Morphologic evidence of interactions between adult ductal epithelium of pancreas and fetal foregut mesenchyme

First, tissue recombinants were used to determine the residual capability of adult endodermal pancreatic ductal epithelium to form islets. Second, adult epithelium was challenged by placing it between adult stroma and fetal mesenchyme to investigate the epithelial response (subsequently called challenge grafts). Trypsin was used to isolate adult mouse (heteroplastic) or rat (homoplastic) pancreatic ductal epithelium and fetal rat mesenchyme. All the adult epithelium was maximally stressed with alloxan to control for beta-cell contamination. Fetal mesenchyme was layered with epithelium in vitro for at least 48 h. Subsequently, the recombinants were grafted into nude mice and allowed to develop for 4-6 wk. To assess development, grafts were removed and fixed for both light and electron microscopy. In general, the grafts became organized into vesicles, tubules, and buds, many of which were in the form of vascularized isletlike structures. Epithelium responded with mitosis and intercellular adhesion. the range of cytodifferentiation varied. Culture time, age, and adhesiveness of the fetal mesenchyme and the amount of adult epithelium used in making the recombinants influenced the degree of differentiation. Analysis of fine structure of isletlike structures revealed cells with small electron-dense granules. These observations provide evidence for activation of endodermal epithelium taken from an adult mammal.     

Expression and role of laminin-1 in mouse pancreatic organogenesis

Previous studies have suggested that basement membrane alone may induce ductal differentiation and morphogenesis in the undifferentiated embryonic pancreas. The mechanism by which this induction occurs has not been investigated. Studies of other organ systems such as the lungs and mammary glands, where differentiation has been shown to be induced by basement membrane, have suggested a major role for laminin as a mediator of ductal or tubular morphogenesis and differentiation. We first defined the ontogeny of laminin-1 in the developing mouse pancreas. To determine the specific role of basement membrane laminin in pancreatic ductal morphogenesis and differentiation, we microdissected 11-day mouse embryonic pancreatic epithelium free from its surrounding mesenchyme and then suspended the explants in a 3-dimensional organ culture to allow us to assay cell differentiation and morphogenesis. When the pancreatic epithelium buds off the foregut endoderm, the pancreatic mesenchyme diffusely expresses laminin-1. This laminin subsequently organizes to the interface between the epithelium and the mesenchyme by E12.5. As gestation progresses, epithelial cells in direct contact with laminin-1 seem to differentiate into ducts and acini, whereas those spared intimate contact with laminin-1 appeared to organize into islets. Although basement membrane gel could induce pancreatic ductal morphogenesis of embryonic pancreatic epithelium, this induction was blocked when we added neutralizing antibodies against any of the following: 1) laminin (specifically laminin-1), 2) the "cross-region" of laminin-1, and 3) the alpha6 moiety of the integrin receptor, which is known to bind laminins. Immunohistochemistry, however, showed that pancreatic duct cell-specific differentiation (carbonic anhydrase II) without ductal morphogenesis was still present, despite the blockage of duct morphogenesis by the anti-laminin-1 neutralizing antibodies. Interestingly, there appeared to be a decrease in carbonic anhydrase II expression over time when the epithelia were grown in a collagen gel, rather than in a basement membrane gel. The pattern of laminin-1 expression in the embryonic pancreas supports the conclusion that laminin-1 is important in the induction of exocrine (ducts and acini) differentiation in the pancreas. Furthermore, our data demonstrate that 1) pancreatic ductal morphogenesis appears to require basement membrane laminin-1 and an alpha6-containing integrin receptor; 2) the cross-region of basement membrane laminin is a biologically active locus of the laminin molecule necessary for pancreatic ductal morphogenesis; 3) duct-specific cytodifferentiation, in the form of carbonic anhydrase II expression, is not necessarily coupled to duct morphogenesis; and 4) the basement membrane gel may contain components (e.g., growth factors) other than laminin-1 that can sustain both carbonic anhydrase II expression and, possibly, the capacity to form ducts, despite the absence of duct structures.     

