Ay Eeng Tan

Human chorion-derived stem cells: changes in stem cell properties during serial passage

BACKGROUND AIMS Fetal membrane from human placenta tissue has been described as a potential source of stem cells. Despite abundant literature on amnion stem cells, there are limited studies on the stem cell properties of chorion-derived stem cells. METHODS The main aim was to determine the stemness properties of serial-passaged human chorion-derived stem cells (hCDSC). Quantitative polymerase chain reaction (PCR) was performed to reveal the following stemness gene expression in serial-passaged hCDSC: Oct-4, Sox-2, FGF-4, Rex-1, TERT, Nanog (3), Nestin, FZD-9, ABCG-2 and BST-1. Cell growth rate was evaluated from passage (P) 1 until P5. The colony-forming unit-fibroblast (CFU-F) frequency of P3 and P5 cells and multilineage differentiation potential of P5 cells were determined. The immunophenotype of hCDSC was compared using the surface markers CD9, CD31, CD34, CD44, CD45, CD73, CD90, CD117, HLA-ABC and HLA-DR, -DP and -DQ. Immunostaining for trophoblast markers was done on P0, P1, P3 and P5 cells to detect the contamination of trophoblasts in culture, while chromosomal abnormality was screened by cytogenetic analysis of P5 cells. RESULTS The surface markers for mesenchymal lineage in hCDSC were more highly expressed at P5 compared with P3 and P0, indicating the increased purity of these stem cells after serial passage. Indeed, all the stemness genes except TERT were expressed at P1, P3 and P5 hCDSC. Furthermore, human chorion contained high clonogenic precursors with a 1:30 CFU-F frequency. Successful adipogenic, chondrogenic and osteogenic differentiation demonstrated the multilineage potential of hCDSC. The karyotyping analysis showed hCDSC maintained chromosomal stability after serial passage. CONCLUSIONS hCDSC retain multipotent potential even at later passages, hence are a promising source for cell therapy in the future.

Value of human amniotic epithelial cells in tissue engineering for cornea

Human amniotic epithelial cells (hAECs) are potentially one of the key players in tissue engineering due to their easy availability. The aim of the present study was to develop an optimal isolation and transportation technique, as well as to determine the immunophenotype and epithelial gene expression of hAECs. Amnion was mechanically peeled off from the chorion and digested with trypsin-ethylenediaminetetraacetic acid. The isolated hAECs were cultured in medium containing 10 ng/mL epidermal growth factor until P4. The epithelial gene expression, cell surface antigen and protein expression of hAECs were analyzed by quantitative polymerase chain reaction, flow cytometry and immunocytochemistry. hAECs were also cultured in adipogenic, osteogenic and neurogenic induction media. The best cell yield of hAECs was seen in the digestion of 15 pieces of amnion (2 × 2 cm) and isolated 30 min after digestion with trypsin. F12:Dulbecco’s modified eagle medium was the best medium for short term storage at 4 °C. hAECs expressed CD9, CD44, CD73 and CD90, and negligibly expressed CD31, CD34, CD45 and CD117. After serial passage, CK3, CK19 and involucrin gene expressions were upregulated, while p63, CK1 and CK14 gene expressions were downregulated. Sustained gene expressions of integrin β1 and CK18 were observed. At initial culture, these cells might have stem-like properties. However, they differentiated after serial passage. Nonetheless, hAECs have epithelial stem cell characteristics and have the potential to differentiate into corneal epithelial cells. Further investigations are still needed to elucidate the mechanism of differentiation involved and to optimize the culture condition for long term in vitro culture.

