Chui Yee Fong

Comparative growth behaviour and characterization of stem cells from human Wharton's jelly

Human embryonic stem cells (hESC) face ethical sensitivities and the problem of teratoma formation. Although Whartons jelly stem cells (WJSC), also of embryonic origin, may not face such ethical concerns, it is not definitely known whether under hESC culture conditions they would be as pluripotent as hESC. WJSC grown on plastic showed two types of morphology (epithelioid and short fibroblastic) in primary culture depending on the culture medium used, and only fibroblastic morphology when passaged. When grown in the presence of hESC medium on mouse feeder cells, they produced atypical colonies containing hESC-like cells with high-nuclear cytoplasmic ratios and prominent nucleoli. They were positive for the hESC markers Tra-1-60, Tra-1-81, SSEA-1, SSEA-4, Oct-4 and alkaline phosphatase, negative for SSEA-3, showed normal karyotypes, developed embryoid body (EB)-like structures, did not produce teratomas in SCID mice and differentiated into neuronal derivatives. They were also positive for the mesenchymal CD markers (CD105, CD90, CD44), negative for CD34 and HLA, and although nine out of 10 embryonic stem cell genomic markers were detectable, these were expressed at low levels. WJSC are thus not as pluripotent as hESC but widely multipotent, and have the advantages of being able to be scaled up easily and not inducing teratomas.

Separation of SSEA-4 and TRA-1–60 Labelled Undifferentiated Human Embryonic Stem Cells from A Heterogeneous Cell Population Using Magnetic-Activated Cell Sorting (MACS) and Fluorescence-Activated Cell Sorting (FACS)

A major concern in human embryonic stem cell (hESC)-derived cell replacement therapy is the risk of tumorigenesis from undifferentiated hESCs residing in the population of hESC-derived cells. Separation of these undifferentiated hESCs from the differentiated derivatives using cell sorting methods may be a plausible approach in overcoming this problem. We therefore explored magnetic activated cell sorting (MACS) and fluorescence activated cell sorting (FACS) to separate labelled undifferentiated hESCs from a heterogeneous population of hESCs and hepatocellular carcinoma cells (HepG2) deliberately mixed respectively at different ratios (10:90, 20:80, 30:70, 40:60 and 50:50) to mimic a standard in vitro differentiation protocol, instead of using a hESC-differentiated cell population, so that we could be sure of the actual number of cells separated. HES-3 and HES-4 cells were labelled in separate experiments for the stem cell markers SSEA-4 and TRA-1–60 using primary antibodies. Anti-PE magnetic microbeads that recognize the PE-conjugated SSEA-4 labelled hESCs was added to the heterogeneous cell mixture and passed through the MACS column. The cells that passed through the column (‘flow-through’ fraction) and those retained (‘labelled’ fraction’) were subsequently analysed using FACS. The maximum efficacy of hESCs retention using MACS was 81.0 ± 2.9% (HES-3) and 83.6 ± 4.2% (HES-4). Using FACS, all the undifferentiated hESCs labelled with the two cell-surface markers could be removed by selective gating. Both hESCs and HepG2 cells in the ‘flow-through’ fraction following MACS separation were viable in culture whereas by FACS separation only the HepG2 cells were viable. FACS efficiently helps to eliminate the undifferentiated hESCs based on their cell-surface antigens expressed.

Human umbilical cord Wharton's jelly stem cells and its conditioned medium support hematopoietic stem cell expansion ex vivo

