Haekwon Kim

Human amniotic fluid-derived stem cells have characteristics of multipotent stem cells

Abstract.u2002 Objectives: To characterize mesenchymal stem cell‐like cells isolated from human amniotic fluid for a new source of therapeutic cells. Materials: Fibroblastoid‐type cells obtained from amniotic fluid at the time of birth. Methods: The ability of ex vivo expansion was investigated until senescence, and stem cell‐like characteristics were analyzed by examining differentiation potential, messenger RNA expression and immunophenotypes. Results and Conclusions: A morphologically homogenous population of fibroblastoid‐type (HAFFTs) cells, similar to mesenchymal stem cells from bone marrow (BM‐MSCs), was obtained at the third passage. The cells became senescent after 27 passages over a period of 8 months while undergoing 66 population doublings. Under appropriate culture conditions, by the 8th passage they differentiated into adipocytes, osteocytes, chondrocytes and neuronal cells, as revealed by oil red O, von Kossa, Alcian blue and anti‐NeuN antibody staining, respectively. Immunophenotype analyses at the 17th passage demonstrated the presence of TRA‐1–60; SSEA‐3 and‐4; collagen types I, II, III, IV and XII; fibronectin; α‐SMA; vimentin; desmin; CK18; CD44; CD54; CD106; FSP; vWF; CD31; and HLA ABC. Reverse transcriptase–polymerase chain reaction analysis of the HAFFTs from passages 6–20 showed consistent expression of Rex‐1, SCF, GATA‐4, vimentin, CK18, FGF‐5 and HLA ABC genes. Oct‐4 gene expression was observed up to the 19th passage but not at the 20th passage. HAFFTs showed telomerase activity at the 5th passage with a decreased level by the 21st passage. Interestingly, BMP‐4, AFP, nestin and HNF‐4α genes showed differential gene expression during ex vivo expansion. Taken together, these observations suggest that HAFFTs are pluripotent stem cells that are less differentiated than BM‐MSCs, and that their gene expression profiles vary with passage number during ex vivo expansion.

Insulin‐Secreting Cells from Human Eyelid‐Derived Stem Cells Alleviate Type I Diabetes in Immunocompetent Mice

Various attempts have been made to develop stem cell‐based therapy to alleviate type I diabetes using animal models. However, it has been a question whether human insulin produced from explanted cells is solely responsible for the normoglycemia of diabetic animals. In this study, we isolated neural crest‐like stem cells from the human eyelid fat and examined their therapeutic potentials for diabetes. The human eyelid adipose‐derived stem cells (HEACs) displayed characteristics of neural crest cells. Using a two‐step culture condition combined with nicotinamide, activin, and/or GLP‐1, we differentiated HEACs into insulin‐secreting cells and examined in vivo effects of differentiated cells by transplantation experiments. Following differentiation in vitro, HEACs released insulin and c‐peptide in a glucose‐dependent manner. Upon their transplantation under kidney capsules of streptozotocin‐treated immunocompetent mice, we observed normalization of hyperglycemia in 10 of 20 recipient mice until sacrifice after 2 months. Only the human, but not the mouse, insulin and c‐peptide were detected in the blood of recipient mice. Removal of the kidneys transplanted with HEACs resulted in a sharp increase of blood glucose level. Removed kidney tissues showed distinct expression of various human genes including insulin, and colocalization of the human insulin and the human nuclear protein in many cells. However, they showed diminished or null expression of some immune‐related genes. In conclusion, human insulin alone produced from eyelid‐derived stem cells following differentiation into insulin‐secreting cells and transplantation could normalize type I diabetes in mice. STEM CELLS 2009;27:1999–2008

Human amnion-derived mesenchymal stem cells are a potential source for uterine stem cell therapy.

Abstract.u2002 Objectives: Human amnion is an easy‐to‐obtain novel source of human mesenchymal stem cells, which poses little or no ethical dilemmas. We have previously shown that human amnion‐derived mesenchymal (HAM) cells exhibit certain mesenchymal stem cell‐like characteristics with respect to expression of stem cell markers and differentiation potentials. Materials and methods: In this study, we further characterized HAM cells’ potential for in vivo therapeutic application. Results: Flow cytometric analyses of HAM cells show that they express several stem cell‐related cell surface markers, including CD90, CD105, CD59, CD49d, CD44 and HLA‐ABC, but not CD45, CD34, CD31, CD106 or HLA‐DR. HAM cells at the 10th passage showed normal karyotype. More interestingly, the AbdB‐like HOXA genes HOXA9, HOXA10 and HOXA11 that are expressed in the mesenchyme of the developing female reproductive tract and pregnant uteri are also expressed in HAM cells, suggesting similarities between these two mesenchymal cell types. Progesterone receptor is also highly expressed in HAM cells and expression of genes or proteins in HAM cells could be manipulated with the aid of lentivirus technology or cell‐permeable peptides. To test potentials of HAM cells for in vivo application, we introduced enhanced green fluorescence protein (EGFP)‐expressing HAM cells to mice by intrauterine infusion (into uteri) or by intravenous injection (into the circulation). Presence of EGFP‐expressing cells within the uterine mesenchyme after intrauterine infusion or in lungs after intravenous injection was noted within 1–4 weeks. Conclusions: Collectively, these results suggest that HAM cells are a potential source of mesenchymal stem cells with therapeutic potential.

