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Fulltext The efficiency of cell sorting and harvesting methods for In vitro expansion of human umbilical cord blood derived CD34+ hematopoietic stem cells

Borojerdi, Mohadese Hashem, Maqbool, Maryam, Zuraidah Yusoff, Vidyadaran, Sharmili, Hwa, Ling King, George, Elizabeth, et al.

Malaysian Journal of Medicine and Health Sciences, 2015;11(2):21-28.
MyJurnal

Introduction: During the last three decades hematopoietic stem cell transplantation (HSCT) has become a well-established treatment for many hematologic malignancies. The most important limitation for HSC transplantation is the low number of hematopoietic stem cells (HSC) that can lead to delayed engraftment or graft failures. Numerous attempts have been made to improve in vitro HSC expansion via optimization of various methods such as isolation techniques, supplementing with growth factors, utilizing stromal cells as feeder layer and other culture conditions. Objective: This project is aimed to decipher the efficiency of an isolation technique and retrieval of culture expanded HSC from feeder layer using two different harvesting methods. Materials and Methods: Hematopoietic stem cells from human umbilical cord blood were isolated via MACS mediated CD34+ double sorting. Then, the cells were cultured onto MSC feeder layer for 3 and 5 days. Culture expanded cells were harvested using two different harvesting method namely cell aspiration and trypsinization methods. Hematopoietic stem cell expansion index were calculated based on harvesting methods for each time point. Results: The numbers of HSC isolated from human umbilical cord blood were 1.64 x 106 and 1.20 x106 cells at single and double sortings respectively. Although the number of sorted cells diminished at the second sorting yet the yield of CD34+ purity has increased from 43.73% at single sorting to 81.40% at double sorting. Employing the trypsinization method, the HSC harvested from feeder layer showed a significant increase in expansion index (EI) as compared to the cell aspiration harvesting method (p≤ 0.05). However, the purity of CD34+ HSC was found higher when the cells were harvested using aspiration method (82.43%) as compared to the trypsinization method (74.13%). Conclusion: A pure population of CD34+ HSC can be retrieved when the cells were double sorted using MACS and expanded in culture after being harvested using cell aspiration method.
Human mesenchymal stem cells protect neutrophils from serum-deprived cell death

Maqbool M, Vidyadaran S, George E, Ramasamy R

Cell Biol Int, 2011 Dec;35(12):1247-51.
PMID: 21649586 DOI: 10.1042/CBI20110070

We have previously shown that human MSC (mesenchymal stem cells) inhibit the proliferation of most of the immune cells. However, there are innate immune cells such as neutrophils and other PMN (polymorphonuclear) cells that do not require an extensive proliferation prior to their effector function. In this study, the effect of MSC on neutrophils in the presence of complete and serum-deprived culture media was investigated. In the presence of MSC, the viability of neutrophils increase as measured in 24 h of incubation at various supplementation of serum concentration. We have utilized Annexin V and PI (propidium iodide) staining to confirm whether the enhancement of neutrophil's viability is due to a reduction in PCD (programmed cell death). MSC significantly rescue neutrophils from apoptosis at 1, 5 and 10% of FBS (fetal bovine serum) supplementation. The fractions of viable and dead cells were increased and decreased respectively in the presence of MSC. Our results indicate MSC rescue neutrophils from nutrient- or serum-deprived cell death. However, whether this effect is exerted through a specific signalling pathway or confining neutrophils in resting state by MSC requires further investigation.
Fulltext Generation and characterization of human Osteoarthritis Cartilage-derived Mesenchymal Stem Cells by modified sample processing and culture method

Algraittee, Satar Jabbar Rahi, Lawal, Hamza, Boroojerdi, Mohadese Hashem, Sarmadi, Vahid Hosseinpour, Maqbool, Maryam, Fahrudin Che Hamzah, et al.

Malaysian Journal of Medicine and Health Sciences, 2018;14(103):1-9.
MyJurnal

Mesenchymal stem cells (MSCs) can be isolated from different tissue sources, and show a high differentiation capacity towards osteogenic, adipogenic, chondrogenic, neurogenic and myogenic lineages upon a specific induction. Although the retrieval of MSCs from normal tissues is very straightforward, yet it could be challenging in degenerative conditions that limit the expansion of stem cells such as osteoarthritis. Thus, this study aimed to establish human MSCs culture from osteoarthritic cartilage (OA hC-MSCs) by optimising the sample processing and culture techniques. Methods: Human osteoarthritis knee cartilage samples were obtained (2-4 g) from 8 patients with a mean age of 62.75 years old during the joint replacement surgery. A conventional culture method carried along with the modified method where the period of enzyme digestion and serial plating culture procedure were incorporated. Results: The modified culture method has significantly increased the number of single cells twice after the sample processing. The time taken to form colonies and achieve confluence was also reduced when samples subjected to the modified method. The number of cell yields after passage 0 for the conventional and modified methods were 3.05±0.31 and 6.10±0.42 million cells, respectively. The adherent cells generated under these two conditions comply with criteria for MSCs in term of immunophenotyping and mesodermal differentiation. Conclusions: The current modified method enhances the production of MSCs and could be opted for samples that known to have reduced or defective stem cell pool which may impede the in vitro cell expansion.
Elevated neutrophil respiratory burst activity in essential hypertensive patients

