Biomedical subjects

Sai-Kiang Lim

Publications and source records attributed to Sai-Kiang Lim.

15 recordsLinked to original sources

PubMed2006

Establishing clonal cell lines with endothelial-like potential from CD9(hi), SSEA-1(-) cells in embryonic stem cell-derived embryoid bodies.

BACKGROUND: Differentiation of embryonic stem cells (ESCs) into specific cell types with minimal risk of teratoma formation could be efficiently directed by first reducing the differentiation potential of ESCs through the generation of clonal, self-renewing lineage-restricted stem cell lines. Efforts to isolate these stem cells are, however, mired in an impasse where the lack of purified lineage-restricted stem cells has hindered the identification of defining markers for these rare stem cells and, in turn, their isolation. METHODOLOGY/PRINCIPAL FINDINGS: We describe here a method for the isolation of clonal lineage-restricted cell lines with endothelial potential from ESCs through a combination of empirical and rational evidence-based methods. Using an empirical protocol that we have previously developed to generate embryo-derived RoSH lines with endothelial potential, we first generated E-RoSH lines from mouse ESC-derived embryoid bodies (EBs). Despite originating from different mouse strains, RoSH and E- RoSH lines have similar gene expression profiles (r(2) = 0.93) while that between E-RoSH and ESCs was 0.83. In silico gene expression analysis predicted that like RoSH cells, E-RoSH cells have an increased propensity to differentiate into vasculature. Unlike their parental ESCs, E-RoSH cells did not form teratomas and differentiate efficiently into endothelial-like cells in vivo and in vitro. Gene expression and FACS analysis revealed that RoSH and E-RoSH cells are CD9(hi), SSEA-1(-) while ESCs are CD9(lo), SSEA-1(+). Isolation of CD9(hi), SSEA-1(-) cells that constituted 1%-10% of EB-derived cultures generated an E-RoSH-like culture with an identical E-RoSH-like gene expression profile (r(2) = 0.95) and a propensity to differentiate into endothelial-like cells. CONCLUSIONS: By combining empirical and rational evidence-based methods, we identified definitive selectable surface antigens for the isolation and propagation of lineage-restricted stem cells with endothelial-like potential from mouse ESCs.

Animals↗

PubMed2006

Derivation of clinically compliant MSCs from CD105+, CD24- differentiated human ESCs.

Adult tissue-derived mesenchymal stem cells (MSCs) have demonstrated therapeutic efficacy in treating diseases or repairing damaged tissues through mechanisms thought to be mediated by either cell replacement or secretion of paracrine factors. Characterized, self-renewing human ESCs could potentially be an invariable source of consistently uniform MSCs for therapeutic applications. Here we describe a clinically relevant and reproducible manner of generating identical batches of hESC-derived MSC (hESC-MSC) cultures that circumvents exposure to virus, mouse cells, or serum. Trypsinization and propagation of HuES9 or H1 hESCs in feeder- and serum-free selection media generated three polyclonal, karyotypically stable, and phenotypically MSC-like cultures that do not express pluripotency-associated markers but displayed MSC-like surface antigens and gene expression profile. They differentiate into adipocytes, osteocytes, and chondrocytes in vitro. Gene expression and fluorescence-activated cell sorter analysis identified CD105 and CD24 as highly expressed antigens on hESC-MSCs and hESCs, respectively. CD105+, CD24- monoclonal isolates have a typical MSC gene expression profiles and were identical to each other with a highly correlated gene expression profile (r(2) > .90). We have developed a protocol to reproducibly generate clinically compliant and identical hESC-MSC cultures.

Adipogenesis↗

PubMed2005

Transplantation of autologous bone marrow-derived cells into the myocardium of patients undergoing coronary bypass.

