4117268
Expansion and conversion of human pancreatic ductal cells into insulin-secreting endocrine cells
Pancreatic islet β-cell insufficiency underlies pathogenesis of diabetes mellitus; thus, functional β-cell replacement from renewable sources is the focus of intensive worldwide effort. However, in vitro production of progeny that secrete insulin in response to physiological cues from primary human cells has proven elusive. Here we describe fractionation, expansion and conversion of primary adult human pancreatic ductal cells into progeny resembling native β-cells. FACS-sorted adult human ductal cells clonally expanded as spheres in culture, while retaining ductal characteristics. Expression of the cardinal islet developmental regulators Neurog3, MafA, Pdx1 and Pax6 converted exocrine duct cells into endocrine progeny with hallmark β-cell properties, including the ability to synthesize, process and store insulin, and secrete it in response to glucose or other depolarizing stimuli. These studies provide evidence that genetic reprogramming of expandable human pancreatic cells with defined factors may serve as a general strategy for islet replacement in diabetes. DOI: http://dx.doi.org/10.7554/eLife.00940.001
2718647
Bi-compartmental communication contributes to the opposite proliferative behavior of Notch1-deficient hair follicle and epidermal keratinocytes
Notch1-deficient epidermal keratinocytes become progressively hyperplastic and eventually produce tumors. By contrast, Notch1-deficient hair matrix keratinocytes have lower mitotic rates, resulting in smaller follicles with fewer cells. In addition, the ratio of melanocytes to keratinocytes is greatly reduced in hair follicles. Investigation into the underlying mechanism for these phenotypes revealed significant changes in the Kit, Tgfβ and insulin-like growth factor (IGF) signaling pathways, which have not been previously shown to be downstream of Notch signaling. The level of Kitl (Scf) mRNA produced by Notch1-deficient follicular keratinocytes was reduced when compared with wild type, resulting in a decline in melanocyte population. Tgfβ ligands were elevated in Notch1-deficient keratinocytes, which correlated with elevated expression of several targets, including the diffusible IGF antagonist Igfbp3 in the dermal papilla. Diffusible stromal targets remained elevated in the absence of epithelial Tgfβ receptors, consistent with paracrine Tgfβ signaling. Overexpression of Igf1 in the keratinocyte reversed the phenotype, as expected if Notch1 loss altered the IGF/insulin-like growth factor binding protein (IGFBP) balance. Conversely, epidermal keratinocytes contained less stromal Igfbp4 and might thus be primed to experience an increase in IGF signaling as animals age. These results suggest that Notch1 participates in a bi-compartmental signaling network that controls homeostasis, follicular proliferation rates and melanocyte population within the skin.
20694251
Murine vibrissae cultured in serum-free medium reinitiate anagen.
Aberrant regulation of the hair cycle has been implicated in human boldness (Reviewed in (Nakamura et al, 2001; Paus and Cotsarelis, 1999)). Although several modulators of mammalian hair follicle cycle have been recently described, discovery of effective treatment for boldness has suffered from the absence of a reliable in vitro culture systems in which the hair cycle can be assessed easily and inexpensively (Stenn and Paus, 2001). Several laboratories have established serum-free culture system where vibrissa can grow and differentiate in vitro (Supplemental Table 1, (Jindo et al, 1993; Robinson et al, 1997; Yano et al, 2001). Although rat vibrissae cultured for 23 days were reported to share histological similarities with catagen or pro-anagen (telogen) stage follicles (Philpott and Kealey, 2000), these follicles did not show any progress beyond pro-anagen phase nor did they produce a new hair shaft (Philpott and Kealey, 2000). In this report, we demonstrate that a simple modification permits murine vibrissae in the current in vitro culture system to reinitiate anagen. This will accelerate the development of screening systems aimed at modifying the hair cycle. 
 
To establish a modified in vitro vibrissa culture system, anagen-stage vibrissae were carefully isolated from 14-day-old mice and the tip of the vibrissa shafts were anchored in a stripe of sterilized silicone grease placed on the culture dish through a 3 ml syringe. The plate was then filled with serum-free medium (Figure 1a, Supplemental Material and Method). Out of 86 cultured vibrissae collected from three pups, 81 vibrissae (94%) showed measurable shaft growth (Table 1, Figure 1b). Two of 81 vibrissae were lost, and 17 developed abnormal (kinked) fiber and were omitted from growth measurement. Of the remaining 62 vibrissae, straight shafts were produced by all at a rate of 0.3–0.5mm/day for the first three days. While some follicles maintained this growth rate through the fifth day of culture (Figure 1b and 1c and data not shown), others began a gradual decline in growth rate (Figure 1b and 1c, ​,2a2a and data not shown). As previously reported (Jindo et al, 1993; Robinson et al, 1997), growth rates of all follicles slowed down considerably after 5 days in culture, indicating independence from culture conditions (Supplemental Table 1). 
 
