Pro-angiogenic potential of human chorion-derived stem cells: in vitro and in vivo evaluation.

Human chorion-derived stem cells (hCDSC) were previously shown to demonstrate multipotent properties with promising angiogenic characteristics in monolayer-cell culture system. In our study, we investigated the angiogenic capability of hCDSC in 3-dimensional (3D) in vitro and in vivo angiogenic models for the purpose of future application in the treatment of ischaemic diseases. Human CDSC were evaluated for angiogenic and endogenic genes expressions by quantitative PCR. Growth factors secretions were quantified using ELISA. In vitro and in vivo vascular formations were evaluated by histological analysis and confocal microscopic imaging. PECAM-1(+) and vWF(+) vascular-like structures were observed in both in vitro and in vivo angiogenesis models. High secretions of VEGF and bFGF by hCDSC with increased expressions of angiogenic and endogenic genes suggested the possible angiogenic promoting mechanisms by hCDSC. The cooperation of hCDSC with HUVECS to generate vessel-like structures in our systems is an indication that there will be positive interactions of hCDSC with existing endothelial cells when injected into ischaemic tissues. Hence, hCDSC is suggested as the novel approach in the future treatment of ischaemic diseases.

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.

Endogenous and induced angiogenic characteristics of human chorion-derived stem cells.

Cell-based therapy using stem cells has emerged as one of the pro-angiogenic methods to enhance blood vessel growth and sprouting in ischaemic conditions. This study investigated the endogenous and induced angiogenic characteristics of hCDSC (human chorion-derived stem cell) using QPCR (quantitative PCR) method, immunocytochemistry and fibrin-matrigel migration assay. The results showed that cultured hCDSC endogenously expressed angiogenic-endogenic-associated genes (VEGF, bFGF, PGF, HGF, Ang-1, PECAM-1, eNOS, Ve-cad, CD34, VEGFR-2 and vWF), with significant increase in mRNA levels of PGF, HGF, Ang-1, eNOS, VEGFR-2 and vWF following induction by bFGF (basic fibroblast growth factor) and VEGF (vascular endothelial growth factor). These enhanced angiogenic properties suggest that induced hCDSC provides a stronger angiogenic effect for the treatment of ischaemia. After angiogenic induction, hCDSC showed no reduction in the expression of the stemness genes, but had significantly higher levels of mRNA of Oct-4, Nanog (3), FZD9, ABCG-2 and BST-1. The induced cells were positive for PECAM-1 (platelet/endothelial cell adhesion molecule 1) and vWF (von Willebrand factor) with immunocytochemistry staining. hCDSC also showed endothelial migration behaviour when cultured in fibrin-matrigel construct and were capable of forming vessels in vivo after implanting into nude mice. These data suggest that hCDSC could be the cells of choice in the cell-based therapy for pro-angiogenic purpose.

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.

An immunohistochemical study of Toll-like receptors 2 and 4 in placenta with and without infection.

OBJECTIVE: The placenta constitutes a physical and immunological barrier against infectious agents. Toll-like receptors (TLRs) are essential components for the induction of innate immunity responses in different human tissues including the placenta. We investigated the expressions of TLR2 and TLR4 in the decidua and amniotic cells in non-inflamed placenta and placenta with infection. MATERIALS AND METHODS: There were a total 74 placentas (37 with infection and 37 without infection- 25 bacterial, 10 viral and 2 toxoplasma). TLR2 and TLR4 expressions were assessed using immunohistochemical technique. Positive cells were indicated by cytoplasmic staining and the percentage of positive in 100 cells was recorded and graded. The grades were 1+ (< 25%), 2+ (25-75%) and 3+ (> 75%). RESULTS: We found significantly higher expression of TLR2 in the amniotic cells and decidua cells in infected placentas as compared to non-inflamed placentas among the preterm placenta. A higher number of cases have TLR4 expression in the amnion of preterm infected placenta than in term placenta. This, however, is not statistically significant. CONCLUSION: Our findings suggest that TLR2 plays a role in the innate immunity in bacterial and viral infection in the placenta, however, their role in protection against toxoplasma may be limited. This study further supports the observations that TLR2 expression was higher in placenta with infection which strengthened the role of TLR2 in the protection of preterm placenta against infection.

Alveolar rhabdomyosarcoma of the left hand with bilateral breast metastases in an adolescent female.

