Promotion of hair follicle development and trichogenesis by Wnt-10b in cultured embryonic skin and in reconstituted skin.

We previously showed that Wnt-10b promoted the differentiation of primary skin epithelial cells (MPSEC) toward hair shaft and inner root sheath of the hair follicle (IRS) cells in vitro. In the present study, we found that Wnt-10b promotes the development of hair follicles using a culture of mouse embryonic skin tissue and trichogenesis using a reconstitution experiment with nude mice. Hair follicle development was observed in skin taken from mouse embryos on embryonic day 10.5 following a 2-day culture with recombinant Wnt-10b (rWnt-10b), however, not without rWnt-10b. Brown hair growth was observed at the site of reconstituted skin in Balb/c nude mice where dermal fibroblasts and keratinocytes, derived from C3H/HeN new born mice, were transplanted with Wnt-10b-producing COS cells (Wnt-COS). Without the co-transplantation of Wnt-COS, no hair growth was observed. Our results suggest an important role of Wnt-10b in the initiation of hair follicle development and following trichogenesis.

Wnt-10b secreted from lymphocytes promotes differentiation of skin epithelial cells.

Wnt-10b was originally isolated from lymphoid tissue and is known to be involved in a wide range of biological actions, while recently it was found to be expressed early in the development of hair follicles. However, few studies have been conducted concerning the role of Wnt-10b with the differentiation of skin epithelial cells. To evaluate its role in epithelial differentiation, we purified Wnt-10b from the supernatant of a concanavalin A-stimulated lymphocyte culture using an affinity column and investigated its effects on the differentiation of adult mouse-derived primary skin epithelial cells (MPSEC). MPSEC cultured with Wnt-10b showed morphological changes from cuboidal to spindle-shaped with inhibited proliferation, and also obtained characteristics of the hair shaft and inner root sheath of the hair follicle, represented by red-colored Ayoub Shklar staining, and reactions to AE-13 and AE-15 as seen with immunocytology. Further, RT-PCR analysis demonstrated the expression of mRNA for keratin 1, keratin 2, loricrin, mHa5, and mHb5, in association with a decreased expression of the basal cell marker keratin 5, in Wnt-10b-treated MPSEC. In addition, involvement of the canonical Wnt signal pathway was demonstrated by a TCF reporter (pTOPFLASH) assay. These results suggest that Wnt-10b promotes the differentiation of MPSEC and may play an important role in hair follicle development by promoting differentiation of epithelial cells.

Wnt-10b promotes differentiation of skin epithelial cells in vitro.

To evaluate the role of Wnt-10b in epithelial differentiation, we investigated the effects of Wnt-10b on adult mouse-derived primary skin epithelial cells (MPSEC). Recombinant Wnt-10b protein (rWnt-10b) was prepared using a gene engineering technique and MPSEC were cultured in its presence, which resulted in morphological changes from cuboidal to spindle-shaped and inhibited their proliferation. Further, involvement of the canonical Wnt signal pathway was also observed. MPSEC treated with rWnt-10b showed characteristics of the hair shaft and inner root sheath of the hair follicle, in results of Ayoub Shklar staining and immunocytochemistry. Further, the cells expressed mRNA for differentiated epithelial cells, including keratin 1, keratin 2, loricrin, mHa5, and mHb5, in association with a decreased expression of the basal cell marker keratin 5. These results suggest that Wnt-10b promotes the differentiation of MPSEC.

Differentiation of embryonic stem cells into insulin-producing cells promoted by Nkx2.2 gene transfer.

AIM: To investigate the ability of a genetically altered embryonic stem (ES) cell line to generate insulin-producing cells in vitro following transfer of the Nkx2.2 gene. METHODS: Hamster Nkx2.2 genes were transferred into mouse ES cells. Parental and Nkx2.2-transfected ES cells were initiated toward differentiation in embryoid body (EB) culture for 5 d and the resulting EBs were transferred to an attached culture system. Dithizone (DTZ), a zinc-chelating agent known to selectively stain pancreatic beta cells, was used to detect insulin-producing cells. The outgrowths were incubated in DTZ solution (final concentration, 100 microg/mL) for 15 min before being examined microscopically. Gene expression of the endocrine pancreatic markers was also analyzed by RT-PCR. In addition, insulin production was determined immunohistochemically and its secretion was examined using an ELISA. RESULTS: DTZ-stained cellular clusters appeared after approximately 14 d in the culture of Nkx2.2-transfected ES cells (Nkx-ES cells), which was as much as 2 wk earlier, than those in the culture of parental ES cells (wt-ES). The frequency of DTZ-positive cells among total cultured cells on day 28 accounted for approximately 1.0% and 0.1% of the Nkx-ES- and wt-ES-derived EB outgrowths, respectively. The DTZ-positive cellular clusters were found to be immunoreactive to insulin, while the gene expressions of pancreatic-duodenal homeobox 1 (PDX1), proinsulin 1 and proinsulin 2 were observed in the cultures that contained DTZ-positive cellular clusters. Insulin secretion was also confirmed by ELISA, whereas glucose-dependent secretion was not demonstrated. CONCLUSION: Nkx2.2-transfected ES cells showed an ability to differentiate into insulin-producing cells.