BACKGROUND: Animal studies suggest that cell transplantation, including bone marrow-derived cells, can ameliorate left ventricular remodeling following myocardial ischemia. Clinical evaluation of the potential benefits of this approach is limited by the lack of safety and feasibility studies. We have assessed the safety and feasibility of intramyocardial transplantation of autologous bone marrow-derived cells in patients undergoing coronary artery bypass graft (CABG) surgery. METHODS AND RESULTS: Between December 2001 and May 2002 7 patients, scheduled for CABG, consented to the trial. All had CABG using hypothermic cardiopulmonary bypass (CPB) and cold cardioplegic arrest. An average of 21 10(6) (8.6 10(6) to 35.1 10(6)) nucleated cells, and 4.2 10(4) (2.5 10(4) to 8.1 10(4)) CD34+ cells were injected into the anterior-lateral wall of the left ventricle, after discontinuation of cardiopulmonary bypass. The end points to assess safety included death, massive bleeding, electrocardiographic or biochemical evidence of myocardial infarction, ventricular dysrhythmia, myocardial perfusion, ventricular function, and the patients' functional status. All patients recovered well without ventricular arrhythmia, bleeding, or other major peri-operative complications. The average intensive care unit (ICU) and hospital stay was 1 and 7 days, respectively. Repeat Technetium-99m myocardial perfusion stress imaging and echocardiography 6 weeks after surgery showed improvement in tissue perfusion, and an average improvement of left ventricular function of 13.5% +/- 11.54% (the mean pre- and post-operative left ventricular EF were 32.5% +/- 15.46% and 46% +/- 18.55%, respectively). Twenty-four hours Holter monitoring showed no significant arrhythmia, 3 months post-operatively. All patients with narrow QRS complex showed no evidence of late potential, on signal-averaged electrocardiogram. At 4 to 9 months after surgery patients were in NYHA functional class "I". CONCLUSIONS: This early clinical experience shows that autologous bone marrow-derived cell transplantation into myocardium is feasible and relatively safe. Further clinical trials to assess the role of cell transplantation for myocardial repair are required.

Aged↗

PubMed2005

Transcriptome profiling of human and murine ESCs identifies divergent paths required to maintain the stem cell state.

Human embryonic stem cells (hESCs) are an important source of stem cells in regenerative medicine, and much remains unknown about their molecular characteristics. To develop a detailed genomic profile of ESC lines in two different species, we compared transcriptomes of one murine and two different hESC lines by massively parallel signature sequencing (MPSS). Over 2 million signature tags from each line and their differentiating embryoid bodies were sequenced. Major differences and conserved similarities between species identified by MPSS were validated by reverse transcription polymerase chain reaction (RT-PCR) and microarray. The two hESC lines were similar overall, with differences that are attributable to alleles and propagation. Human-mouse comparisons, however, identified only a small (core) set of conserved genes that included genes known to be important in ESC biology, as well as additional novel genes. Identified were major differences in leukemia inhibitory factor, transforming growth factor-beta, and Wnt and fibroblast growth factor signaling pathways, as well as the expression of genes encoding metabolic, cytoskeletal, and matrix proteins, many of which were verified by RT-PCR or by comparing them with published databases. The study reported here underscores the importance of cross-species comparisons and the versatility and sensitivity of MPSS as a powerful complement to current array technology.

Animals↗

PubMed2004

Embryonic cell lines with endothelial potential: an in vitro system for studying endothelial differentiation.

OBJECTIVE: Endothelial differentiation is a fundamental process in angiogenesis and vasculogenesis with implications in development, normal physiology, and pathology. To better understand this process, an in vitro cellular system that recapitulates endothelial differentiation and is amenable to experimental manipulations is required. METHODS AND RESULTS: Embryonic cell lines that differentiate exclusively into endothelial cells were derived from early mouse embryos using empirical but reproducible culture techniques without viral or chemical transformation. The cells were not pluripotent and expressed reduced levels of Oct 4 and Rex-1. They were non-tumorigenic with a population doubling time of approximately 15 hours. When plated on matrigel, they readily differentiated to form patent tubular structures with diameters of 30 to 150 microm. The differentiated cells endocytosed acetylated low-density lipoprotein (LDL) and began to express endothelial-specific markers such as CD34, CD31, Flk-1, TIE2, P-selectin, Sca-1, and thy-1. They also expressed genes essential for differentiation and maintenance of endothelial lineages, eg, Flk-1, vascular endothelial growth factor (VEGF), and angiopoietin-1. When transplanted into animal models, these cells incorporated into host vasculature. CONCLUSIONS: These cell lines can undergo in vitro and in vivo endothelial differentiation that recapitulated known endothelial differentiation pathways. Therefore, they are ideal for establishing an in vitro cellular system to study endothelial differentiation.