The induction of new ductal growth in adult prostatic epithelium in response to an embryonic prostatic inductor

Tissue recombinants were prepared with a single epithelial ductal tip from adult prostate and mesenchyme from either the embryonic urogenital sinus or adult urinary bladder. Recombinants were grown in vivo beneath the renal capsule of male hosts. After 4 weeks of in vivo growth, extensive growth of arborizing ducts was apparent in recombinants composed of urogenital sinus mesenchyme and a single adult prostatic ductal tip. One-dimensional polyacrylamide gel electrophoresis indicated that these recombinants contained many of the proteins of the mature prostate. Heterospecific recombinants (rat urogenital sinus mesenchyme and mouse prostatic epithelium) showed the ductal tissue to be derived solely from the prostatic epithelium. In recombinants of a prostatic ductal tip with mesenchyme from the urinary bladder, ductal growth was absent, the ductal tip was maintained as a single, discrete, epithelial structure, and the protein composition of these recombinants more closely resembled that of the bladder. The results demonstrate that the epithelial cells of the adult prostate can participate in new ductal growth in response to an embryonic prostatic inductor. These data provide experimental evidence to support the hypothesis that human benign prostatic hyperplasia may result from the anomalous reactivation of embryonic growth potential in the adult prostate.     

Stromal-epithelial interactions: II. Regulation of prostatic growth by embryonic urogenital sinus mesenchyme

Embryonic urogenital sinus mesenchyme (UGM) has been demonstrated previously to be a potent inductor of prostatic morphogenesis and functional differentiation when associated with either embryonic urogenital sinus epithelium (UGE) or urothelium derived from adult urinary bladder (ABLE) and grown in male hosts. To determine the role of mesenchyme in prostatic acinar growth, homotypic tissue recombinants of 16-day-old embryonic mouse urogenital sinus mesenchyme and epithelium (UGM_mouse + UGE_mouse) were prepared in which the relative amounts of UGM to UGE were varied from approximately 0.1:1 to 100: 1. Recombinants were grown for 1 month in intact male hosts after which the amount of acinar growth was assessed by histological analysis and by determination of wet weight and DNA content. The latter criteria are valid measures of acinar content of histologically normal prostatic tissue because the rodent prostate is composed of greater than 80% ductalacinar tissue. From this analysis the amount of acinar growth was found to be determined by the amount of mesenchymal tissue and was independent of the amount of epithelium utilized to prepare the tissue recombinants. Similarly, the magnitude of growth in heterospecific rat-mouse tissue recombinants prepared with urogenital sinus epithelium or adult bladder epithelium (ABLE) and urogenital sinus mesenchyme (UGM_mouse + UGE_rat, UGM_mouse + ABLE_rat UGM_rat + UGE_mouse, or UGM_rat + ABLE_mouse) is determined by the source of UGM. Although the initial DNA content per UGM is comparable between mouse and rat, the overall acinar growth induced by UGM_rat is about 10-fold greater than that induced by UGM_mouse when recombined reciprocally with UGE_mouse and UGE_rat, respectively. These results suggest that UGM is of fundamental importance as a regulator of prostatic epithelial growth. The relevance of this finding to the development of human benign prostatic hyperplasia is discussed.     

Pancreatic alpha-cell differentiation by mesenchyme-to-epithelial transition: implications for cell-based therapies in children

Purpose

Stem cell-derived tissue may provide a curative treatment for children with type 1 diabetes. Using an avian model, we have previously shown that foregut mesenchyme is able to differentiate into insulin-positive β-cell islets (B islets). Successful clinical islet transplantation, however, is reliant on graft tissue containing both insulin- and glucagon-secreting cells. Therefore, in this study, we assessed the ability of foregut mesenchyme to differentiate into glucagon-positive α-cell islets (A islets).

Methods

Chimeric recombinants (n = 14) were constructed using chick pancreatic epithelium combined with quail stomach mesenchyme from day 4 avian embryos and then cultured in 3 dimensions for 7 days. Cryosectioned recombinants were analyzed using immunocytochemistry against glucagon, insulin, and the quail-specific nucleolar antigen. The A islets and B islets were determined to be of solely epithelial, solely mesenchymal, or mixed origin according to the coexpression of the quail-specific nucleolar antigen.

