Endothelial cells (ECs) represent the major component of the embryonic pancreatic niche and play a key role in the differentiation of insulin-producing β cells in vivo. However, it is unknown if ECs promote such differentiation in vitro. We investigated whether interaction of ECs with mouse embryoid bodies (EBs) in culture promotes differentiation of pancreatic progenitors and insulin-producing cells and the mechanisms involved. We developed a co-culture system of mouse EBs and human microvascular ECs (HMECs). An increase in the expression of the pancreatic markers PDX-1, Ngn3, Nkx6.1, proinsulin, GLUT-2, and Ptf1a was observed at the interface between EBs and ECs (EB-EC). No expression of these markers was found at the periphery of EBs cultured without ECs or those co-cultured with mouse embryonic fibroblasts (MEFs). At EB-EC interface, proinsulin and Nkx6.1 positive cells co-expressed phospho-Smad1/5/8 (pSmad1/5/8). Therefore, EBs were treated with HMEC conditioned media (HMEC-CM) suspecting soluble factors involved in bone morphogenetic protein (BMP) pathway activation. Upregulation of PDX-1, Ngn3, Nkx6.1, insulin-1, insulin-2, amylin, SUR1, GKS, and amylase as well as down-regulation of SST were detected in treated EBs. In addition, higher expression of BMP-2/-4 and their receptor (BMPR1A) were also found in these EBs. Recombinant human BMP-2 (rhBMP-2) mimicked the effects of the HMEC-CM on EBs. Noggin (NOG), a BMP antagonist, partially inhibited these effects. These results indicate that the differentiation of EBs to pancreatic progenitors and insulin-producing cells can be enhanced by ECs in vitro and that BMP pathway activation is central to this process.Electronic supplementary materialThe online version of this article (doi:10.1007/s12015-011-9232-z) contains supplementary material, which is available to authorized users.

The prevalence of androgenic alopecia (AGA) increases with age and it affects both men and women. Patients diagnosed with AGA may experience decreased quality of life, depression, and feel self-conscious. There are a variety of therapeutic options ranging from prescription drugs to non-prescription medications. Currently, AGA involves an annual global market revenue of US$4 billion and a growth rate of 1.8%, indicating a growing consumer market. Although natural and synthetic ingredients can promote hair growth and, therefore, be useful to treat AGA, some of them have important adverse effects and unknown mechanisms of action that limit their use and benefits. Biologic factors that include signaling from stem cells, dermal papilla cells, and platelet-rich plasma are some of the current therapeutic agents being studied for hair restoration with milder side effects. However, most of the mechanisms exerted by these factors in hair restoration are still being researched. In this review, we analyze the therapeutic agents that have been used for AGA and emphasize the potential of new therapies based on advances in stem cell technologies and regenerative medicine.

Angiogenesis is a dynamic process that requires an interaction of pro-and antiangiogenic factors. It is known that the cytokine leptin stimulates endothelial cell growth and angiogenesis, but further quantitative analysis is necessary to understand leptin angiogenic effects. The quail chorioallantoic membrane ͑CAM͒ assay has been used to study angiogenesis in vivo by focusing on morphometric parameters that quantify vascular complexity and density. We quantify the angiogenic activity of leptin using the CAM assay by digital morphometry and a computer-assisted image analysis to evaluate more precisely vessel length, diameter, branching, and tortuousity. CAM images are obtained from ex ovo cultures of E8-E9 quail embryos. MATLAB® and custom software are used for our analysis. The effects of leptin, vascular endothelial growth factor-165 ͑VEGF 165 ͒, and their corresponding neutralizing antibodies are compared. Our results show that CAM treated with leptin and VEGF 165 has a significant increase in vascular complexity and density. A corresponding decrease is observed using neutralizing antibodies. Notably, leptin induced more significant changes than VEGF in vessel length and tortuousity. Conversely, VEGF induced a greater increase in vessel branching than leptin. These results underscore the importance of using multiparametric quantitative methods to assess several aspects of angiogenesis and enable us to understand the proangiogenic effects of leptin.