Animals↗

PubMed2003

Hepatic expression of PPARalpha, a molecular target of fibrates, is regulated during inflammation in a gender-specific manner.

Dyslipidemia, inflammation and gender are major risk factors in cardiovascular disease. Here we show that hepatic expression of Peroxisome proliferator-activated receptor alpha (PPARalpha), a nuclear receptor that regulates lipid metabolism and inflammation, is regulated in a gender-specific manner during lipopolysaccharide (LPS)-induced systemic inflammation. Immediately following LPS-induced systemic inflammation, hepatic PPARalpha mRNA level decreased dramatically in mice. It was restored to baseline within 24 h in females but remained below baseline for >72 h in male mice. In gonadectomized mice of both sexes, PPARalpha mRNA level was restored to baseline within 48 h after the initial decrease.

Animals↗

PubMed2003

Expression of major HDL-associated antioxidant PON-1 is gender dependent and regulated during inflammation.

Paraoxonase 1, an HDL-associated enzyme that confers antioxidant activity on HDL, and its activity in serum have been correlated with protection against atherosclerosis, an oxidative disease. However, serum PON-1 activity is highly variable and its regulation is complex, involving both genetic and environmental factors. It is influenced by gender and inflammation, two important factors in atherosclerosis. Serum PON-1 activity has been shown to be lower in male mice and is decreased in male Syrian hamster during inflammation. Here we show that male mice had lower hepatic PON-1 mRNA that increased by 170% after castration. Our data also suggested that this effect was testes but not plasma testosterone dependent. Ovariectomy had no effect on PON-1 mRNA in female mice. LPS caused hepatic PON-1 mRNA to decrease further in male mice, and to increase moderately in female mice. Anti-inflammatory dexamethasone enhanced PON-1 mRNA level by 2-fold in male and female LPS-treated mice, and increased PON-1 expression by 8-fold in Hepa cell, a mouse hepatoma cell line. Therefore, antioxidant PON-1 is regulated at the mRNA level in a gender-specific manner by proinflammatory LPS and anti-inflammatory dexamethasone.

Animals↗

PubMed2003

Freund adjuvant induces TLR2 but not TLR4 expression in the liver of mice.

Freund adjuvants are used extensively to establish experimental animal models of autoimmune diseases and to produce antibodies. However, studies on their mechanisms of action have been largely neglected, particularly their effects on liver, the primary target organ for host-microbe interaction. Here we show that treatment with either complete (CFA) or incomplete (IFA) Freund adjuvant induced a 5-10-fold increase in toll-like receptor (TLR) 2 mRNA but not TLR4 mRNA in livers of mice. Since CFA is essentially made of killed Mycobacterium tuberculosis bacilli (Mtb) dissolved in IFA, it is the solvent in CFA that induced an increase in TLR2 expression. As TLR2 is the receptor activated by killed Mtb, this solvent-mediated increase in TLR2 expression will result in enhanced recognition of killed Mtb by hepatocytes during CFA administration. We propose that the potency of Freund adjuvant in eliciting an immune response lies in their ability to induce expression of the appropriate TLR, TLR2, for the active ingredient, killed Mtb, in CFA.

Animals↗

PubMed2002

Quinacrine abolishes increases in cytoplasmic phospholipase A2 mRNA levels in the rat hippocampus after kainate-induced neuronal injury.

The present investigation was carried out to study the possible effects of quinacrine in modulating cytoplasmic phospholipase A(2) (cPLA(2)) mRNA levels in rat hippocampus after kainate treatment. Injections of kainate into the right lateral ventricle resulted in significant increases in cPLA(2) mRNA levels in the hippocampus, at 3 days and 7 days after injection. The elevation in cPLA(2) mRNA levels is consistent with previous observations of increased cPLA(2) immunoreactivity in degenerating neurons and astrocytes at these times. Rats that received once daily intraperitoneal injections of quinacrine (5 mg/kg) after the intracerebroventricular kainate injections showed almost complete attenuation of increased cPLA(2) expression, at both 3 and 7 days after kainate injection. These results show that in addition to its well-known effect of inhibition of PLA(2) activity, quinacrine could also inhibit cPLA(2) expression, and further supports a role for PLA(2) in kainate-induced neuronal injury.