Stemness and angiogenic gene expression changes of serial-passage human amnion mesenchymal cells

BACKGROUND Particular attention has been directed towards human amnion mesenchymal stem cells (HAMCs) due to their accessibility, availability and immunomodulatory properties. Therefore, the aim of the present study was to determine the temporal changes of stemness and angiogenic gene expressions of serial-passage HAMCs. METHODS HAMCs were isolated from human term placenta and cultured in serial passages in culture medium supplemented with 10% fetal bovine serum. Morphological analysis, growth kinetic and CFU-F assay of HAMCs were assessed. In vitro differentiation and the immunophenotype of HAMCs at P5 were also analyzed. Quantitative PCR was used to determine the stemness, angiogenic and endothelial gene expression of cultured HAMCs after serial passage. RESULTS Cultured HAMCs displayed intermediate epitheloid-fibroblastoid morphology at an initial culture and the fibroblastoid features became more pronounced in later passages. They showed high clonogenic activity and faster proliferation at later passages with colony forming efficiency of 0.88%. HAMCs were successfully differentiated into adipocytes, osteocytes and neuron-like cells. Most HAMCs expressed CD9, CD44, CD73, CD90 and HLA-A,B,C but negligibly expressed CD31, CD34, CD45, CD117 and HLA-DR,DP,DQ. After serial passage, stemness genes Oct-3/4, Sox-2, Nanog3, Rex-1, FGF-4 and FZD-9 expressions significantly decreased. Of the angiogenic genes PECAM-1, bFGF, eNOS, VEGFR-2, VEGF, and vWF expressions also decreased significantly except angiopoietin-1 which significantly increased. No significant differences were observed in ABCG-2, BST-1, nestin, PGF and HGF expressions after serial passage. CONCLUSION These results suggested that cultured HAMCs could be an alternative source of stem cells and may have the potential for angiogenesis and hence its use in stem-cell based therapy.

Effects of epidermal growth factor on the proliferation and cell cycle regulation of cultured human amnion epithelial cells.

Human amnion epithelial cells (HAECs) hold great promise in tissue engineering for regenerative medicine. Large numbers of HAECs are required for this purpose. Hence, exogenous growth factor is added to the culture medium to improve epithelial cells proliferation. The aim of the present study was to determine the effects of epidermal growth factor (EGF) on the proliferation and cell cycle regulation of cultured HAECs. HAECs at P1 were cultured for 7 days in medium containing an equal volume mix of HAMs F12: Dulbeccos Modified Eagles Medium (1:1) supplemented with different concentrations of EGF (0, 5, 10, 20, 30 and 50 ng/ml EGF) in reduced serum. Morphology, growth kinetics and cell cycle analysis using flow cytometry were assessed. Quantitative gene expression for cell cycle control genes, pluripotent transcription factors, epithelial genes and neuronal genes were also determined. EGF enhanced HAECs proliferation with optimal concentration at 10 ng/ml EGF. EGF significantly increased the proportion of HAECs at S- and G2/M-phase of the cell cycle compared to the control. At the end of culture, HAECs remained as diploid cells under cell cycle analysis. EGF significantly decreased the mRNA expression of p21, pRb, p53 and GADD45 in cultured HAECs. EGF also significantly decreased the pluripotent genes expression: Oct-3/4, Sox2 and Nanog; epithelial genes expression: CK14, p63, CK1 and Involucrin; and neuronal gene expression: NSE, NF-M and MAP 2. The results suggested that EGF is a strong mitogen that promotes the proliferation of HAECs through cell cycle regulation. EGF did not promote HAECs differentiation or pluripotent genes expression.

Umbilical artery resistance index in diabetic pregnancies: The associations with fetal outcome and neonatal septal hypertrophic cardiomyopathy

Aim: To determine the incidence of an abnormal umbilical artery resistance index (UARI) in diabetic pregnancies and the relation to fetal outcome and the development of neonatal septal hypertrophic cardiomyopathy.

