoid cell lines. In contrast,
MTRNR2, EEF1A1, PTMA, HLA-DR, C6orf115, PBX3, ENPP4, SELL, and IL3Ra were expressed
more than 2-fold higher in B-lymphoid compared to myeloid cell lines studied here. Conclusion: Thus,
B-lymphoid leukaemia cell lines here exhibited active, proliferating characteristics closer to GP genes.
Higher expression of several genes in B-lymphoid compared to myeloid leukaemia cell lines may be
useful markers to study biological differences including drug resistance between lineages.
Neoplasms
9.Molecular responses during chemotherapy in acute myeloid leukemias in predicting poor-response to standard chemotherapy.
Maha, Abdullah ; Cheong, Soon-Keng ; Leong, Chooi-Fun ; Seow, Heng-Fong
The Malaysian Journal of Pathology 2009;31(2):81-91
Signal transduction pathways are constitutively expressed in leukaemic cells resulting in aberrant survival of the cells. It is postulated that in cells of chemo-sensitive patients, chemotherapy induces apoptotic signals leading to cell death while survival signals are maintained in cells of chemo-resistant patients. There is very little information currently, on the expression of these mediators in patients immediately after chemotherapy initiation. We examined the expression pattern of proinflammatory cytokines, signaling molecules of the PI3K and MAPK pathways molecules and death receptor, DR5 on paired samples at diagnosis and during chemotherapy in acute myeloid leukaemia patients treated with cytosine arabinoside and daunorubicin. The results were correlated with remission status one month after chemotherapy. We found that in chemo-sensitive patients, chemotherapy significantly increased the percentage of cases expressing TNF-alpha (p = 0.025, n = 9) and IL-6 (p = 0.002, n = 11) compared to chemo-resistant cases. We also observed an increased percentage of chemo-sensitive cases expressing DR5 and phosphorylated p38, and Jnk. Thus, expression of TNF-alpha, IL-6, DR5, phospho-p38 and phospho-Jnk may regulate cell death in chemo-sensitive cases. In contrast, a significantly higher percentage of chemo-resistant cases expressed phospho-Bad (p = 0.027, n = 9). IL-beta and IL-18 were also found to be higher in chemo-resistant cases at diagnosis and during chemotherapy. Thus, expression of various cellular molecules in leukaemic blasts during chemotherapy may be useful in predicting treatment outcome. These cellular molecules may also be potential targets for alternative therapy.
10.Dendritic cell distribution in lymphomas.
Hussin, Hamidah Noor ; Zulkifli, Fadzilah Noor ; Phang, Koon Song ; Cheong, Soon Keng
The Malaysian Journal of Pathology 2009;31(2):105-12
Dendritic cells (DC) are specialized antigen presenting cells (APC) that have important roles in host defenses and in generating anti-tumour immune response. Altered frequency and maturation of DC have been reported in malignant tumours. We studied the distribution and maturation status of DC by immunohistochemistry, on the formalin-fixed, paraffin-embedded lymph node tissues of 32 histologically diagnosed lymphomas and 40 inflammatory conditions that were retrieved from the Department of Pathology, UKM Medical Centre, Kuala Lumpur. Our study showed a significant reduction in the total DC counts in the lymphoma tissues compared to the inflammatory conditions. The mature and immature DC counts were both significantly reduced (p = 0.008 and 0.001 respectively), although a greater reduction was observed in mature DC numbers. We also observed compartmentalization of DC where the immature DC were seen within the tumour tissues and the mature DC were more in peri-tumoural areas. Our findings were similar to other reports, suggesting that reduced numbers of DC appears to be a factor contributing to lack of tumour surveillance in these cases.