Ramasamy R, Maqbool M, Mohamed AL, Noah RM

Cell Immunol, 2010;263(2):230-4.
PMID: 20471005 DOI: 10.1016/j.cellimm.2010.04.004

Neutrophils play a significant role in maintaining the integrity of innate immunity via their potent respiratory burst activity. However, the uncontrolled activation of respiratory burst in neutrophils also attributes to chronic diseases such as primary hypertension and atherosclerosis. In our study, we have investigated the activation of respiratory burst function of neutrophils harvested from essential hypertensive patients. In the presence of stimuli PMA and opsonized zymosan (OZ), hypertensive patients' neutrophils secrete significantly higher amount of superoxide anions compared to normotensive control. Although the magnitude of activation varies between both groups, yet the kinetics of activation is similar. When normotensive control's neutrophils were pre-treated with hypertensive serum, the cells failed to migrate toward fMLP which indicates the impairment of the migration property. In conclusion, the respiratory burst activity of neutrophils is affected by hypertension and their elevated superoxide anions production could be an aggravating factor in hypertension-related complication.
Fulltext Characterization and Expression of Senescence Marker in Prolonged Passages of Rat Bone Marrow-Derived Mesenchymal Stem Cells

Ridzuan N, Al Abbar A, Yip WK, Maqbool M, Ramasamy R

Stem Cells Int, 2016;2016:8487264.
PMID: 27579045 DOI: 10.1155/2016/8487264

The present study is aimed at optimizing the in vitro culture protocol for generation of rat bone marrow- (BM-) derived mesenchymal stem cells (MSCs) and characterizing the culture-mediated cellular senescence. The initial phase of generation and characterization was conducted using the adherent cells from Sprague Dawley (SD) rat's BM via morphological analysis, growth kinetics, colony forming unit capacity, immunophenotyping, and mesodermal lineage differentiation. Mesenchymal stem cells were successfully generated and characterized as delineated by the expressions of CD90.1, CD44H, CD29, and CD71 and lack of CD11b/c and CD45 markers. Upon induction, rBM-MSCs differentiated into osteocytes and adipocytes and expressed osteocytes and adipocytes genes. However, a decline in cell growth was observed at passage 4 onwards and it was further deciphered through apoptosis, cell cycle, and senescence assays. Despite the enhanced cell viability at later passages (P4-5), the expression of senescence marker, β-galactosidase, was significantly increased at passage 5. Furthermore, the cell cycle analysis has confirmed the in vitro culture-mediated cellular senescence where cells were arrested at the G0/G1 phase of cell cycle. Although the currently optimized protocols had successfully yielded rBM-MSCs, the culture-mediated cellular senescence limits the growth of rBM-MSCs and its potential use in rat-based MSC research.
Fulltext Generation and characterisation of human umbilical cord derived mesenchymal stem cells by explant method

Yusoff Z, Maqbool M, George E, Hassan R, Ramasamy R

Med J Malaysia, 2016 Jun;71(3):105-10.
PMID: 27495882 MyJurnal

Mesenchymal stem cells (MSCs) derived from human umbilical cord (UC) have been considered as an important tool for treating various malignancies, tissue repair and organ regeneration. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) are better alternative to MSCs that derived from bone marrow (BM-MSCs) as they are regarded as medical waste with little ethical concern for research and easily culture-expanded. In this present study, the foetal distal end of human UC was utilised to generate MSC by explant method. Upon in vitro culture, adherent cells with fibroblastic morphology were generated with rapid growth kinetics. Under the respective inductive conditions, these cells were capable of differentiating into adipocytes and osteocytes; express an array of standard MSC's surface markers CD29, CD73, CD90, CD106 and MHC-class I. Further assessment of immunosuppression activity revealed that MSCs generated from UC had profoundly inhibited the proliferation of mitogen-activated T lymphocytes in a dosedependent manner. The current laboratory findings have reinforced the application of explant method to generate UCMSCs thus, exploring an ideal platform to fulfil the increasing demand of MSCs for research and potential clinical use.
Fulltext Human mesenchymal stem cells inhibit the differentiation and effector functions of monocytes

Maqbool M, Algraittee SJR, Boroojerdi MH, Sarmadi VH, John CM, Vidyadaran S, et al.