Cronkhite-Canada syndrome with colon cancer, portal thrombosis, high titer of antinuclear antibodies, and membranous glomerulonephritis.

A 64-year-old man, who came to us with diarrhea, presented with ectodermal changes such as hyperpigmentation, alopecia, and onychatrophy, and was affected by polyposis in the colorectum and stomach. The polyps were histologically consistent with those in Cronkhite-Canada syndrome (CCS). Interestingly, the patient also had colon cancer, as well as portal thrombosis and a high concentration of antinuclear antibody. Treatment with prednisolone ameliorated the symptoms and the gastrointestinal polyposis, while the cancer was successfully treated with a hemicolectomy. Six months after the surgery, the patient developed nephropathy, with nephrotic-range proteinuria, without recurrence of the cancer. The biopsied renal specimen showed membranous glomerulonephritis. This is a rare case of CCS associated with various complications such as colon cancer, portal vein thrombosis, a high titer of antinuclear antibodies, and membranous glomerulonephritis. Although the pathogenesis of CCS is essentially unknown, these complications might have been indicative of an underlying immunological abnormality.

Expressions of hepatobiliary organic anion transporters and bilirubin-conjugating enzyme in differentiating embryonic stem cells.

Mouse embryonic stem (ES) cells are clonal cell lines derived from the inner cell mass of developing blastocysts and have multi-lineage differentiation ability. We previously reported that ES cells can be made to differentiate into hepatocytes possessing high metabolic activities by transfection of hepatocyte nuclear factor-3beta (HNF-3beta). In the present study, we investigated the expression of hepatobiliary organic anion transporters and bilirubin uridine diphosphate glucuronosyltransferase (ugt1a1) in undifferentiated and differentiating HNF-3beta-transfected ES (HNF-3beta-ES) cells. The expression of organic anion transporting polypeptide 1 (oatp1), multidrug resistance-associated protein 1 (mrp1), mrp2, mrp3, and ugt1a1 was not seen in the undifferentiated HNF-3beta-ES cells by RT-PCR, whereas all were expressed in differentiating HNF-3beta-ES cells. Protein expression for oatp1, mrp1, mrp2, mrp3, and ugt1a1 was also observed in the differentiating HNF-3beta-ES cells by Western blotting. An immunofluorescence examination revealed that oatp1 was co-located with desmoplakin, a marker for the basolateral (sinusoidal) membrane, and mrp2 was co-localized with CD26, a marker for the apical (canalicular) membrane, though they were both expressed throughout most of the cell membranes.

In vitro differentiation of hepatocyte-like cells from embryonic stem cells promoted by gene transfer of hepatocyte nuclear factor 3 beta.

We investigated the ability of a genetically altered embryonic stem (ES) cell line to promote endodermal differentiation toward hepatocytes in vitro by transfecting the hepatocyte nuclear factor-3beta (HNF-3beta) gene. Parental and HNF-3beta-transfected ES cells were initiated toward differentiation in embryoid bodies (EBs) for 5 days and the resulting EBs were transferred to an attached culture system. Albumin production was observed using an immuno-cytochemical method 7 days after induction of differentiation in almost all differentiating HNF-3beta-transfected ES cells, whereas scant immuno-reactivity against albumin was found on the same day in the cultures of differentiating parental ES cells. An analysis using a reverse transcriptase polymerase chain reaction revealed the HNF-4alpha expression in the HNF-3beta-transfected ES cells and also demonstrated that the expression of endodermal and hepatocyte-related markers, such as transthyretin, alpha-fetoprotein, albumin, alpha-1 antitrypsin, tryptophan-2,3-dioxygenase and phosphoenol-pyruvate carboxykinase, could be observed at an early stage in the outgrowths of HNF-3beta-transfected ES cells compared to the parental ES cells. These results suggest that HNF-3beta-transfected ES cells may be useful for the efficient induction of hepatocytes in vivo.