Results

Forty-eight A islets and 34 B islets were analyzed. Eighty-five percent of the A islets were solely derived from the epithelium, but, notably, 5% were solely derived from the mesenchyme and 10% were of mixed origin. A-islet differentiation from foregut mesenchyme was reduced as compared with B islets (P = .03).

Conclusion

We demonstrate that foregut mesenchyme is able to differentiate into both α and β cells, albeit with quantitative differences. These findings may have important implications for the derivation of islet tissue from mesenchymal stem cells to cure juvenile-onset diabetes.     

Evidence of stem cells in the adult prostatic epithelium based upon responsiveness to mesenchymal inductors

Ductal tips approximately 300 μm in length from adult rat dorsal (DP), lateral type 1 (L1), and lateral type 2 (L2) prostates were combined with mesenchyme from the embryonic urogenital sinus (UGM), neonatal seminal vesicle (SVM), or neonatal bulbourethral gland (BUGM) and grafted underneath the renal capsule of syngeneic male hosts. Following 1 month of in vivo growth, all tissue recombinants formed large masses of prostatic ductal tissue, which represented massive growth of the original population of prostatic epithelial cells. Examination of secretory protein expression in these tissue recombinants indicated that each mesenchyme influenced secretory function in the adult prostatic epithelium in a characteristic way. SVM maintained expression of DP-1 and probasin in prostatic ducts of DP, L1, and L2, which normally express these proteins. BUGM induced expression of C3 in prostatic ducts of the DP, L1, and L2, which normally do not express C3. UGM induced the expression of DP-1, probasin, and C3 in prostatic ducts from all dorsal-lateral lobes. Mesenchymal induction of massive epithelial growth, new ductal branching morphogenesis, and change in prostatic lobe identity are indicative of the presence of stem cells in adult prostatic epithelium because high proliferative capacity, tissue regeneration, and pluripotency (change in functional differentiation) are hallmarks of stem cells. © 1996 Wiley-Liss, Inc.     

Androgenic induction of DNA synthesis in prostatic glands induced in the urothelium of testicular feminized (Tfm/Y) mice

It has been shown previously that wild-type urogenital sinus mesenchyme can induce the formation of prostate-like glandular structures in urinary bladder epithelium derived from adult Tfm (testicular feminization) mice. Total DNA synthesis within these tissue recombinants has been shown biochemically to be androgen sensitive. To determine which tissue (epithelium or stroma) accounts for this androgen-dependent DNA synthesis, an autoradiographic study was performed with tissue recombinants composed of rat wild-type urogenital sinus mesenchyme (UGM) associated with bladder epithelium from either wild-type (BLE) or Tfm mice (Tfm BLE). Both types of recombinants were grown under the kidney capsule of male athymic nude mice for 4 weeks. The hosts were then castrated, and 2 weeks later were treated with either testosterone propionate (TP), TP plus cyproterone acetate (CA), or oil vehicle for 3 days. DNA synthetic activity was measured through analysis of labelling index (LI) after incorporation of 3[H]-thymidine in vivo. For both UGM + BLE and UGM + Tfm BLE recombinants thymidine incorporation in epithelial cells greatly exceeded that of the stromal cells. TP stimulated epithelial LI to a similar degree (about 50- to 200-fold greater than controls) in both UGM + BLE and UGM + Tfm BLE recombinants; CA antagonized the effect of TP. Nuclear 3H-DHT binding was observed autoradiographically within the epithelial cells of the induced epithelium of UGM + wild-type BLE recombinants, but not within epithelium of UGM + Tfm BLE recombinants. Wild-type mesenchymal cells in both tissue recombinants showed specific nuclear 3H-DHT uptake. Thus, the proliferative effect of androgens upon prostatic epithelium is not a direct effect mediated by intra-epithelial androgen receptors, but rather it appears to be elicited indirectly via regulatory influences from androgen-receptor-positive stromal cells.     

The competency of foregut mesenchyme in islet mesenchyme-to-epithelial transition during embryonic development

Background/Purpose

Potential for curative stem-cell treatments of juvenile-onset diabetes has focussed research into pancreatic islet development. Islets were previously thought to originate solely from embryonic pancreatic epithelium, but we have demonstrated that islets can originate from mesenchyme, that is, islet mesenchyme-to-epithelial transition. The aim of this study was to establish the competence of foregut mesenchyme during mesenchymal islet development.