Potential of Human Decidua Stem Cells for Angiogenesis and Neurogenesis

BACKGROUND AND AIMS Placenta as a fetomaternal organ is a potential source of fetal as well as maternal stem cells. This present study describes novel properties of the cells isolated from the maternal part of term placenta membrane, the decidua basalis. METHODS Colony-forming unit-fibroblast (CFU-F) frequency and immunophenotype of human decidua-derived stem cells (hDSC) was carried out using flow cytometry. Quantitative polymerase chain reaction was performed to reveal the stemness, angiogenic- and endothelial cell-associated genes expression in serial-passage hDSC. Adipogenic and osteogenic differentiation potential of passage 5 (P5) cells were determined. We also performed immunostaining of common angiogenic/endogenic (CD31 and vWF) and neurogenic markers (GFAP, NF, NSE, vimentin and nestin) on hDSC at P5. RESULTS HDSC contains high clonogenic precursor with 1:25 CFU-F frequency. Mesenchymal stem cell-associated markers CD90, CD9, CD44, CD73 and HLA ABC were highly expressed in P0 and P5 hDSC. The specific lineage markers CD117, CD45, CD34, CD31 and HLA DR DP DQ were scarcely expressed. HDSC expressed all the stem cell-associated genes and the expression was maintained until P5. Also, the cells are capable of differentiating into adipogenic and osteogenic lineage. Positive expressions of angiogenic/endogenic markers (CD31, vWF) and neurogenic markers (GFAP, NF, NSE, vimentin and nestin) were demonstrated by hDSC. CONCLUSIONS Human decidua contains stem cells with great proliferation capacity and mesenchymal properties. Expressions of angiogenic/endogenic and neurogenic markers support the conclusion that hDSC is a promising stem cell source for neurogenesis as well as angiogenesis.

The role of serum insulin-like growth factor I (IGF-I) in neonatal outcome

Aims: To determine the role of serum insulin-like growth factor I (IGF-I) in predicting the occurrence of septal hypertrophic cardiomyopathy in infants of mothers with diabetes. Methods/materials: In this prospective study, 100 pregnant women (50 with diabetes and 50 controls), matched for age and race, were studied. One intrapartum blood sample was taken at 28 weeks of gestation from both groups of mothers and another sample at delivery. All samples were analysed for maternal IGF-I by an enzyme linked immunosorbent assay method. A chest radiograph and an electrocardiogram were performed on the babies of the mothers with diabetes within the first 24 hours of life. An echocardiogram was performed in the first 3 days of life to look for septal hypertrophy and to measure the myocardial thickness. Results: In the six cases of neonatal septal hypertrophic cardiomyopathy, all the mothers had greatly raised IGF-I concentrations of more than 400 ng/ml at the time of delivery compared with a mean (SD) of 302 (25) ng/ml in control mothers. Conclusions: In the present study a crude analysis revealed that increased IGF-I concentrations correlate with neonatal septal hypertrophic cardiomyopathy.

Organotypic culture of human amnion cells in air-liquid interface as a potential substitute for skin regeneration

BACKGROUND AIMS The aim of the present study was to evaluate the effects of air-liquid interface on the differentiation potential of human amnion epithelial cells (HAECs) to skin-like substitute in organotypic culture. METHODS HAECs at passage 1-2 were seeded onto a fibrin layer populated with human amnion mesenchymal cells to form the organotypic cultures. The organotypic HAECs were then cultured for 7, 14 and 21 d in two types of culture system: the submerged culture and the air-liquid interface culture. Cell morphogenesis was examined under the light and electron microscopes (transmission and scanning) and analyzed by immunohistochemistry. RESULTS Organotypic HAECs formed a single layer epithelium after 3 wk in submerged as well as air-liquid interface cultures. Ultrastructurally, desmosomes were observed in organotypic HAECs cultured in the air-liquid interface but not in the submerged culture. The presence of desmosomes marked the onset of early epidermal differentiation. Organotypic HAECs were positive against anti-CK18 and anti-CK14 in both the submerged and the air-liquid interface cultures. The co-expression of CK14 and CK18 suggested that differentiation of HAECs into skin may follow the process of embryonic skin development. However, weak expression of CK14 was observed after 2 and 3 wk of culture in air-liquid interface. CK10, involucrin, type IV collagen and laminin-5 expression was absent in organotypic HAECs. This observation reflects the initial process of embryonic epidermal differentiation and stratification. CONCLUSIONS Results from the present study suggest that the air-liquid interface could stimulate early differentiation of organotypic HAECs to epidermal cells, with a potential use for skin regeneration.

