Trophoblast differentiation and early placental development are essential for the establishment of pregnancy, yet these critical events are not readily investigated in human pregnancy. We used embryoid bodies (EBs) prepared from human embryonic stem (hES) cells as an in vitro model of early human development. The levels of human chorionic gonadotropin (hCG), progesterone, and estradiol-17beta in medium from hES cell-derived EBs grown in suspension culture for 1 wk were higher than unconditioned culture medium or medium from undifferentiated hES cells or spontaneously differentiated hES cell colonies. EBs were explanted into Matrigel (MG) "rafts" and cultured for up to 53 d. During the first 7-10 d of three-dimensional growth in MG, small protrusions appeared on the outer surface of EBs, some of which subsequently extended into multicellular outgrowths. The secretion of hCG, progesterone, and estradiol-17beta began to increase on approximately d 20 of MG culture and remained dramatically elevated over the next 30 d. EBs maintained in suspension culture failed to demonstrate this elevation in hormone secretion. Suspension-cultured and MG-embedded EBs exhibited widespread expression of cytokeratins 7/8, demonstrating extensive epithelial differentiation as well as consistent hCG expression. We propose that hES cell-derived EBs may be a useful model for investigation of human trophoblast differentiation and placental morphogenesis.

The advent of regenerative medicine has brought us the opportunity to regenerate, modify and restore human organs function. Stem cells, a key resource in regenerative medicine, are defined as clonogenic, self-renewing, progenitor cells that can generate into one or more specialized cell types. Stem cells have been classified into three main groups: embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult/postnatal stem cells (ASCs). The present review focused the attention on ASCs, which have been identified in many perioral tissues such as dental pulp, periodontal ligament, follicle, gingival, alveolar bone and papilla. Human dental pulp stem cells (hDPSCs) are ectodermal-derived stem cells, originating from migrating neural crest cells and possess mesenchymal stem cell properties. During last decade, hDPSCs have received extensive attention in the field of tissue engineering and regenerative medicine due to their accessibility and ability to differentiate in several cell phenotypes. In this review, we have carefully described the potential of hDPSCs to differentiate into odontoblasts, osteocytes/osteoblasts, adipocytes, chondrocytes and neural cells.

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Conceptuses of ruminant ungulates produce large amounts of a type I interferon, interferon-tau (IFNtau), which is the signal for maternal recognition of pregnancy. Induction of cellular Mx proteins is an important component of the response to type I interferon in the immune system, but Mx regulation and function have not been studied in the uterus. This study examined temporal and spatial alterations in ovine uterine Mx expression during the cycle and early pregnancy using immunohistochemistry, in situ hybridization, and Northern and slot-blot analysis. Sheep uterine endometrium expressed a single approximately 2.5-kilobase Mx mRNA transcript that was detectable at all stages of the estrous cycle and early pregnancy examined. In cyclic ewes, mRNA abundance in endometrium increased from Day 1 to peak levels at Day 13 and then declined to Day 15. In pregnant ewes, steady-state levels of Mx mRNA were first detected above the level in cyclic ewes at Day 13 postmating, were greater than 10-fold higher at Day 15, and remained elevated at Day 19. Expression of Mx mRNA in the myometrium did not change during the estrous cycle but increased approximately 23-fold between Days 11 and 15 of pregnancy. Immunohistochemical and in situ hybridization analysis revealed a similar temporal pattern of Mx expression. In cyclic ewes, Mx protein and mRNA were initially localized to the luminal epithelium at Days 1 and 3, increased from Days 5 to 13, especially in the shallow uterine glands, and then declined at Day 15. Pregnancy resulted in up-regulation of Mx expression in the luminal and glandular epithelium, stroma, and myometrium. Punctate Mx immunostaining and Mx mRNA concentrations were greatest when progesterone production was maximal during the estrous cycle and were strongly up-regulated by the conceptus across the entire uterine wall. It is suggested that a cascade of induction of Mx gene expression proceeds from the luminal epithelium to the outer longitudinal myometrium and that transcriptional activation of the promoter may involve both soluble cytokines (i.e., IFNtau) and steroid hormones (i.e., progesterone).

Diabetic foot ulcers (DFUs) are chronic wounds, with 20% of cases resulting in amputation, despite intervention. A recently approved tissue engineering product—a cell‐free collagen‐glycosaminoglycan (GAG) scaffold—demonstrates 50% success, motivating its functionalization with extracellular matrix (ECM). Induced pluripotent stem cell (iPSC) technology reprograms somatic cells into an embryonic‐like state. Recent findings describe how iPSCs‐derived fibroblasts (“post‐iPSF”) are proangiogenic, produce more ECM than their somatic precursors (“pre‐iPSF”), and their ECM has characteristics of foetal ECM (a wound regeneration advantage, as fetuses heal scar‐free). ECM production is 45% higher from post‐iPSF and has favorable components (e.g., Collagen I and III, and fibronectin). Herein, a freeze‐dried scaffold using ECM grown by post‐iPSF cells (Post‐iPSF Coll) is developed and tested vs precursors ECM‐activated scaffolds (Pre‐iPSF Coll). When seeded with healthy or DFU fibroblasts, both ECM‐derived scaffolds have more diverse ECM and more robust immune responses to cues. Post‐iPSF‐Coll had higher GAG, higher cell content, higher Vascular Endothelial Growth Factor (VEGF) in DFUs, and higher Interleukin‐1‐receptor antagonist (IL‐1ra) vs. pre‐iPSF Coll. This work constitutes the first step in exploiting ECM from iPSF for tissue engineering scaffolds.

