Evaluation of a xeno-free protocol for long-term cryopreservation of cord blood cells.

Cord blood is regarded as a powerful source for adult stem cells. Cord blood transplants have been used successfully to treat children and adults in autologous and allogeneic settings. Nevertheless, in many cases, the clinically relevant cell number (CD34+ cells and total leukocytes) is a limiting factor. To enable standardized cell banking and future in vitro expansion of adult stem/progenitor cells, elimination of serum, which inevitably differs from lot to lot and donor to donor, is highly desirable. Here, we demonstrate the feasibility of a xeno-free, chemically defined cryopreservation procedure for cord blood-derived cells over a period of 1 year. Cell recoveries with respect to retrieval of clinically relevant CD34+ cells, colony-forming units, and in vitro cultures of erythroid progenitor cells under standardized conditions were analyzed after 1 week or 1 year of cryopreservation and found to be very high and similar to the samples before freezing. The established xeno-free procedure is an important step toward using the full potential of adult stem cells from cord blood, enabling the elimination of serum-derived factors negatively influencing proliferation, differentiation, and survival of hematopoietic stem cells.

Oct-4 expression in human endometrium.

The transcription factor Oct-4 is crucial for the maintenance of cell pluripotency and is known to be expressed in embryonic stem cells, germ cells and whole embryos at various stages of development. Oct-4 regulates cell fate in a dose-dependent manner and plays a key role in germ-cell tumours. In the past, several stem-cell markers have been detected, and their role in the pathogenesis of diseases has been discussed frequently. Thus, we investigated the expression of Oct-4 comparing its occurrence in endometrium of healthy and diseased women using immunohistochemistry (IHC) and RT-PCR. IHC demonstrated Oct-4 expression in 25 of 60 sections (42%), respectively in 11 out of 25 patients (44%). Oct-4 mRNA was detected by RT-PCR in all tested samples (9 of 9) of endometrium, although the levels of expression varied. To our knowledge, this is the first study demonstrating Oct-4 expression in human endometrium.

Erythroid progenitor renewal versus differentiation: genetic evidence for cell autonomous, essential functions of EpoR, Stat5 and the GR.

The balance between hematopoietic progenitor commitment and self-renewal versus differentiation is controlled by various transcriptional regulators cooperating with cytokine receptors. Disruption of this balance is increasingly recognized as important in the development of leukemia, by causing enhanced renewal and differentiation arrest. We studied regulation of renewal versus differentiation in primary murine erythroid progenitors that require cooperation of erythropoietin receptor (EpoR), the receptor tyrosine kinase c-Kit and a transcriptional regulator (glucocorticoid receptor; GR) for sustained renewal. However, mice defective for GR- (GR(dim/dim)), EpoR- (EpoR(H)) or STAT5ab function (Stat5ab(-/-)) show no severe erythropoiesis defects in vivo. Using primary erythroblast cultures from these mutants, we present genetic evidence that functional GR, EpoR, and Stat5 are essential for erythroblast renewal in vitro. Cells from GR(dim/dim), EpoR(H), and Stat5ab(-/-) mice showed enhanced differentiation instead of renewal, causing accumulation of mature cells and gradual proliferation arrest. Stat5ab was additionally required for Epo-induced terminal differentiation: differentiating Stat5ab(-/-) erythroblasts underwent apoptosis instead of erythrocyte maturation, due to absent induction of the antiapoptotic protein Bcl-X(L). This defect could be fully rescued by exogenous Bcl-X(L). These data suggest that signaling molecules driving leukemic proliferation may also be essential for prolonged self-renewal of normal erythroid progenitors.

Cultured human epithelium: human umbilical cord blood stem cells differentiate into keratinocytes under in vitro conditions.