Effects of keratinocyte growth factor on skin epithelial differentiation of human amnion epithelial cells

The aim of the present study was to determine the effects of KGF on the differentiation of cultured human amnion epithelial cells (HAECs) towards skin keratinocyte. HAECs at passage 1 were cultured in medium HAMs F12: Dulbeccos Modified Eagles Medium (1:1) supplemented with different concentrations of KGF (0, 5, 10, 20, 30 and 50 ng/ml KGF). Dose-response of KGF on HAECs was determined by morphological assessment; growth kinetic evaluation; immunocytochemical analysis; stemness and epithelial gene expression quantification with two step real time RT-PCR. KGF promotes the proliferation of HAECs with maximal effect observed at 10 ng/ml KGF. However, KGF decreased the stemness genes expression: Oct-3/4, Sox-2, Nanog3, Rex-1, FGF-4, FZD-9 and BST-1. KGF also down-regulates epithelial genes expression: CK3, CK18, CK19, Integrin-β1, p63 and involucrin in cultured HAECs. No significant difference on the gene expression was detected for each Nestin, ABCG-2, CK1 and CK14 in KGF-treated HAECs. Immunocytochemical analysis for both control and KGF-treated HAECs demonstrated positive staining against CK14 and CK18 but negative staining against involucrin. The results suggested that KGF stimulates an early differentiation of HAECs towards epidermal cells. Differentiation of KGF-treated HAECs to corneal lineage is unfavourable. Therefore, further studies are needed to elucidate the roles of KGF in the differentiation of HAECs towards skin keratinocytes.

65 Placental IGF-I Receptor and Septal Hypertrophic Cardiomyopathy in Infants of Diabetic Mothers

Background/Aims Septal hypertrophic cardiomyopathy (sHCM) is a characteristic anomaly of the infant of diabetic mother (IDM). Insulin-like growth factor-I (IGF-I) has been identified to contribute to the various tissue overgrowth in these infants. We have previously shown that maternal IGF-I levels were significantly elevated among neonates with sHCM. IGF-I does not cross the placenta and its physiologic action is mediated through the receptor, IGF-IR. IGF-IR are present in various tissues including the heart muscle. We have thus investigated whether there is an association between placental IGF-IR in IDMs with and without sHCM.Methods A cohort of 50 diabetic and 50 normal pregnancies booked in the Hospital UKM, Kuala Lumpur, over a period of two years (Jan. 2001- Dec. 2002) were enrolled into a larger related study. The placentae of six diabetic pregnancies with neonatal sHCM and six randomly selected diabetic pregnancies without neonatal sHCM were compared to the placentae from six normal pregnancies for IGF-IR expression by immunohistochemistry. The staining for IGF-IR in the decidua, cytotrophoblast, syncytiotrophoblast and fetal endothelium for these 18 samples were assessed randomly by the pathologist who was blinded to the respective diagnoses. The intensities of IGF-IR staining were classified as 0 (negative), 1+ (weak), 2+ (medium), 3+ (strong).Results Placental IGF-IR staining was negative in the fetal endothelium for all three groups. In the normal controls, and diabetic group without neonatal sHCM, IGF-IR were 3+ in decidua, 2+ in cytotrophoblast and 2+ in syncytiotrophoblast. In contrast, IGF-IR were all 2+ in the decidua, cytotrophoblast and syncytiotrophoblast from the diabetic group with neonatal sHCM.Conclusions Reduced placental IGF-IR expression in the decidua of diabetic pregnancies suggests that IGF-IR is down-regulated in response to elevated maternal IGF or mediators of neonatal sHCM. Modulation of activity at the IGF-IR level may be a target for therapy against the development of fetal/neonatal sHCM in diabetic pregnancies.