Methods

Embryonic chick pancreatic epithelium of gestational stage Hamburger-Hamilton (HH) 22 (J Morphol. 1951;88:49-92) was combined with quail stomach mesenchyme of increasing gestation (stage HH22 [n = 6], HH26 [n = 6], HH28 [n = 4], or HH31 [n = 6]). Recombinants were cultured and analysed by immunocytochemistry for coexpression of insulin and quail-specific antigen to determine the embryonic origin of islets.

Results

Recombinants constructed using stage HH22 mesenchyme yielded 34 islets, of which 35% were mesenchymal. However, when recombinants were constructed using stage HH26 mesenchyme, 24% of 25 islets were mesenchymal. When using mesenchyme, 13% of 15 islets were mesenchymal. All islets (n = 35) in recombinants constructed using stage HH31 mesenchyme were epithelial derived. Islet mesenchyme-to-epithelial transition diminished significantly with increasing mesenchymal gestational stage (P = .002).

Conclusions

These data show foregut mesenchyme is competent to form islets between stages HH22 and HH28. Developmental competence of foregut mesenchyme in islet mesenchyme-to-epithelial transition diminishes as gestation increases. This may have important implications for identifying stem cells to treat juvenile-onset diabetes.     

The importance of pancreatic embryonic epithelium for mesenchyme-to-epithelial transition during islet development

Stem or progenitor cells are a promising potential alternative source of pancreatic islets for transplantation in the treatment of juvenile-onset diabetes. However, to derive islets from such cells, it is important to elucidate the mechanisms of normal pancreatic development. Previous work in our laboratory has shown that, contrary to previous thinking, pancreatic mesenchyme when combined with pancreatic epithelium can contribute cells to islets. However, the signals and role of individual tissues involved in this mesenchyme-to-epithelial transition (MET) have yet to be elucidated. The aim of this study was to investigate whether MET can occur in the absence of pancreatic epithelium. Chick and quail eggs were incubated for 4 days and the dorsal pancreatic buds and stomach rudiments were microdissected. Mesenchyme and epithelium of the organ rudiments were separated after collagenase treatment. Separated pancreatic mesenchyme were cultured alone and in combination with stomach (nonpancreatic). After 7 days of culture, the specimens were analysed using immunohistochemistry for quail-specific nucleolar antigen (QCPN), insulin, and islet precursor cell marker (ISL-1). Pancreatic mesenchyme when cultured in the absence of epithelium did not differentiate into islets, but differentiated into fibroblast-like cells. When pancreatic mesenchyme were cultured in combination with stomach epithelium, there was no evidence of mesenchymally derived islets. We have demonstrated that pancreatic mesenchyme require pancreatic epithelium to differentiate into islet cells. These findings further increase our understanding of normal pancreatic islet development and may help to elucidate the molecular mechanisms of MET in islet development.     

Regional differences in the inductive activity of the mesenchyme of the embryonic mouse urogenital sinus

Embryonic urogenital sinuses (UGS) of 16-day-old mice were divided into two or three zones (cranial half and caudal half; or cranial third, intermediate, and caudal thirds). Following tryptic digestion, these zones were separated into their mesenchymal (UGM) and epithelial (UGE) components. The cranial and caudal zones of the UGM were recombined separately with the different zones of the UGE or with adult mouse urinary bladder epithelium (BLE). Following four weeks of growth in syngeneic male hosts, tissue recombinants of cranial UGM + caudal UGE contained numerous prostatic ducts and urethral glands. Conversely, recombinants composed of caudal UGM + cranial UGE developed into fibromuscular tissue covered with urethral epithelium and did not contain prostate, but occasionally contained urethral glands. Urethral glands were usually present in grafts of cranial UGM + cranial UGE or in grafts of the intact intermediate third of the UGS. In heterotypic recombinants of the UGM + adult BLE, prostatic glands were induced only when cranial UGM was utilized. Urethral glands were not observed in tissue recombinants prepared with adult BLE. These data suggest that regional differences in mesenchymal inductive ability and epithelial responsiveness play a role in the harmonious morphogenesis of the male lower genitourinary tract.     