Bone marrow mesenchymal stromal cells (BMMSCs) have been used as feeder support for the ex vivo expansion of hematopoietic stem cells (HSCs) but have the limitations of painful harvest, morbidity, and risk of infection to the patient. This prompted us to explore the use of human umbilical cord Whartons jelly MSCs (hWJSCs) and its conditioned medium (hWJSC‐CM) for ex vivo expansion of HSCs in allogeneic and autologous settings because hWJSCs can be harvested in abundance painlessly, are proliferative, hypoimmunogenic, and secrete a variety of unique proteins. In the presence of hWJSCs and hWJSC‐CM, HSCs put out pseudopodia‐like outgrowths and became highly motile. Time lapse imaging showed that the outgrowths helped them to migrate towards and attach to the upper surfaces of hWJSCs and undergo proliferation. After 9 days of culture in the presence of hWJSCs and hWJSC‐CM, MTT, and Trypan blue assays showed significant increases in HSC numbers, and FACS analysis generated significantly greater numbers of CD34+ cells compared to controls. hWJSC‐CM produced the highest number of colonies (CFU assay) and all six classifications of colony morphology typical of hematopoiesis were observed. Proteomic analysis of hWJSC‐CM showed significantly greater levels of interleukins (IL‐1a, IL‐6, IL‐7, and IL‐8), SCF, HGF, and ICAM‐1 compared to controls suggesting that they may be involved in the HSC multiplication. We propose that cord blood banks freeze autologous hWJSCs and umbilical cord blood (UCB) from the same umbilical cord at the same time for the patient for future ex vivo HSC expansion and cell‐based therapies. J. Cell. Biochem. 113: 658–668, 2012.

Fertilization, cleavage, and cytogenetics of 48-hour zona-intact and zona-free human unfertilized oocytes reinseminated with donor sperm

Objective To examine the fertilization rates of 48-hour unfertilized oocytes inseminated with fertile donor sperm and to evaluate the cleavage and cytogenetics of ensuing embryos. Design Prospective. Setting Assisted reproductive technology (ART) program. Patients Four hundred ninety-seven unfertilized oocytes from 97 ART patients were categorized into four groups. A (zona-intact) and B (zona-free) were from patients with partial fertilization failure, whereas C (zona-intact) and D (zona-free) were total fertilization failures. Results Fertilization rates in groups A and B were significantly higher than C and D (33.2% to 60.9% versus 20.0% to 48.1%; P Conclusions One cause of total fertilization failure appears to lie in intrinsic oocyte problems confined to the zona and oolemma. The fertilization of 48-hour unfertilized oocytes may be of some value in diagnosing fertilization failure in ART patients.

Hyaluronan Receptor LYVE-1-Expressing Macrophages Maintain Arterial Tone through Hyaluronan-Mediated Regulation of Smooth Muscle Cell Collagen

SUMMARY The maintenance of appropriate arterial tone is critically important for normal physiological arterial function. However, the cellular and molecular mechanisms remain poorly defined. Here, we have shown that in the mouse aorta, resident macrophages prevented arterial stiffness and collagen deposition in the steady state. Using phenotyping, transcriptional profiling, and targeted deletion of Csf1r, we have demonstrated that these macrophages—which are a feature of blood vessels invested with smooth muscle cells (SMCs) in both mouse and human tissues—expressed the hyaluronan (HA) receptor LYVE‐l. Furthermore, we have shown they possessed the unique ability to modulate collagen expression in SMCs by matrix metalloproteinase MMP‐9‐dependent proteolysis through engagement of LYVE‐1 with the HA pericellular matrix of SMCs. Our study has unveiled a hitherto unknown homeostatic contribution of arterial LYVE‐1+ macrophages through the control of collagen production by SMCs and has identified a function of LYVE‐1 in leukocytes. Graphical Abstract Figure. No caption available. HighlightsLYVE‐1+ macrophages coat murine and human blood vessels harboring smooth muscle cellsDeficiency in LYVE‐1+ macrophages induces arterial stiffness and collagen depositionLYVE‐1+ macrophages degrade collagen on smooth muscle cells via pericellular MMP‐9LYVE‐1 on macrophage engages HA on smooth muscle for collagen degradation &NA; Macrophages are essential to maintain tissue homeostasis. Lim and colleagues demonstrate that perivascular LYVE‐1‐expressing macrophages prevent arterial stiffness by controlling the expression of collagen in vascular smooth muscle cells, a process dependent on the engagement of LYVE‐1 with hyaluronan on smooth muscle cells.