Rhabdomyosarcoma is a common extramammary malignancy in pediatric age groups, but it rarely metastasizes to the breast. Breast rhabdomyosarcomas are commonly metastatic, with possible primary locations at the head and neck, trunk, extremities, retroperitoneum and perianal region. We report a case of primary alveolar rhabdomyosarcoma of the upper extremities in a 17-year-old adolescent female who presented with bilateral lower limb weakness and bilateral breast lumps.

Insulin-like growth factor-1 receptor expression in the placentae of diabetic and normal pregnancies.

BACKGROUND: Septal hypertrophic cardiomyopathy (sHCM) is a characteristic anomaly of the infant of diabetic mother (IDM). Insulin-like growth factor-1 (IGF-1) has been identified as a mediator of tissue overgrowth and we have previously shown that maternal IGF-1 levels were significantly elevated among neonates with asymmetrical sHCM. IGF-1 does not cross the placenta; it exerts physiologic action through binding to the IGF-1 receptor (IGF-1R). Localisation and expression of IGF-1R in term diabetic pregnancies are largely unclear. We have studied IGF-1R in the placentae of diabetic and normal pregnancies and this receptor expression in association with neonates with sHCM. METHODS: IGF-1R localization and expression in the placentae of six diabetic pregnancies associated with neonatal sHCM were compared with six each of randomly selected diabetic and normal pregnancies without neonatal sHCM by immunohistochemistry. The staining for IGF-1R in the deciduas, cytotrophoblasts, syncytiotrophoblasts and villous endothelium for these 18 samples were assessed and scored by two pathologists who were blinded to the respective diagnoses. RESULTS: Placental IGF-1R staining was negative in the villous endothelium for all three groups. IGF-1R staining was present in deciduas, cytotrophoblasts and syncytiotrophoblasts but the staining was weaker in the entire group of infants with sHCM compared to those without sHCM. CONCLUSIONS: IGF-1R is localized in all cell types of the placenta except in villous endothelium. Weaker placental IGF-1R staining in the placentae of diabetic pregnancies associated with sHCM suggests reduced expression of IGF-1R. This may be a down-regulatory response to elevated maternal IGF with neonatal sHCM outcome.

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 HAM's F12: Dulbecco's 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-ß1, 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.

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 HAM's F12: Dulbecco's 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.

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.

Low perinatal autopsy rate in Malaysia: time for a change.

Our objectives were to determine the perinatal autopsy rate in a tertiary hospital in Malaysia and to quantify the value of the perinatal autopsy. All stillbirths, miscarriages, therapeutic abortions, and neonatal deaths between January 1, 2004, and August 31, 2009, were identified from the archives. The autopsy findings were compared with the clinical diagnoses. The autopsy reports were also reviewed to determine if it would be possible to improve the quality of the autopsies. There were 807 perinatal deaths, of which 36 (4.5%) included an autopsy. There were ethnic differences in the rate of autopsy, with the lowest rate among the Malays. The autopsy provided the diagnosis, changed the clinical diagnosis, or revealed additional findings in 58.3% of cases. Ancillary testing, such as microbiology, chromosomal analysis, and biochemistry, could improve the quality of the autopsy. This study provides further data on the perinatal autopsy rate from an emerging and developing country. It reaffirms the value of the perinatal autopsy. Attempts must be made to improve on the low autopsy rate while recognizing that the performance of autopsies can be enhanced through the use of ancillary testing.

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 ß1 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.

An immunohistochemical study of CD1a and CD83-positive infiltrating dendritic cell density in cervical neoplasia.

Cervical carcinoma is the second leading cancer in women in Malaysia, after breast cancer. Human papillomavirus (HPV) has been implicated in the development of dysplasia or cervical intraepithelial neoplasia and progression to squamous cell carcinoma. Because of the confinement of the human papillomavirus infection within the epithelial layer, the presence of dentritic cells or Langerhans cells in epithelial layer of the ectocervix is paramount in producing immune response. The mature dentritic cells express CD83 and high CD40/80/86, whereas the immature cells express CD1a and low CD40/80/86. By identifying CD1a and CD83, theoretically, both immature and mature dentritic cell populations can be studied. In view of the facts, we investigated the infiltrating cell density of mature and immature dentritic cells in cervical neoplasia.