Differentiation of human multipotent dermal fibroblasts into islet-like cell clusters

Background  

We have previously obtained a clonal population of cells from human foreskin that is able to differentiate into mesodermal, ectodermal and endodermal progenies. It is of great interest to know whether these cells could be further differentiated into functional insulin-producing cells.     

Pancreatic islet-like clusters from periosteum-derived progenitor cells

Recent studies comparing the insulin-producing cell (IPC) differentiation capacity of mesenchymal stem cells (MSCs) derived from four different sources (bone marrow, Wharton’s jelly, adipose tissue, and the periosteum) demonstrated that IPC differentiation of periosteum-derived progenitor cells (PDPCs) progressed faster than any other MSCs within 7 days, indicating that PDPCsare most suited to IPC differentiation. Here, two different cell culture methods, adhesion and cluster culture, were assessed for their ability to support in vitro IPC differentiation. The induction of IPC differentiation was confirmed by RTqPCR analysis of insulin gene expression levels and immunofluorescence analysis of insulin protein. An enzyme-linked immunosorbent assay was used to quantify secreted insulin. PDPC-derived IPCs from cluster cultures demonstrated a significantly increased expression of insulin and an enhanced secretion of insulin of insulin protein in response to glucose compared to IPCs derived from adhesion cultures. Thus, pancreatic islet-like cluster cultures appear to provide the optimal conditions such as cluster culture for IPC differentiation of PDPCs.     

Generation of induced pluripotent stem cells from newborn marmoset skin fibroblasts

Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine. For the application of iPSCs to forms of autologous cell therapy, suitable animal models are required. Among species that could potentially be used for this purpose, nonhuman primates are particularly important, and among these the marmoset offers significant advantages. In order to demonstrate the feasibility of the application of iPSC technology to this species, here we derived lines of marmoset iPSCs. Using retroviral transduction with human Oct4, Sox2, Klf4 and c-Myc, we derived clones that fulfil critical criteria for successful reprogramming: they exhibit typical iPSC morphology; they are alkaline phosphatase positive; they express high levels of NANOG, OCT4 and SOX2 mRNAs, while the corresponding vector genes are silenced; they are immunoreactive for Oct4, TRA-1-81 and SSEA-4; and when implanted into immunodeficient mice they produce teratomas that have derivatives of all three germ layers (endoderm, α-fetoprotein; ectoderm, βIII-tubulin; mesoderm, smooth muscle actin). Starting with a population of 4 × 10⁵ newborn marmoset skin fibroblasts, we obtained ～ 100 colonies with iPSC-like morphology. Of these, 30 were expanded sufficiently to be cryopreserved, and, of those, 8 were characterized in more detail. These experiments provide proof of principle that iPSC technology can be adapted for use in the marmoset, as a future model of autologous cell therapy.     

Transplantation of islet-like cell clusters derived from human dental pulp stem cells restores normoglycemia in diabetic mice

Background aimsThe success of islet transplantation for diabetes depends on the availability of an adequate number of allogeneic or autologous islets. Postnatal stem cells are now considered for the generation of physiologically competent, insulin-producing cells. Our group showed earlier that it is possible to generate functional islets from human dental pulp stem cells by using a serum-free cocktail in a three-step protocol.MethodsWe compared the yield of generated islet-like cell clusters (ICCs) from stem cells from pulps of human exfoliated deciduous teeth (SHED) and dental pulp stem cells from permanent teeth (DPSCs). ICCs derived from SHED were packed in immuno-isolatory biocompatible macro-capsules and transplanted into streptozotocin (STZ)-induced diabetic mice. Non-diabetic and diabetic controls were transplanted with macro-capsules with or without islets.ResultsSHED were superior to DPSCs. STZ diabetic mice alone and mice transplanted with empty macro-capsules exhibited hyperglycemia throughout the experiment, whereas mice transplanted with macro-capsules containing ICCs were restored to normoglycemia within 3–4 weeks, which persisted for >60 days.ConclusionsOur results demonstrate for the first time that ICCs derived from SHED reverse STZ diabetes in mice without immunosuppression and offer an autologous and non-controversial source of human tissue that could be used for stem cell therapy in diabetes.     

Removal of glycosaminoglycans from cultures of human skin fibroblasts.