Potential use of embryonic stem cells for the treatment of mouse parkinsonian models: improved behavior by transplantation of in vitro differentiated dopaminergic neurons from embryonic stem cells.

BACKGROUND: and Aims. The purpose of the present study was to examine the efficacy of transplantation of mouse embryonic-stem-(ES)-cell-derived tyrosine hydroxylase-positive (TH(+)) cells into Parkinsonian mice using behavioral tests and immunohistochemical evaluation. METHODS: Undifferentiated ES cells carrying the enhanced green fluorescent protein (EGFP) gene were differentiated into a cell population containing TH(+) neurons using a five-step in vitro differentiation method. These ES-cell-derived cells were used as allografts in Parkinsonian mice, made by administering injections of 6-hydroxydopamine (6-OHDA). Fifteen hemiparkinsonian mice were divided into three groups. Four weeks after 6-OHDA injection, mice in groups 1, 2, and 3 received phosphate-buffered saline, 1 x 10(4) graft cells, and 1 x 10(5) graft cells, respectively, into their dopamine-denervated striata. RESULTS: Improved rotational behavior was observed in the graft-transplanted groups (groups 2 and 3) 2 weeks after transplantation. Mice in group 2 displayed a continuous maintenance of reduced rotational behavior, while those in group 3 showed ipsilateral rotation toward the lesioned side at 4, 6, and 8 weeks after transplantation. Tumor formation was observed in one mouse in group 3. TH(+) cells were found at the grafted sites 8 weeks after transplantation in mice in groups 2 and 3, some of which were immunopositive to GFP, demonstrating the presence of dopaminergic neurons derived from the ES cells. CONCLUSION: Transplantation of in vitro differentiated ES cells changed rotational behavior in Parkinsonian mice. Our results suggest the potential availability of ES cells for Parkinson's disease.

Development of hepatocytes from ES cells after transfection with the HNF-3beta gene.

We have attempted to generate embryonic stem (ES) cell-derived hepatocytes expressing liver-specific functional properties by use of ES cell technology. It was found that ES cells are allowed to differentiate into hepatocytes possessing high metabolic activities when hepatocyte nuclear factor (HNF)-3beta-transfected ES cells are cultured in alpha-MEM medium supplemented with 10% fetal bovine serum (FBS) and fibroblast growth factor (FGF)-2 in the three-dimensional cell culture system at 5% CO2. The differentiated cells induced albumin, triacylglycerol, urea, and glycogen synthesis as well as further expression of metabolic proteins and serum factors as markers of hepatocytic differentiation for at least 4 months. The cells differentiated from HNF-3beta-transfected ES cells also had hepatocyte-like ultrastructural characteristics, including several endoplasmic reticula, mitochondrion, and glycogen. Our findings indicate that generation of hepatocytes maintaining high metabolic functions developed from mouse ES cells will facilitate the study of the basic mechanism for hepatogenesis and will certainly provide new opportunities for tissue transplantation.

Identification of insulin-producing cells derived from embryonic stem cells by zinc-chelating dithizone.

BACKGROUND AND AIMS: Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages in vitro. We have recently identified the emergence of cellular clusters within differentiated ES cell cultures by staining with dithizone (DTZ). DTZ is a zinc-chelating agent known to selectively stain pancreatic beta cells because of their high zinc content. The aim of the present study was to investigate the characteristics of DTZ-stained cellular clusters originating from ES cells. METHODS: Embryoid bodies (EBs), formed by a 5-day hanging drop culture of ES cells, were allowed to form outgrowths in the culture. The outgrowths were incubated in DTZ solution (final concentration, 100 microg/ml ) for 15 minutes before being examined microscopically. The gene expression of endocrine pancreatic markers was also analyzed by reverse transcriptase-polymerase chain reaction. In addition, insulin production was examined immunohistochemically, and its secretion was examined using enzyme-linked immunosorbent assay. RESULTS: DTZ-stained cellular clusters appeared after approximately 16 days in the EB culture and became more apparent by day 23. They were found to be immunoreactive to insulin and expressed pancreatic-duodenal homeobox 1 (PDX1), proinsulin 1, proinsulin 2, glucagon, pancreatic polypeptide, glucose transporter-2 (GLUT2), and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) mRNA. They were also able to secrete detectable amounts of insulin. CONCLUSIONS: ES cell-derived DTZ-positive cellular clusters possess characteristics of the endocrine pancreas, including insulin secretion. Further, DTZ staining is a useful method for the identification of differentiated pancreatic islets developed from EBs in vitro.