Opposing Roles of c-Jun NH2-Terminal Kinase and p38 Mitogen-Activated Protein Kinase in the Cellular Response to Ionizing Radiation in Human Cervical Cancer Cells

Exposure of cells to ionizing radiation induces activation of multiple signaling pathways that play critical roles in determining cell fate. However, the molecular basis for cell death or survival signaling in response to radiation is unclear at present. Here, we show opposing roles of the c-jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) pathways in the mitochondrial cell death in response to ionizing radiation in human cervical cancer cells. Ionizing radiation triggered Bax and Bak activation, Bcl-2 down-regulation, and subsequent mitochondrial cell death. Inhibition of JNK completely suppressed radiation-induced Bax and Bak activation and Bcl-2 down-regulation. Dominant-negative forms of stress-activated protein kinase/extracellular signal-regulated kinase kinase 1 (SEK-1)/mitogen-activated protein kinase kinase-4 (MKK-4) inhibited JNK activation. Radiation also induced phosphoinositide 3-kinase (PI3K) activation. Interestingly, inhibition of PI3K effectively attenuated radiation-induced mitochondrial cell death and increased clonogenic survival. Inhibition of PI3K also suppressed SEK-1/MKK-4 and JNK activation, Bax and Bak activation, and Bcl-2 down-regulation. In contrast, inhibition of p38 MAPK led to enhanced Bax and Bak activation and mitochondrial cell death. RacN17, a dominant-negative form of Rac1, inhibited p38 MAPK activation and increased Bax and Bak activation. Exposure of cells to radiation also induced selective activation of c-Src among Src family kinases. Inhibition of c-Src by pretreatment with Src family kinase inhibitor PP2 or small interfering RNA targeting of c-Src attenuated radiation-induced p38 MAPK and Rac1 activation and enhanced Bax and Bak activation and cell death. Our results support the notion that the PI3K-SEK-1/MKK-4-JNK pathway is required for the mitochondrial cell death in response to radiation, whereas the c-Src-Rac1-p38 MAPK pathway plays a cytoprotective role against mitochondrial cell death. (Mol Cancer Res 2008;6(11):1718–31)

Abundance of ADAM-8, -9, -10, -12, -15 and -17 and ADAMTS-1 in mouse uterus during the oestrous cycle

The aim of the present study was to determine whether a disintegrin and metalloproteinase (ADAM)-8, -9, -10, -12, -15 and -17 and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-1 are involved in the remodelling process of the mouse uterus during the oestrous cycle. The mRNA expression of ADAM was observed in all uterine tissues throughout the entire cycle. The levels of ADAM-8 mRNA were maximal at pro-oestrus, whereas the expression of ADAM-9 and ADAMTS-1 mRNA was maximal at oestrus. The minimum mRNA level of all ADAM genes always occurred at dioestrus. The mRNA levels of ADAM-10, -12, -15 and -17 did not vary significantly, regardless of the stage of the oestrous cycle. Immunoblot analyses demonstrated the presence of all ADAM proteins throughout the cycle. In terms of protein intensities, ADAM-8, -12 and -17 were maximal at pro-oestrus, whereas ADAM-10 and ADAMTS-1 were maximal at metoestrus and ADAM-9 was maximal at oestrus. Regardless of the ADAM species, minimal protein expression always occurred at dioestrus. Immunohistochemical studies showed ADAM protein expression in luminal and glandular epithelial layers, but not in the stromal layer. Moreover, ADAM proteins were found to be heterogeneously localised and their individual localisations depended on the stage of the oestrous cycle. From these observations, we suggest that the ADAM genes play an important role in mouse uterine tissue remodelling during the oestrous cycle.

Transplantation of insulin-secreting cells differentiated from human adipose tissue-derived stem cells into type 2 diabetes mice.

Currently, there are limited ways to preserve or recover insulin secretory capacity in human pancreas. We evaluated the efficacy of cell therapy using insulin-secreting cells differentiated from human eyelid adipose tissue-derived stem cells (hEAs) into type 2 diabetes mice. After differentiating hEAs into insulin-secreting cells (hEA-ISCs) in vitro, cells were transplanted into a type 2 diabetes mouse model. Serum levels of glucose, insulin and c-peptide were measured, and changes of metabolism and inflammation were assessed in mice that received undifferentiated hEAs (UDC group), differentiated hEA-ISCs (DC group), or sham operation (sham group). Human gene expression and immunohistochemical analysis were done. DC group mice showed improved glucose level, and survival up to 60 days compared to those of UDC and sham group. Significantly increased levels of human insulin and c-peptide were detected in sera of DC mice. RT-PCR and immunohistochemical analysis showed human gene expression and the presence of human cells in kidneys of DC mice. When compared to sham mice, DC mice exhibited lower levels of IL-6, triglyceride and free fatty acids as the control mice. Transplantation of hEA-ISCs lowered blood glucose level in type 2 diabetes mice by increasing circulating insulin level, and ameliorating metabolic parameters including IL-6.