Animals↗

PubMed2002

Nitric oxide synthesis is involved in arterial haptoglobin expression after sustained flow changes.

The acute phase protein haptoglobin is highly expressed in arteries after sustained flow changes and involved in cell migration and arterial restructuring. In the liver, haptoglobin expression is mainly regulated by interleukin-6 (IL-6). In the artery, shear stress and NO influence IL-6 expression. In the present study, we demonstrate that NO synthesis is involved in the regulation of arterial haptoglobin expression after sustained flow changes. Decreased haptoglobin expression after NO inhibition coincided with decreased IL-6 levels. However, IL-6 knockout mice had normal arterial haptoglobin expression levels after sustained flow changes suggesting that other mediators may provide compensatory mechanisms for the regulation of arterial haptoglobin expression.

Animals↗

PubMed2002

Whole bone marrow transplantation induces angiogenesis following acute ischemia.

Several recent studies have shown that purified subsets of bone marrow (BM) cells can differentiate into endothelial, cardiac, and other cell types. During coronary artery bypass graft (CABG) surgery, sternal BM is routinely discarded. To determine if this BM can be used to induce angiogenesis and augment perfusion of the cardiac tissues after CABG, a simplified and more practical approach of using whole BM extract was tried to determine whether it would be adequate for the induction of BM-derived angiogenesis in experimental acute limb ischemia. BM was prepared from FVB/N-TgN(TIE2 lacZ)182 Sato (Tie2-lacZ) or B6.129S7-Gtrosa 26 (Rosa 26) mice that express beta-galactosidase (beta-gal) in endothelial cells and most adult tissues, respectively. Acute limb ischemia was induced in either C57BL6/J or FVB/N mice by double ligation of the left femoral artery just distal to the profunda femoral artery branch. Occlusion of the ligated artery was verified by angiography. The study group (n = 31) received an intramuscular injection of 50 micro l containing 1 x 10(6) BM cells, 5 mm proximal to the site of ligation. Experimental controls (n = 21) had an intramuscular injection of 50 micro l of saline. Angiogenesis in the mice was assessed by histological analysis. BM-derived beta-gal(+) cells were observed to aggregate in the vicinity of the ligated artery and not in the injected musculature BM-derived endothelial cells were incorporated within capillaries and small size blood vessels near the site of ligation. Generation of BM-derived blood vessels in experimental acute limb ischemia does not require purification of specific subset of cells. The elimination of cell purification will enhance the ease of using BM transplantation in generating blood vessels.

Acute Disease↗

PubMed2002

AFP(+), ESC-derived cells engraft and differentiate into hepatocytes in vivo.

A major problem in gene therapy and tissue replacement is accessibility of tissue-specific stem cells. One solution is to isolate tissue-specific stem cells from differentiating embryonic stem (ES) cells. Here, we show that liver progenitor cells can be purified from differentiated ES cells using alpha-fetoprotein (AFP) as a marker. By knocking the green fluorescent protein (GFP) gene into the AFP locus of ES cells and differentiating the modified ES cells in vitro, a subpopulation of GFP(+) and AFP-expressing cells was generated. When transplanted into partially hepatectomized lacZ-positive ROSA26 mice, GFP(+) cells engrafted and differentiated into lacZ-negative and albumin-positive hepatocytes. Differentiation into hepatocytes also occurred after transplantation of GFP(+) cells in apolipoprotein-E- (ApoE) or haptoglobin-deficient mice as demonstrated by the presence of ApoE-positive hepatocytes and ApoE mRNA in the liver of ApoE-deficient mice or by haptoglobin in the serum and haptoglobin mRNA in the liver of haptoglobin-deficient mice. This study describes the first isolation of ES-cell-derived liver progenitor cells that are viable mediators of liver-specific functions in vivo.