Normal and abnormal development of the male urogenital tract. Role of androgens, mesenchymal-epithelial interactions, and growth factors.

Androgen-dependent male urogenital development occurs via mesenchymal-epithelial interactions in which mesenchyme induces epithelial morphogenesis, regulates epithelial proliferation, and evokes expression of tissue-specific secretory proteins. Mesenchymal-epithelial interactions continue to be important into adulthood. For example, mesenchyme of the urogenital sinus (UGM) and seminal vesicle (SVM) induce dramatic morphologic and functional changes in various adult epithelia. Since adult epithelial cells are unquestionably responsive to mesenchymes that can elicit expression of alternative morphologic and functional phenotypes, established carcinomas might also be influenced by their connective tissue environment. In this regard, Dunning prostatic tumor has been induced by UGM or SVM to differentiate into tall columnar secretory epithelial cells. This change in cytodifferentiation is associated with a reduction in growth rate and loss of tumorigenesis. The role of soluble growth factors in the mechanism of mesenchymal-epithelial interactions is discussed.     

Tissue Recombination Techniques for Mouse Embryonic Mammary Glands

This review gives detailed technical protocols for dissection of embryonic mammary rudiments and preparation of tissue recombinants composed of embryonic mouse mammary mesenchyme and epithelium. This experimental protocol was used in several seminal experiments that have greatly increased our understanding of embryonic mammary gland development, including the finding that mammary mesenchyme induces and specifies mammary epithelial identity. Analysis of mesenchymal-epithelial interactions has facilitated identification of molecular mediators of cell-cell interactions, in particular the tissue-specific roles of genes expressed in mesenchyme and epithelium during embryonic development.     

成人胰腺干细胞分离及转分化为胰岛的研究

目的 通过对成人胰腺干细胞分离和转分化为胰岛过程的研究以便更进一步了解及改进胰腺干细胞分离、培养、鉴定方法。方法 成人胰腺组织以胶原酶消化，密度梯度离心法获得纯化的胰腺外分泌细胞、导管上皮细胞和胰岛。导管上皮细胞在体外共培养27d，观察细胞形态学变化及干细胞特异性转录基因PDX—1，CK—19蛋白等的表达。结果 上述方法可获得大量胰腺导管上皮细胞。体外培养第1天即可见PDX—1，CK—19阳性细胞，胰腺导管上皮细胞迅速分裂增殖并转变为有分化能力的干细胞继而转分化为三维结构的胰岛细胞。培养27d后，平均每克胰腺组织可生成760个胰岛。结论 用改进的方法可获得大量成人胰腺导管上皮细胞，并可在体外转分化为大量具有内分泌功能的胰岛，可能为克服胰岛移植的供体短缺提供一条新途径。     

成人胰腺干细胞转分化为胰岛的研究

目的：通过对成人胰腺干细胞转分化为胰岛过程的研究以便更深入了解及改进胰腺干细胞分离、培养、鉴定方法。方法：成人胰腺组织经胶原酶消化后,用密度梯度离心法将胰腺外分泌细胞、导管上皮细胞和胰岛分离、纯化,导管上皮细胞即具有转分化潜能的干细胞,在体外先后以CMRL1066和无血清DMEM／F12培养液共培养27d,在培养的不同时间点取样本于光镜和电镜下观察细胞形态学变化及干细胞特异性转录基因PDX－1,CK－19蛋白等单抗的免疫组化染色,并测定培养液中的淀粉酶和胰岛素含量。结果：上述方法可获得大量以往在胰岛分离时丢弃的胰腺导管上皮细胞。经体外一定条件的培养后,第1天即可见PDX－1,CK－19阳性细胞,胰腺导管上皮细胞迅速分裂增殖并转变为有分化能力的干细胞继而转分化为三维结构的胰岛细胞。培养27d后,平均每克胰腺组织可生成760个胰岛。结论：成人胰腺的导管上皮具有干细胞潜能并可在体外转分化为大量具有内分泌功能的胰岛,用此方法获得大量的胰岛可能为克服胰岛移植的供体短缺提供一条新的途径。     