BACKGROUND: Stem cells have the capacity to renew or to give rise to a specialized cell types. Human umbilical cord blood (HUCB) has been explored as an alternative source of stem cells. However, its potential to differentiate into cells of other tissues is still under discussion. The aim of our study was to evaluate if HUCB stem cells could differentiate into epithelial cells under in vitro conditions. METHODS: Human keratinocytes derived from adult female skin donors, were isolated and cultured on fibrin glue/fibroblast gels-control group. In the umbilical cord blood cell group, male umbilical cord blood cells were added at a 1:10 ratio to keratinocytes and co-cultured on the fibrin glue/fibroblasts gel. After 15 days of culture, the sheets were analyzed by use of histochemistry and FISH. DNA was extracted and evaluated by use of polymerase chain reaction (PCR) for detection of Y-chromosome-specific sequences. RESULTS: In both groups a regular epithelial sheet consisting of three to four layers of cells was formed. Using PCR and FISH, in the umbilical cord blood cell group the presence of Y-chromosome-specific sequences in the cultured keratinocytes could be detected. In the control group, no Y-chromosome-specific sequences could be detected. CONCLUSION: Our findings indicate that umbilical cord blood stem cells differentiate into epithelial cells under in vitro conditions and thereby, might serve as a starting material for isolation and expansion of cells for transplantation in patients with large skin defects.

A predictive model for endometriosis.

BACKGROUND: Aromatase is the key enzyme in the process of estrogen biosynthesis from the precursor androgen. Recently, aromatase has been found to be aberrantly expressed in eutopic endometrium of patients suffering from endometriosis. This finding has prompted speculation about the contribution of this enzyme to the prediction of this disease. METHODS: We prospectively aimed to evaluate whether endometrial biopsy, prior to laparoscopy in symptomatic women to screen for the presence of aromatase by real-time RT-PCR and immunohistochemistry, combined with select patients' characteristics, is of value to predict endometriosis. RESULTS: Of 48 consecutive symptomatic and eligible patients, 25 (52.1%) exhibited endometriosis and 23 (47.9%) were disease-free. A multiple logistic regression model revealed that 95.5% of patients whose eutopic endometrium was found to be positive for aromatase mRNA as well as immunohistochemically detected protein and who were additionally suffering from moderate to severe dysmenorrhoea (visual analogue scale score >4/10) exhibited endometriosis at laparoscopy. CONCLUSIONS: These findings provide direct evidence that screening for eutopic endometrial aromatase in combination with clinical data could be of discriminative value in the prediction of disease.

Mammary gland development and lactation are controlled by different glucocorticoid receptor activities.

OBJECTIVE: Regulation of physiological processes by glucocorticoids is achieved by binding to the glucocorticoid receptor (GR) and subsequent modulation of gene expression, either by DNA binding-dependent mechanisms or via protein-protein interaction with other transcription factors. The purpose of this study was to define the molecular mechanism of GR underlying the control of mammary gland development and lactation. DESIGN: To dissect the mechanism of GR action in the mammary gland, we used genetically modified mice carrying a DNA binding-defective GR. These mice retain the ability to regulate transcription by protein-protein interaction but fail to control gene expression by DNA binding-dependent mechanisms. Thus, they allow the study of the mode of GR action in vivo. METHODS: The development of the mammary gland and milk protein synthesis during lactation were studied using histological and biochemical methods. RESULTS: Our findings demonstrated that the lack of the DNA binding function of GR impairs the ductal development of the mammary gland in virgin females and that this can presumably be accounted for by reduced proliferation of epithelial cells. In contrast, lactating females have normally differentiated mammary glands and are fully capable of milk protein production. This is in good agreement with the demonstration that the DNA binding-defective GR is still able to interact with phosphorylated Stat5 proteins, suggesting that transcriptional regulation by protein-protein interaction forms the basis of glucocorticoid action in this process. CONCLUSIONS: The present study has demonstrated that GR plays an important role in the mammary gland and that it uses different molecular modes of action to control development and milk protein synthesis.

Organotypic cocultures with genetically modified mouse fibroblasts as a tool to dissect molecular mechanisms regulating keratinocyte growth and differentiation.

Organotypic cocultures of keratinocytes and fibroblasts generate a normal epidermis irrespective of the species and tissue origin of fibroblasts. The use of mouse fibroblasts and human keratinocytes facilitates the identification of the origin of compounds involved in epidermal tissue reconstitution and growth regulation. Moreover, the functional significance for the keratinocyte phenotype of genetically modified fibroblasts from transgenic or knockout mice, even those exhibiting an embryonic lethal phenotype, can be studied in such heterologous in vitro tissue equivalents. Here we communicate results of such studies revealing the antagonistic function of mouse fibroblasts defective in the AP-1 constituents c-Jun and JunB, respectively, on human keratinocyte growth and differentiation. Furthermore, the hematopoietic growth factor granulocyte macrophage-colony stimulating factor has been identified as a novel regulator of keratinocyte growth and differentiation. As will be reported in detail elsewhere both granulocyte macrophage-colony stimulating factor and keratinocyte growth factor have been identified as major mediators of fibroblast-keratinocyte interactions and their expression is induced via AP-1 by interleukin-1 released by the epithelial cells. Thus, these heterologous cocultures provide a novel promising tool for elucidating molecular mechanisms of epithelial-mesenchymal interactions and their consequences on epithelial cell proliferation and differentiation.

c-Jun and JunB antagonistically control cytokine-regulated mesenchymal-epidermal interaction in skin.