Insulin-like growth factor 2 enhances insulinogenic differentiation of human eyelid adipose stem cells via the insulin receptor.

Objectives:u2002 Previously, we have isolated stem cells (HEAC) from human eyelid adipose tissue and functionally differentiated them into insulin‐secreting cells. In the present study, we examined whether insulin family members might influence insulinogenic differentiation of HEAC.

Bovine Follicular Fluid and Serum Share a Unique Isoform of Matrix Metalloproteinase-2 That Is Degraded by the Oviductal Fluid

Abstract Whereas the mammalian fertilization environment consists of possible products of the mutual interaction between oviductal and follicular fluids in addition to both fluid components, little is known regarding the interaction. In the present study, we have demonstrated that a mutual interaction occurs, resulting in the biochemical changes of follicular fluid components. Gelatin zymographic analyses of bovine follicular fluid (bFF) showed consistently a distinct, gelatinolytic activity having a molecular weight of 110 kDa (GA110) in addition to other gelatinases, whereas bovine oviductal fluid (bOF) showed a lack of GA110. Surprisingly, when bFF was mixed with bOF before zymography, the GA110 of bFF mostly disappeared at a 1:1 (v/v) mixture, completely disappeared at a 1:10 mixture, as fast as within 30 min after mixing. Other bFF gelatinase activities were not affected by bOF at 1:1 or 10:1 mixtures. Addition of EDTA or phenanthroline, but not of phenylmethylsulfonyl fluoride or trypsin inhibitor, to the mixture greatly increased the gelatinolytic activity of bFF GA110. The increased activity of bFF GA110 by EDTA was again abolished by subsequent bOF treatment. Addition of aminophenylmercuric acetate to the EDTA-treated bFF also abolished GA110; however, this was accompanied by the disappearance of other gelatinases, except the 62-kDa gelatinase, the activity of which increased as the treatment continued up to 24 h. Addition of EDTA or phenanthroline to the gelatin gel incubation buffer after electrophoresis abolished almost all gelatinases of bFF, except those of 88–84 kDa, demonstrating that they were indeed gelatinases or isoforms. Bovine serum and fetal bovine serum also showed the presence of GA110, the activity of which was increased by EDTA. However, ovarian granulosa cell homogenate did not exhibit GA110. Immunoblot experiments using antibodies against matrix metalloproteinase (MMP)-2 and MMP-9 demonstrated that bFF GA110 was an isoform of MMP-2, and that the 62-kDa form was an active form of MMP-2. Disappearance of immunoreactive GA110 of bFF and serum by bOF was also observed. Based on these observations, we conclude that bFF and bovine serum share a unique isoform of MMP-2, and that bOF can specifically degrade the isoform, suggesting that a mutual interaction between bFF and bOF could occur at the time of ovulation.

Character comparison of abdomen‐derived and eyelid‐derived mesenchymal stem cells

While most human adipose tissues, such as those located in the abdomen, hip and thigh, are of mesodermal origin, adipose tissues located in the face are of ectodermal origin. The present study has compared stem cell‐related features of abdomen‐derived adult stem cells (A‐ASCs) with those of eyelid‐derived adult stem cells (E‐ASCs).

Cells with dysfunctional telomeres are susceptible to reactive oxygen species hydrogen peroxide via generation of multichromosomal fusions and chromosomal fragments bearing telomeres

During genotoxic stress, reactive oxygen species hydrogen peroxide (H(2)O(2)) is a prime mediator of the DNA damage response. Telomeres function both to assist in DNA damage repair and to inhibit chromosomal end-to-end fusion. Here, we show that telomere dysfunction renders cells susceptible to H(2)O(2), via generation of multichromosomal fusion and chromosomal fragments. H(2)O(2) caused formation of multichromosomal end-to-end fusions involving more than three chromosomes, preferentially when telomeres were erosive. Interestingly, extensive chromosomal fragmentation (yielding small-sized fragments) occurred only in cells exhibiting such multichromosomal fusions. Telomeres were absent from fusion points, being rather present in the small fragments, indicating that H(2)O(2) cleaves chromosomal regions adjacent to telomeres. Restoration of telomere function or addition of the antioxidant N-acetylcysteine prevented development of chromosomal aberrations and rescued the observed hypersensitivity to H(2)O(2). Thus, chromosomal regions adjacent to telomeres become sensitive to reactive oxygen species hydrogen peroxide when telomeres are dysfunctional, and are cleaved to produce multichromosomal fusions and small chromosomal fragments bearing the telomeres.