 
 
Figure 1 
 
Vibrissae produce hair shafts and reinitiate anagen in serum-free culture medium 
 
 
 
 
 
Figure 2 
 
Histological and immunohistochemical characteristics of new anagen follicles 
 
 
 
 
 
Table 1 
 
Summary of the in vitro vibrissa culture 
 
 
 
Hair shaft elongation rate decreases and eventually stops when follicles enter catagen/telogen phase in vivo (Alonso and Fuchs, 2006). Several days after cessation of hair shaft growth, cultured vibrissae follicles continuously changed their morphology, at times visible through their collagen capsules (Figure 1d). Strikingly, dissection and removal of collagen capsules from 21-day-cultured vibrissae revealed that all vibrissae produced a second shaft (Figures 1d and 2a–h, arrows; Figure S1), readily distinguishable from the original shaft whose proximal end formed club-end indicative of a completed catagen (Figures 1 and ​and22 closed arrowheads). Histological and immunohistochemical analyses using AE13, a marker for cortex/cuticle specific keratins, confirmed the formation of new hair shafts (Figures 2c–f and S1). The new shafts were produced in proximal bulbs that appeared smaller in size than the original ones before culture. Since the original hair growth stopped within the first 5 days of culture, and since a new shaft emerges from a secondary bulb, we concluded that vibrissa follicles from CD1 mice reinitiated anagen phase by regenerating hair bulbs (Figure 2). Staining for alkaline phosphatase activity identified an intact (but small) dermal papilla within the new bulb (Figure 1d, open arrowhead; Figure 2g, alkaline phosphatase), as seen in the early anagen phase of vibrissae in vivo (Oshima et al, 2001; Young and Oliver, 1976). Moreover, the hair matrix cells were positive for Ki-67, a proliferation marker, even after 13 days in culture (Figure 2h), indicating that the formation of new shaft was the result of active proliferation and differentiation of matrix cells in cultured vibrissae. Similar cycling patterns were also observed with vibrissae from 14-day-old C57BL/6 mice (Figure S2), indicating that hair cycle in vitro is independent of mouse strains. 
 
To better visualize the hair cycle process, we next performed vibrissae culture after careful removal of their collagen capsules (Figure 2i, Supplemental Material and Method). Within 2 days, the proximal end of the vibrissa keratinized, forming a club hair, at which time dermal papilla structures were no longer discernable (Figure 2i and data not shown). A day later, however, a bud-like structure emerged from the proximal part of the bulb in eight of ten cultured vibrissae. Of these, one vibrissa continued growing outwards, eventually forming a new bulb producing a vibrissa thinner than the club hair (Figure 2i). Strikingly, we observed that this vibrissa appeared to enter a third anagen, producing a new bud from the second bulb (Figure 2i, open arrow). Collectively, the data presented here indicate that in our in vitro culture system, vibrissa follicles enter a second anagen phase and it may be possible for a few to enter a third cycle in culture. 
 