Interactions between mesenchymal and epithelial cells are responsible for organogenesis and tissue homeostasis. This mutual cross-talk involves cell surface proteins and soluble factors, which are mostly the result of regulated transcription. To elucidate dimer-specific functions of the AP-1 family of transcription factors, we reconstituted skin by combining primary human keratinocytes and mouse wild-type, c-jun(-/-), and junB(-/-) fibroblasts. We have discovered an antagonistic function of these AP-1 subunits in the fibroblast-mediated paracrine control of keratinocyte proliferation and differentiation, and traced this effect to the IL-1-dependent regulation of KGF and GM-CSF. These data suggest that the relative activation state of these AP-1 subunits in a non-cell-autonomous, transregulatory fashion directs regeneration of the epidermis and maintenance of tissue homeostasis in skin.

c-Jun-dependent CD95-L expression is a rate-limiting step in the induction of apoptosis by alkylating agents.

Mouse 3T3 fibroblasts derived from fetuses lacking c-Jun were used to define an essential role of c-Jun, a main component of the transcription factor AP-1, in the cellular response to the alkylating agent methyl methanesulfonate (MMS). MMS represents the most potent and selective activator of the stress-induced kinases JNK/SAPK and p38, resulting in very efficient induction of c-Jun hyperphosphorylation and c-jun transcription. This agent induced apoptosis with high efficiency in wild-type cells but not in c-jun(-/-) cells. Resistance to apoptosis was accompanied by impaired expression of CD95 ligand (CD95-L), a well-known inducer of apoptosis. The addition of recombinant CD95-L restored apoptosis sensitivity in c-jun(-/-) fibroblasts. MMS-induced apoptosis in wild-type fibroblasts or human lymphocytes was strongly reduced by neutralizing CD95-L antibodies or transdominant negative FADD, confirming the importance of CD95 signalling in MMS-induced apoptosis. The loss-of-function approach in fibroblasts allowed the identification and dissection of c-Jun-dependent and -independent processes upstream or downstream of CD95 activation. We have found that c-Jun can act as a proapoptotic regulator in cells exposed to DNA damage via induction of CD95-L. Once activated, CD95-induced death signalling is not affected by the loss of c-Jun, demonstrating that only the initiation and not the execution of stress-induced apoptosis depends on c-Jun.

Control of cell cycle progression by c-Jun is p53 dependent.

The c-jun proto-oncogene encodes a component of the mitogen-inducible immediate-early transcription factor AP-1 and has been implicated as a positive regulator of cell proliferation and G1-to-S-phase progression. Here we report that fibroblasts derived from c-jun-/- mouse fetuses exhibit a severe proliferation defect and undergo a prolonged crisis before spontaneous immortalization. The cyclin D1- and cyclin E-dependent kinases (CDKs) and transcription factor E2F are poorly activated, resulting in inefficient G1-to-S-phase progression. Furthermore, the absence of c-Jun results in elevated expression of the tumor suppressor gene p53 and its target gene, the CDK inhibitor p21, whereas overexpression of c-Jun represses p53 and p21 expression and accelerates cell proliferation. Surprisingly, protein stabilization, the common mechanism of p53 regulation, is not involved in up-regulation of p53 in c-jun-/- fibroblasts. Rather, c-Jun regulates transcription of p53 negatively by direct binding to a variant AP-1 site in the p53 promoter. Importantly, deletion of p53 abrogates all defects of cells lacking c-Jun in cell cycle progression, proliferation, immortalization, and activation of G1 CDKs and E2F. These results demonstrate that an essential, rate-limiting function of c-Jun in fibroblast proliferation is negative regulation of p53 expression, and establish a mechanistic link between c-Jun-dependent mitogenic signaling and cell-cycle regulation.