Innate Immun, 2020 07;26(5):424-434.
PMID: 32635840 DOI: 10.1177/1753425919899132

Although monocytes represent an essential part of the host defence system, their accumulation and prolonged stimulation could be detrimental and may aggravate chronic inflammatory diseases. The present study has explored the less-understood immunomodulatory effects of mesenchymal stem cells on monocyte functions. Isolated purified human monocytes were co-cultured with human umbilical cord-derived mesenchymal stem cells under appropriate culture conditions to assess monocytes' vital functions. Based on the surface marker analysis, mesenchymal stem cells halted monocyte differentiation into dendritic cells and macrophages and reduced their phagocytosis functions, which rendered an inability to stimulate T-cell proliferation. The present study confers that mesenchymal stem cells exerted potent immunosuppressive activity on monocyte functions such as differentiation, phagocytosis and Ag presentation; hence, they promise a potential therapeutic role in down-regulating the unwanted monocyte-mediated immune responses in the context of chronic inflammatory diseases.
Fulltext Preliminary study on overproduction of reactive oxygen species by neutrophils in diabetes mellitus

Ridzuan N, John CM, Sandrasaigaran P, Maqbool M, Liew LC, Lim J, et al.

World J Diabetes, 2016 Jul 10;7(13):271-8.
PMID: 27433296 DOI: 10.4239/wjd.v7.i13.271

To assess the amount and pattern of reactive oxygen species (ROS) production in diabetic patient-derived neutrophils.
Directional capacity of human mesenchymal stem cells to support hematopoietic stem cell proliferation in vitro

Hashem Boroojerdi M, Hosseinpour Sarmadi V, Maqbool M, Ling KH, Safarzadeh Kozani P, Safarzadeh Kozani P, et al.

Gene, 2022 Feb 05;820:146218.
PMID: 35134469 DOI: 10.1016/j.gene.2022.146218

OBJECTIVES: Hematopoietic stem cells (HSCs) reside in a specialised microenvironment in the bone marrow, which is majorly composed of mesenchymal stem cells (MSCs) and its' derivatives. This study aimed to investigate the regulatory role of MSCs to decipher the cellular and humoral communications on HSCs' proliferation, self-renewal, and differentiation at the transcriptomic level.
MATERIALS AND METHODS: Microarray assay was employed to analyse the gene expression profile of HSCs that imparted by MSCs during co-culture.
RESULTS: The proliferation of human umbilical cord blood-derived HSCs (hUC-HSCs) markedly propagated when MSCs were used as the feeder layer, without disturbing the undifferentiated state of HSCs, and reduced the cell death of HSCs. Upon co-culture with MSCs, the global microarray analysis of HSCs disclosed 712 differentially expressed genes (DEGs) (561 up-regulated and 151 down-regulated). The dysregulations of various transcripts were enriched for cellular functions such as cell cycle (including CCND1), apoptosis (including TNF), and genes related to signalling pathways governing self-renewal, as well as WNT5A from the Wnt signalling pathway, MAPK, Hedgehog, FGF2 from FGF, Jak-STAT, and PITX2 from the TGF-β signalling pathway. To concur this, real-time quantitative PCR (RT-qPCR) was utilised for corroborating the microarray results from five of the most dysregulated genes.
CONCLUSION: This study elucidates the underlying mechanisms of the mitogenic influences of MSCs on the propagation of HSCs. The exploitation of such mechanisms provides a potential means for achieving larger quantities of HSCs in vitro, thus obviating the need for manipulating their differentiation potential for clinical application.
Fulltext Human Mesenchymal Stem Cells-mediated Transcriptomic Regulation of Leukemic Cells in Delivering Anti-tumorigenic Effects

Sarmadi VH, Ahmadloo S, Boroojerdi MH, John CM, Al-Graitte SJR, Lawal H, et al.

Cell Transplant, 2020 2 7;29:963689719885077.
PMID: 32024378 DOI: 10.1177/0963689719885077

Treatment of leukemia has become much difficult because of resistance to the existing anticancer therapies. This has thus expedited the search for alternativ therapies, and one of these is the exploitation of mesenchymal stem cells (MSCs) towards control of tumor cells. The present study investigated the effect of human umbilical cord-derived MSCs (UC-MSCs) on the proliferation of leukemic cells and gauged the transcriptomic modulation and the signaling pathways potentially affected by UC-MSCs. The inhibition of growth of leukemic tumor cell lines was assessed by proliferation assays, apoptosis and cell cycle analysis. BV173 and HL-60 cells were further analyzed using microarray gene expression profiling. The microarray results were validated by RT-qPCR and western blot assay for the corresponding expression of genes and proteins. The UC-MSCs attenuated leukemic cell viability and proliferation in a dose-dependent manner without inducing apoptosis. Cell cycle analysis revealed that the growth of tumor cells was arrested at the G0/G1 phase. The microarray results identified that HL-60 and BV173 share 35 differentially expressed genes (DEGs) (same expression direction) in the presence of UC-MSCs. In silico analysis of these selected DEGs indicated a significant influence in the cell cycle and cell cycle-related biological processes and signaling pathways. Among these, the expression of DBF4, MDM2, CCNE2, CDK6, CDKN1A, and CDKN2A was implicated in six different signaling pathways that play a pivotal role in the anti-tumorigenic activity exerted by UC-MSCs. The UC-MSCs perturbate the cell cycle process of leukemic cells via dysregulation of tumor suppressor and oncogene expression.