Early-passage human skin fibroblasts were grown as monolayers for 2-3 days in minimum essential medium containing [35S]sulphate, [3H]glucosamine, [3H]fucose, [3H]proline or [3H]leucine to label proteoglycans, glycoproteins or collagen and other proteins. A crude enzyme preparation obtained from a supernatant from sonicated freeze-dried Flavobacter heparinum was added to the cell monolayers. This treatment removed most of the 35S-labelled glycosaminoglycans, with no appreciable removal of the 3H-labelled proteins or 3H-labelled glycoproteins. The cells remained attached and viable as a monolayer. The formation of 35S-labelled glycosaminoglycans was examined after pretreating cultures with crude F. heparinum enzyme, followed by addition of fresh growth medium containing [35S]sulphate. The F. heparinum enzyme did not significantly alter the amount or type of 35S-labelled glycosaminoglycans produced. Thus F. heparinum enzyme can be used to provide cultured-cell monolayers depleted of surface glycosaminoglycans. These cells remain attached, viable and subsequently synthesize normal amounts and type of glycosaminoglycans.     

Origin of insulin secreted from islet-like cell clusters derived from murine embryonic stem cells

Islet-like cell clusters (ILCCs) were derived from murine embryonic stem cells using a slightly modified version of the protocol originally described by Lumelsky et al. in 2001. Analysis with enzyme-linked immunosorbent assays (ELISAs) that distinguish human from murine insulin demonstrated that insulin released from these ILCCs, upon initial in vitro glucose challenge, was of non-murine origin and in fact corresponded to the species of insulin, human or bovine, that had been added to the culture media used to derive ILCCs. This finding convincingly supports the hypothesis that ILCCs are not synthesizing insulin de novo, but rather simply regurgitating insulin taken up during tissue culture. In further experiments, ILCCs were derived in media in which insulin had been replaced by IGF-I with which it shares a common signaling pathway. These ILCCs failed to release any detectable insulin. In contrast, ILCCs produced by various protocols stained positive (dithizone and immunoselective antibodies) for intracellular insulin and, in some cases, C-peptide. Despite the presence of at least some level of de novo, synthesized insulin in ILCCs, the majority of insulin released by ILCCs was sequestered from the exogenous medium.     

Molecular organization of heparan sulphate from human skin fibroblasts.

The molecular structure of human skin fibroblast heparan sulphate was examined by specific chemical or enzymic depolymerization and high-resolution separation of the resulting oligosaccharides and disaccharides. Important features of the molecular organization, disaccharide composition and O-sulphate disposition of this heparan sulphate were identified. Analysis of the products of HNO2 hydrolysis revealed a polymer in which 53% of disaccharide units were N-acetylated and 47% N-sulphated, with an N-/O-sulphate ratio of 1.8:1. These two types of disaccharide unit were mainly located in separate domains. Heparitinase and heparinase scission indicated that the iduronate residues (37% of total hexuronate) were largely present in contiguous disaccharide sequences of variable size that also contained the majority of the N-sulphate groups. Most of the iduronate residues (approx. 70%) were non-sulphated. About 8-10% of disaccharide units were cleaved by heparinase, but only a minority of these originated from contiguous sequences in the intact polymer. Trisulphated disaccharide units [alpha-N-sulpho-6-sulphoglucosaminyl-(1----4)-iduronate 2-sulphate], which are the major structural units in heparin, made up only 3% of the disaccharide units in heparan sulphate. O-Sulphate groups (approx. 26 per 100 disaccharide units) were distributed almost evenly among C-6 of N-acetylglucosamine, C-2 of iduronate and C-6 of N-sulphated glucosamine residues. The results indicate that the sulphated regions of heparan sulphate have distinctive and potentially variable structural characteristics. The high content of non-sulphated iduronate in this heparan sulphate species suggests a conformational versatility that could have important implications for the biological properties of the polymer.     

Differentiation of mesenchymal stem cells derived from human bone marrow and subcutaneous adipose tissue into pancreatic islet-like clusters in vitro

Although stem cells are present in various adult tissues and body fluids, bone marrow has been the most popular source of stem cells for treatment of a wide range of diseases. Recent results for stem cells from adipose tissue have put it in a position to compete for being the leading therapeutic source. The major advantage of these stem cells over their counterparts is their amazing proliferative and differentiation potency. However, their pancreatic lineage transdifferentiation competence was not compared to that for bone marrow-derived stem cells. This study aims to identify an efficient source for transdifferentiation into pancreatic islet-like clusters, which would increase potential application in curative diabetic therapy. The results reveal that mesenchymal stem cells (MSC) derived from bone marrow and subcutaneous adipose tissue can differentiate into pancreatic islet-like clusters, as evidenced by their islet-like morphology, positive dithizone staining and expression of genes such as Nestin, PDX1, Isl 1, Ngn 3, Pax 4 and Insulin. The pancreatic lineage differentiation was further corroborated by positive results in the glucose challenge assay. However, the results indicate that bone marrow-derived MSCs are superior to those from subcutaneous adipose tissue in terms of differentiation into pancreatic islet-like clusters. In conclusion, bone marrow-derived MSC might serve as a better alternative in the treatment of diabetes mellitus than those from adipose tissue.     