Endothelial cells (ECs) are essential for pancreas differentiation, endocrine specification, and endocrine function. They are also involved in the physiopathology of type 1 and type 2 diabetes. During embryogenesis, aortic ECs provide specific factors that maintain the expression of key genes for pancreas development such as pancreatic and duodenal homeobox-1. Other unknown factors are also important for pancreatic endocrine specification and formation of insulin-producing beta cells. Endocrine precursors proliferate interspersed with ductal cells and exocrine precursors and, at some point of development, these endocrine precursors migrate to pancreatic mesenchyme and start forming the islets of Langerhans. By the end of the gestation and close to birth, these islets contain immature beta cells with the capacity to express vascular endothelial growth factor and therefore to recruit ECs from the surrounding microenvironment. ECs in turn produce factors that are essential to maintain insulin secretion in pancreatic beta cells. Once assembled, a cross talk between endocrine cells and ECs maintain the integrity of islets toward an adequate function during the whole life of the adult individual. This review will focus in the EC role in the differentiation and maturation of pancreatic beta cells during embryogenesis as well as the current knowledge about the involvement of endothelium to derive pancreatic beta cells in vitro from mouse or human pluripotent stem cells.

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Endothelial cells (ECs) provide inductive signals for cell differentiation in vivo. However, it is unknown if these cells promote such differentiation in vitro and the signals involved. We investigated whether ECs are able to enhance the differentiation of the three germ layers and the underlying mechanisms. We established a coculture system of mouse embryoid bodies (EBs) and ECs. Then, we analyzed the expression of markers representative of the three germ layers, such as PDX-1, proinsulin, insulin1 (endoderm), nestin, neurofilament light (ectoderm), CD31, cardiotin, and cardiac troponin I (mesoderm) in EBs cultured alone (controls) or with ECs. A significant increase of these markers was observed in EBs cocultured with ECs compared to controls. The cocultured EBs also exhibited more robust vascular networks similar to those EBs treated with bone morphogenetic protein-2 or -4 (BMP-2 or -4). Therefore, the role of these peptides in the differentiation was investigated. We found a significant upregulation of BMP-2/-4 and BMP receptor 1A in EBs treated with EC conditioned medium (EC-CM) at early or middle stages of EB development. Recombinant human BMP-2 and BMP-4 exerted similar effects than EC-CM in the expression of BMPs or in the upregulation of the three germ layer specific markers. BMP-2/-4 antagonists, such as noggin and chordinlike-1, respectively inhibited the EC-CM inductive effects. These results demonstrate that ECs enhance the differentiation in vitro of cells that derived from the three germ layers and that BMP-2/-4 play a central role in this process.

Here we document three highly reproducible protocols: 1) A culture system for the derivation of human oigodendrocites (OLs) from human induced pluripotent stem cells (hiPS) and their further maturation; Key features are the concomitant fate restriction, and lineage specification of hiPS towards the neutral and OL phenotypes. The use of very precise amounts of fresh additives and factors also contributes to the shorter periods of time (56days vs. /200days/75days) necessary to obtain hiPS-derived OLs. Our protocol generates viral and integration free OLs that efficiently commit and move forward in the OL lineage recapitulating all the steps known to occur during in vivo development. 2) Protocols for the isolation, propagation and maintenance of neural stem cells (NSCs); and 3) Protocols for the production, isolation, and maintenance of OLs from perinatal rodent and human brain-derived NSCs. Our unique culture systems rely on a series of chemically defined media, specifically designed and carefully characterized for each developmental stage of OL as they advance from OL progenitors to mature, myelinating cells. We are confident that these protocols bring our field a step closer to efficient autologous cell replacement therapies and disease modeling.

Avian chorioallantoic membrane (CAM) has been used as a model to explore angiogenesis and to study the microvasculature of transplanted tissues. Because CAM provides a vascular bed, cells can be implanted, and their development can be monitored and modified. We used the CAM model to study the differentiation process of embryoid bodies (EBs) derived from mouse embryonic stem cells (ESCs) influenced by the CAM vascular bed. After EBs were incubated in CAM for 5 days, they underwent further differentiation and became tissue masses (TMs) of different morphologies from those that grew outside CAM. Immunohistochemical analysis of TMs demonstrated tissue-specific markers such as neurofilament light, CD34, collagen IV, cardiac myosin heavy chain (MHC), and cardiotin. Differentiated mouse blood vessels stained with anti-CD31 were found within the TMs, as well as blood vessels stained positive for QH1 and QCPN, markers for quail endothelial cells and perinuclear quail antigen, respectively. Quail erythrocytes inside mouse blood vessels suggested a connection between existing quail vessels and blood vessels growing inside the TMs as a result of EB differentiation. Therefore, CAM could be a suitable model to trigger and study the differentiation of EBs in close interaction with surrogate quail blood vessels.