We are not certain which modification in our culture system permits vibrissae to cycle. We do not think this is caused by differences in media composition, mouse strain, or frequency of media change. Our attempt to grow vibrissae at the air-fluid interface as in (Jindo et al, 1993) showed that one of five vibrissae formed a new hair shaft (Figure S3, Arrows), indicating that hair cycle is not strictly dependent on culture media or on the frequency of media change. However, we observed that on a solid substrate most vibrissae gradually lost their hair matrix possibly due to a pressure caused by friction generated while extending a hair shaft on a solid surface or in the absence of sebum (Sundberg et al, 2000) (Figure S3, Arrowheads). Although not proven, we speculate that the critical modification was our method of immobilizing the hair shaft in silicon grease that provided friction-free environment; anchoring the tip of shafts may have allowed gravity to facilitate the separation of club hairs from the hair matrix and that supports new hair shaft formation. In addition, the smaller size of mouse vibrissae, in comparison with human or rat vibrissae, could also be a contributing factor in the anagen reinitiation in vitro As this culture system is the first to support reinitiation of anagen in culture dish, we hope this improvement will expedite the testing and development of hair cycle modifying pharmaceuticals.
140203938
T2*-based fiber orientation mapping
Recent MRI studies at high field have observed that, in certain white matter fiber bundles, the signal in T(2)*-weighted MRI (i.e. MRI sensitized to apparent transverse relaxivity) is dependent on fiber orientation θ relative to B(0). In this study, the characteristics of this dependency are quantitatively investigated at 7 T using ex-vivo brain specimens, which allowed a large range of rotation angles to be measured. The data confirm the previously suggested variation of R(2)* (=1/T(2)*) with θ and also indicate that this dependency takes the shape of a combination of sin2θ and sin4θ functions, with modulation amplitudes (=ΔR(2)*) reaching 6.44±0.15 Hz (or ΔT(2)*=2.91±0.33 ms) in the major fiber bundles of the corpus callosum. This particular dependency can be explained by a model of local, sub-voxel scale magnetic field changes resulting from magnetic susceptibility sources that are anisotropic. As an illustration of a potential use of the orientation dependence of R(2)*, the feasibility of generating fiber orientation maps from R(2)* data is investigated.
207620666
Conditional deletion of the focal adhesion kinase FAK alters remodeling of the blood-brain barrier in glioma.
Gliomas generally infiltrate the surrounding normal brain parenchyma, a process associated with increased vascular permeability (VP) and dysregulation of the blood-brain barrier (BBB). However, the molecular mechanisms underlying glioma-induced VP in the brain remain poorly understood. Using a conditional, endothelium-specific deletion of the focal adhesion kinase (FAK) in the mouse (FAK CKO), we show that FAK is critical for destabilization of the tumor endothelium in tumor-bearing mice, with mutant mice exhibiting a relatively normalized vasculature compared with wild-type mice (FAK WT). Tumor vessels in the FAK CKO mice displayed reduced VP compared with FAK WT mice, resulting in reduced tumor growth. Additionally, FAK CKO mice displayed partial restoration of cell-cell junction proteins in the tumor vessels and astrocyte-endothelium interactions in tumors, revealing an additional role of astrocytes in mediating tumor-induced VP. Together, these results provide genetic evidence that FAK is a mediator of tumor-induced VP in the brain. Our findings may help understand how therapeutics might be used to regulate specific cell-type interactions to restore BBB structure/function in cancer and perhaps other pathologic conditions.
205354931
Heritable Imprinting Defect Caused by Epigenetic Abnormalities in Mouse Spermatogonial Stem Cells1
Abstract Male germ cells undergo dynamic epigenetic reprogramming during fetal development, eventually establishing spermatogonial stem cells (SSCs) that can convert into pluripotent stem cells. However, little is known about the developmental potential of fetal germ cells and how they mature into SSCs. We developed a culture system for fetal germ cells that proliferate for long periods of time. Male germ cells from embryos 12.5–18.5 days postcoitum could expand by glial cell line-derived neurotrophic factor, a self-renewal factor for SSCs. These cells did not form teratomas, but repopulated seminiferous tubules and produced spermatogenesis, exhibiting spermatogonia potential. However, the offspring from cultured cells showed growth abnormalities and were defective in genomic imprinting. The imprinting defect persisted in both the male and female germlines for at least four generations. Moreover, germ cells in the offspring showed abnormal histone modifications and DNA methylation patterns. These results indicate that fetal germ cells have a limited ability to become pluripotent cells and lose the ability to undergo epigenetic reprogramming by in vitro culture.
36669058
Underexpression of TIM-3 and Blunted Galectin-9-Induced Apoptosis of CD4+ T Cells in Rheumatoid Arthritis
The aim of this study is to compare the expression of TIM-3 from CD4+ T cells from rheumatoid arthritis (RA) patients and healthy controls and to evaluate the effect of galectin-9 (Gal-9) on apoptosis of CD4+ T cells in these patients. CD4+ T cells from RA patients and healthy controls were isolated from peripheral blood mononuclear cells and were activated. The expression of TIM-3 mRNA in CD4+ T cells was measured using real-time polymerase chain reaction. CD4+ T cells were activated in the presence of graded doses of Gal-9 or control, and Gal-9-induced cytotoxicity and apoptotic activity of CD4+ T cells were analyzed using MTT assays and annexin-V staining, respectively. TIM-3 mRNA expression was significantly lower in CD4+ T cells from RA patients compared with those in healthy controls (p = 0.028). CD4+ T cell survival as measured by MTT assay when incubated with Gal-9 (15 nM) was significantly higher in RA patients than in healthy controls (p = 0.002). Apoptotic activity of CD4+ T cells from healthy controls as measured by annexin staining increased with graded doses of Gal-9 (0 nM vs. 30 nM, 0 nM vs. 90 nM, p = 0.016 each). However, apoptotic activity of CD4+ T cells from RA patients did not change despite the stimulation with Gal-9. Gal-9-mediated apoptosis of CD4+ T cells is dysfunctional in RA patients. Blunted Gal-9-mediated apoptosis may be exerted through underexpression of TIM-3 that negatively regulates Th1 response. Our data suggest that TIM-3 and its interaction with Gal-9 may play an important role in the pathogenesis of RA and may represent a potential therapeutic target.
8565377
Quantitative Imaging of Cerebral Blood Flow Velocity and Intracellular Motility using Dynamic Light Scattering–Optical Coherence Tomography
This paper describes a novel optical method for label-free quantitative imaging of cerebral blood flow (CBF) and intracellular motility (IM) in the rodent cerebral cortex. This method is based on a technique that integrates dynamic light scattering (DLS) and optical coherence tomography (OCT), named DLS–OCT. The technique measures both the axial and transverse velocities of CBF, whereas conventional Doppler OCT measures only the axial one. In addition, the technique produces a three-dimensional map of the diffusion coefficient quantifying nontranslational motions. In the DLS–OCT diffusion map, we observed high-diffusion spots, whose locations highly correspond to neuronal cell bodies and whose diffusion coefficient agreed with that of the motion of intracellular organelles reported in vitro in the literature. Therefore, the present method has enabled, for the first time to our knowledge, label-free imaging of the diffusion-like motion of intracellular organelles in vivo. As an example application, we used the method to monitor CBF and IM during a brief ischemic stroke, where we observed an induced persistent reduction in IM despite the recovery of CBF after stroke. This result supports that the IM measured in this study represent the cellular energy metabolism-related active motion of intracellular organelles rather than free diffusion of intracellular macromolecules.