Hyaluronidase expression in human skin fibroblasts

Hyaluronidase activity has been detected for the first time in normal human dermal fibroblasts (HS27), as well as in fetal fibroblasts (FF24) and fibrosarcoma cells (HT1080). Enzymatic activity was secreted predominantly into the culture media, with minor amounts of activity associated with the cell layer. In both classes of fibroblasts, hyaluronidase expression was confluence-dependent, with highest levels of activity occurring in quiescent, post-confluent cells. However, in the fibrosarcoma cell cultures, expression was independent of cell density. The enzyme had a pH optimum of 3.7 and on hyaluronan substrate gel zymography, activity occurred as a single band corresponding to an approximate molecular size of 57 kDa. The enzyme could be immunoprecipitated in its entirety using monoclonal antibodies raised against Hyal-1, human plasma hyaluronidase. PCR confirmed that fibroblast hyaluronidase was identical to Hyal-1. The conclusion by previous investigators using earlier technologies that fibroblasts do not contain hyaluronidase activity should be reevaluated.     

Stiffening of human skin fibroblasts with age

Changes in mechanical properties are an essential characteristic of the aging process of human skin. Previous studies attribute these changes predominantly to the altered collagen and elastin organization and density of the extracellular matrix. Here, we show that individual dermal fibroblasts also exhibit a significant increase in stiffness during aging in vivo. With the laser-based optical cell stretcher we examined the viscoelastic biomechanics of dermal fibroblasts isolated from 14 human donors aged 27 to 80. Increasing age was clearly accompanied by a stiffening of the investigated cells. We found that fibroblasts from old donors exhibited an increase in rigidity of ∼60% with respect to cells of the youngest donors. A FACS analysis of the content of the cytoskeletal polymers shows a shift from monomeric G-actin to polymerized, filamentous F-actin, but no significant changes in the vimentin and microtubule content. The rheological analysis of fibroblast-populated collagen gels demonstrates that cell stiffening directly results in altered viscoelastic properties of the collagen matrix. These results identify a new mechanism that may contribute to the age-related impairment of elastic properties in human skin. The altered mechanical behavior might influence cell functions involving the cytoskeleton, such as contractility, motility, and proliferation, which are essential for reorganization of the extracellular matrix.     

Cultured human skin fibroblasts absorb65Zn

The radioactive isotope⁶⁵Zn was used to study the incorporation of zinc by cultured human skin fibroblasts. The development of the method for studying cell uptake of⁶⁵Zn in a minimal synthetic medium is presented. Kinetics carried out on control cultures up to 240 min indicated that zinc uptake occurred in three phases, the first being the most rapid. Temperature and pH affect zinc uptake, in favor of an active transport process. In addition, the rate of incorporation is considerably decreased during the first phases after adding potassium cyanide, during the last phases after adding sodium iodoacetate, and during all the phases if dithioerythritol is used. A hypothesis is therefore proposed according to which several types of mechanisms would be involved in zinc uptake by fibroblasts. At least a part of these mechanisms is energy-dependent.     

Phosphorylation-activated chloride channels in human skin fibroblasts

A chloride-selective channel has been found using patch-clamp electrophysiology in human skin fibroblasts and it exhibits many of the biophysical properties of the Cl- channel found in airway epithelia. As in the case of epithelial Cl- channels, Cl- channels in fibroblasts are activated at depolarized membrane potentials in excised patches, rectifying in an outward direction with a unit conductance of 33 pS at 0 mV. Furthermore, the agonists forskolin and prostaglandin E2 evoke Cl- channel activity in cell-attached patches. The effect of these agonists can be mimicked by direct application of catalytic subunit of protein kinase A with ATP and Mg2+ to the internal membrane surface of excised, inside-out patches. The Cl- channel is also sensitive to inhibition by the stilbene derivative, DIDS. These results indicate that fibroblasts may provide a convenient and available model for the study of epithelial Cl- channel regulation and accelerate efforts to determine the regulatory defect expressed in cystic fibrosis.     

Collagenase production by human skin fibroblasts.

Normal human skin fibroblasts, when cultured in serum free medium, produce collagenase in an inactive form. The enzyme in the crude medium can be activated by a brief preincubation with trypsin or by autoactivation. Once activated, the fibroblast collagenase is identical in its mechanism of action to human skin collagenase obtained from organ cultures. In addition, an inhibitor of collagenase is also present in the medium of fibroblast cultures. The inhibitor appears to be produced by the cells and its molecular weight is slightly higher than that of the enzyme. The presence of this inhibitor may account for previous inability to detect collagenase in human skin fibroblast cultures. It is also possible that some of the inactive enzyme exists in the medium in the form of a proenzyme.