Developmental comparisons of murine secretory amelogenesis in vivo,as xenografts on the chick chorio-allantoic membrane,and in vitro

Summary The temporal, spatial, and cytological characteristics of secretory amelogenesis in developing mouse mandibular first molar tooth organs have been compared during in vivo odontogenesis (from the Cap Stage in Theiler stage 25 C57BL/6 embryos to 10-day-old postnatal mice), as xenografts on the chick chorioallantoic membrane (CAM) for periods up to 7 days, and as explants in chemically defined medium without serum or antibiotics for periods up to 21 days in vitro. Tooth morphogenesis and cytodifferentiation proceeded in each environmental condition in the same sequence albeit at different rates of development. In vivo and CAM xenografts were remarkably comparable in their respective expressions of dentinogenesis and amelogenesis, whereas those explants cultured in a chemically defined medium without serum or antibiotics developed at a much slower rate (e.g., 0.5 days in vivo is equivalent to 1 day in vitro). In each experimental group, secretory amelogenesis was typically first detected along the mesial-buccal cusp of the molar organ independent of which environment was evaluated. Tooth morphogenesis in vitro and as xenografts on the CAM was routinely smaller than in situ odontogenesis. In each environmental condition a “stippled” precursor ultrastructural form of enamel matrix preceded mineralization, except during in vitro cultures of tooth organs. In vitro secretory amelogenesis or dentinogenesis did not indicate morphological characteristics of mineralization; both dentine and enamel matrices did not mineralize under the permissive environmental conditions used in these experiments. Calcium hydroxyapatite crystal formations within dentine and along the dentinoenamel junction during initial enamel matrix formation were not observed during in vitro tooth organogenesis, even for periods up to 21 days in vitro. We conclude that cap stage mandibular first molar tooth organs (enamel organ epithelium and adjacent dental papilla mesenchyme) from Theiler stage 25 embryos contain all of the necessary developmental instructions to express morphogenesis and cytodifferentiation except cues for the serum-containing factors for mineralization.     

Elucidation of a Role for Stromal Steroid Hormone Receptors in Mammary Gland Growth and Development Using Tissue Recombinants

The use of tissue recombinants in conjunction with steroid receptor deficient mice is described as a tool to dissect the complex paracrine pathways of sex-hormone-regulated epithelial growth and ductal morphogenesis in the mammary gland and other hormone target organs. The basic methodology involves the construction of the four possible tissue recombinants composed of epithelium (E)⁶ and stroma (S) from wild-type (wt) and knock-out (KO) mice: wt-S + wt-S, wt-S + KO-E, KO-S + KO-E, and KO-S + wt-E. All tissue recombinants are grown as subrenal capsule grafts in nude mice. Following appropriate hormonal challenge epithelial growth can be studied in the four types of tissue recombinants. Such studies using estrogen receptor, androgen receptor and progesterone receptor knockout mice demonstrate that epithelial steroid receptors are neither necessary nor sufficient for hormonal regulation of epithelial proliferation. Instead, hormonal regulation of epithelial proliferation is a paracrine event mediated by hormone-receptor-positive stromal cells.     

ISL-1 is induced in stomach mesenchyme in the presence of pancreatic epithelia

Background/Purpose

β-Cell replacement offers a potential cure for type 1 diabetes mellitus in children. We have previously shown that stomach mesenchyme (SM) is competent to derive islet tissue by mesenchymal-to-epithelial transition (iMET). The aim of this study was to further characterize the developmental fate of this SM in the presence of pancreatic epithelia (PE) in SM/PE recombinants. The homeobox ISL-1 was examined in these recombinants because this gene is restricted to the dorsal pancreatic mesenchyme and endocrine cells in early pancreatic development.

Methods

Chick-quail recombinants of SM + PE (n = 15) and whole stomach controls (n = 8) were cultured for 7 days. In addition, organ blocks were examined after normal development at days 4 to 10 (n = 4 for each stage). Tissues were analyzed using immunochemistry against quail-specific antigen and ISL-1.

Results

Thirteen of 15 SM + PE recombinants expressed the ISL-1 protein in cells from SM origin. Nine of 15 of these recombinants showed iMET and coexpression of insulin, and ISL-1 was recorded.