Animals↗

PubMedDate not supplied

Increased renal hypertrophy in diabetic mice genetically modified at the haptoglobin locus.

BACKGROUND: The human haptoglobin (Hp) gene is polymorphic with two functional classes of alleles, denoted 1 and 2. We have demonstrated in three longitudinal studies and several cross-sectional studies that the Hp genotype is an independent risk factor for diabetic vascular disease. These studies have presented a compelling argument that diabetic individuals homozygous for the Hp 1 allele are at decreased risk of vascular complications as compared to diabetic individuals with the Hp 2 allele. METHODS: The naturally occurring (wild type) mouse Hp is a class 1 Hp allele. We examined renal hypertrophy in wild-type mice, Hp knockout mice (Hp 0), and in mice with the Hp 2 allele (Hp 2) with and without diabetes. RESULTS: In the absence of diabetes, we found that renal hypertrophy was significantly increased in Hp 0 mice and that this could be prevented with vitamin E. There was no difference between wild type and Hp 2 mice with regard to renal hypertrophy in the absence of diabetes. However, in the presence of diabetes, Hp 2 mice demonstrated a significant increase in renal hypertrophy as compared to wild-type mice. CONCLUSIONS: These results support a direct linkage between diabetic vascular disease and the Hp genotype. These Hp-modified mice may serve as a platform on which to test a variety of pharmacological agents in order to decrease diabetic vascular disease.

Animals↗

PubMedDate not supplied

Myocardial infarction in the C57BL/6J mouse: a quantifiable and highly reproducible experimental model.

INTRODUCTION: The laboratory mouse is a powerful tool in cardiovascular research. In this report, we describe a method for a reproducible mouse myocardial infarction model that would allow subsequent comparative and quantitative studies on molecular and pathophysiological variables. METHODS: (A) The distribution of the major coronary arteries including the septal artery in the left ventricle of the C57BL/6J mice (n=20) was mapped by perfusion of latex dye or fluorescent beads through the aorta. (B) The territory of myocardial infarction after the ligation of the most proximal aspect of the left anterior descending (LAD) coronary artery was quantified. (C) The consistency in the histological changes parallel to the infarction at different time points was analyzed. RESULTS: (A) The coronary artery tree of the mouse is different from human and, particularly, in regard to the blood supply of the septum. (B) Contrary to previous belief, the septal coronary artery in the mouse is variable in origin. (C) A constant ligation of the LAD immediately below the left auricular level ensures a statistically significant reproducible infarct size. (D) The ischemic changes can be monitored at a histological level in a way similar to what is described in the human. CONCLUSION: We illustrate a method for maximal reproducibility of experimental acute myocardial infarction in the mouse model, due to a consistent loss of perfusion in the lower half of the left ventricle. This will allow the study of molecular and physiological variables in a controlled and quantifiable experimental model environment.

Animals↗

PubMedDate not supplied

Generation of hybrid cell lines with endothelial potential from spontaneous fusion of adult bone marrow cells with embryonic fibroblast feeder.

We have previously isolated mouse embryonic cell lines with endothelial potential using a simple empirical approach. In an attempt to isolate similar cell lines from adult mouse bone marrow (BM), BM cells were cultured on mitotically inactive mouse embryonic fibroblast (MEF) feeder cells. Several cell lines with putative endothelial potential were generated. They expressed endothelial-specific genes and formed vascular-like structures when plated on matrigel. When transplanted into appropriate mouse models, they incorporated into the endothelium of the vasculature. Similar cell lines were also obtained using human or porcine BM. None of these lines induced tumor formation when transplanted into immunodeficient Rag1-/- mice. However, all the lines were aneuploid with genetic markers from BM samples and the MEF feeder, suggesting that they resulted from a non-species-specific fusion of a BM cell and mitotically inactive MEF. Together, these lines demonstrated for the first time that BM cells can also undergo fusion with commonly used mitotically inactive feeder cells. Although these fusion cell lines were culture artifacts, their derivation would be useful in understanding fusion of BM cells with other cell types, and their endothelial potential will also be useful in characterizing endothelial differentiation.

Aneuploidy↗