Clinical barriers to stem-cell therapy include the need for efficient derivation of histocompatible stem cells and the zoonotic risk inherent to human stem-cell xenoculture on mouse feeder cells. We describe a system for efficiently deriving induced pluripotent stem (iPS) cells from human and mouse amniocytes, and for maintaining the pluripotency of these iPS cells on mitotically inactivated feeder layers prepared from the same amniocytes. Both cellular components of this system are thus autologous to a single donor. Moreover, the use of human feeder cells reduces the risk of zoonosis. Generation of iPS cells using retroviral vectors from short- or long-term cultured human and mouse amniocytes using four factors, or two factors in mouse, occurs in 5–7 days with 0.5% efficiency. This efficiency is greater than that reported for mouse and human fibroblasts using similar viral infection approaches, and does not appear to result from selective reprogramming of Oct4+ or c-Kit+ amniocyte subpopulations. Derivation of amniocyte-derived iPS (AdiPS) cell colonies, which express pluripotency markers and exhibit appropriate microarray expression and DNA methylation properties, was facilitated by live immunostaining. AdiPS cells also generate embryoid bodies in vitro and teratomas in vivo. Furthermore, mouse and human amniocytes can serve as feeder layers for iPS cells and for mouse and human embryonic stem (ES) cells. Thus, human amniocytes provide an efficient source of autologous iPS cells and, as feeder cells, can also maintain iPS and ES cell pluripotency without the safety concerns associated with xenoculture.

Diabetic foot ulcers (DFUs) are a major complication of diabetes, and there is a critical need to develop novel cell- and tissue-based therapies to treat these chronic wounds. Induced pluripotent stem cells (iPSCs) offer a replenishing source of allogeneic and autologous cell types that may be beneficial to improve DFU wound-healing outcomes. However, the biologic potential of iPSC-derived cells to treat DFUs has not, to our knowledge, been investigated. Toward that goal, we have performed detailed characterization of iPSC-derived fibroblasts from both diabetic and nondiabetic patients. Significantly, gene array and functional analyses reveal that iPSC-derived fibroblasts from both patients with and those without diabetes are more similar to each other than were the primary cells from which they were derived. iPSC-derived fibroblasts showed improved migratory properties in 2-dimensional culture. iPSC-derived fibroblasts from DFUs displayed a unique biochemical composition and morphology when grown as 3-dimensional (3D), self-assembled extracellular matrix tissues, which were distinct from tissues fabricated using the parental DFU fibroblasts from which they were reprogrammed. In vivo transplantation of 3D tissues with iPSC-derived fibroblasts showed they persisted in the wound and facilitated diabetic wound closure compared with primary DFU fibroblasts. Taken together, our findings support the potential application of these iPSC-derived fibroblasts and 3D tissues to improve wound healing.-Kashpur, O., Smith, A., Gerami-Naini, B., Maione, A. G., Calabrese, R., Tellechea, A., Theocharidis, G., Liang, L., Pastar, I., Tomic-Canic, M., Mooney, D., Veves, A., Garlick, J. A. Differentiation of diabetic foot ulcer-derived induced pluripotent stem cells reveals distinct cellular and tissue phenotypes.

Diabetic foot ulcers (DFUs) are a serious complication of diabetes. Previous exposure to hyperglycemic conditions accelerates a decline in cellular function through metabolic memory despite normalization of glycemic control. Persistent, hyperglycemia-induced epigenetic patterns are considered a central mechanism that activates metabolic memory; however, this has not been investigated in patient-derived fibroblasts from DFUs. We generated a cohort of patient-derived lines from DFU fibroblasts (DFUF), and site-and age-matched diabetic foot fibroblasts (DFF) and nondiabetic foot fibroblasts (NFF) to investigate global and genome-wide DNA methylation patterns using liquid chromatography/mass spectrometry and the Illumina Infinium HumanMethylation450K array. DFFs and DFUFs demonstrated significantly lower global DNA methylation compared to NFFs (p D 0.03). Hierarchical clustering of differentially methylated probes (DMPs, p D 0.05) showed that DFFs and DFUFs cluster together and separately from NFFs. Twenty-five percent of the same probes were identified as DMPs when individually comparing DFF and DFUF to NFF. Functional annotation identified enrichment of DMPs associated with genes critical to wound repair, including angiogenesis (p D 0.07) and extracellular matrix assembly (p D 0.035). Identification of sustained DNA methylation patterns in patient-derived fibroblasts after prolonged passage in normoglycemic conditions demonstrates persistent metabolic memory. These findings suggest that epigenetic-related metabolic memory may also underlie differences in wound healing phenotypes and can potentially identify therapeutic targets.