Conclusions

Pancreatic epithelium is able to reprogram SM to a more caudal pancreatic fate when cocultured. Islet tissue by mesenchymal-to-epithelial transition observed in recombinants showed coexpression of insulin and ISL-1. These experiments are important to identify the molecular mechanisms behind iMET for potential therapeutic use for treating children with diabetes.     

Studies of the isolation and viability of human islets of Langerhans

Pancreas obtained from 34 adult human cadaver organ donors was divided into proximal and distal segments, and the duct to each segment was cannulated. Collagenase was injected into the proximal duct of 7 glands and into the distal duct of 7 others; the duct of the opposite segment was perfused with collagenase. The pancreas was then dispersed by teasing, trituration, and passage through filters. Perfused proximal and distal segments released 1461 +/- 287 and 2728 +/- 797 islets/g (+/- SEM) versus 710 +/- 149 (P less than 0.05) and 1950 +/- 636 after injection. Twenty other pancreases were perfused with collagenase warmed rapidly to 39 degrees C (n = 4) or warmed slowly to 37 degrees C (n = 6) or 39 degrees C (n = 10): the yield was 1625 +/- 632, 1320 +/- 116, and 2009 +/- 277 islets/g respectively. Total yields from the latter were 76 X 10(3) large (greater than 100 microns) and 85 X 10(3) small (less than 100 microns) islets with recoveries of 61% and 42%, respectively, after Ficoll density gradient purification. Histology showed highly purified islets. Perifusion with glucose elicited a biphasic release of insulin with the mean response (microU/islet/min) rising to a first peak of 0.5 and constant second phase secretion of 0.25, followed by a return to baseline. Reduced response was observed for islets from pancreas stored greater than 6 hr and tissue obtained from multiple centers. Less insulin was produced by freshly isolated islets, islets less than 100 microns, and after Ficoll separation. Secretion was similar for islets derived from proximal or distal segments. Perfusion of collagenase via the ducts of human pancreas improves islet isolation and Ficoll gradient separation yields highly purified islets. Important factors influencing insulin secretion are the source of donor tissue, cold storage of pancreas, Ficoll purification, islet size, and tissue culture.     

Effects of transforming growth factor beta-1 and all-trans-retinoic acid on androgen-induced development of neonatal mouse bulbourethral glands in vitro

Effects of transforming growth factor beta-1 (TGF-beta1) and all-trans-retinoic acid (All-trans-RA) on development of bulbourethral glands (BUGs) of neonatal mice were investigated in vitro. BUGs from 0-day-old male mice were cultured for 6 days in serum-free, chemically defined medium containing transferrin and bovine serum albumin, supplemented with 5alpha-dihydrotestosterone (DHT; 10-8 M) and insulin (10 microg/mL) alone or in combination. Prior to culture, BUGs from 0-day-old mice consisted of a simple epithelial rudiment encapsulated by mesenchyme. Epithelial growth and ductal branching occurred in BUGs cultured in medium containing DHT and insulin or DHT alone, but epithelial branching did not occur in BUGs cultured in the presence of insulin alone. Addition of TGF-beta1 at concentrations of > 5 ng/mL (0.2 x 10-9 M) to medium containing both insulin and DHT, inhibited the expected increase in overall size of BUGs, epithelial area and ductal branching in a dose-dependent manner. TGF-beta1 also decreased [3H]-thymidine labelling indices of both epithelium and mesenchyme. TGF-beta1 at 10 ng/mL elicited these inhibitory effects on BUGs cultured in medium containing DHT alone. Addition of All-trans-RA (10-8 to 10-6 M) to the medium containing DHT plus insulin, or DHT alone did not exert significant effects on either overall size of BUGs or epithelial growth and ductal branching. All-trans-RA at 10-6 M decreased the [3H]-thymidine labelling index of mesenchyme of BUGs cultured in medium with DHT plus insulin or DHT alone, but did not decrease the [3H]-thymidine labelling index of epithelium. The present results indicate that TGF-beta1 inhibits androgen-induced epithelial and mesenchymal growth as well as epithelial morphogenesis of BUGs from neonatal mice. Such an inhibitory effect of TGF-beta1 is not mimicked by All-trans-RA at physiological concentrations.