[Show abstract][Hide abstract] ABSTRACT: To explore restoration of ovarian function using epigenetically-related, induced pluripotent stem cells (iPSCs), we functionally evaluated the epigenetic memory of novel iPSC lines, derived from mouse and human ovarian granulosa cells (GCs) using c-Myc, Klf4, Sox2 and Oct4 retroviral vectors. The stem cell identity of the mouse and human GC-derived iPSCs (mGriPSCs, hGriPSCs) was verified by demonstrating embryonic stem cell (ESC) antigen expression using immunocytochemistry and RT-PCR analysis, as well as formation of embryoid bodies (EBs) and teratomas that are capable of differentiating into cells from all three germ layers. GriPSCs' gene expression profiles associate more closely with those of ESCs than of the originating GCs as demonstrated by genome-wide analysis of mRNA and microRNA. A comparative analysis of EBs generated from three different mouse cell lines (mGriPSCs; fibroblast-derived iPSC, mFiPSCs; G4 embryonic stem cells, G4 mESCs) revealed that differentiated mGriPSC-EBs synthesize 10-fold more estradiol (E2) than either differentiated FiPSC- or mESC-EBs under identical culture conditions. By contrast, mESC-EBs primarily synthesize progesterone (P4) and FiPSC-EBs produce neither E2 nor P4. Differentiated mGriPSC-EBs also express ovarian markers (AMHR, FSHR, Cyp19a1, ER and Inha) as well as markers of early gametogenesis (Mvh, Dazl, Gdf9, Boule and Zp1) more frequently than EBs of the other cell lines. These results provide evidence of preferential homotypic differentiation of mGriPSCs into ovarian cell types. Collectively, our data support the hypothesis that generating iPSCs from the desired tissue type may prove advantageous due to the iPSCs' epigenetic memory.
PLoS ONE 03/2015; 10(3):e0119275. DOI:10.1371/journal.pone.0119275 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Embryonic stem (ES) cells provide a potentially useful in vitro model for the study of in vivo tissue differentiation. We used mouse and human ES cells to investigate whether the lens regulatory genes Pax6 and Six3 could induce lens cell fate in vitro. To help assess the onset of lens differentiation, we derived a new mES cell line (Pax6-GFP mES) that expresses a GFP reporter under the control of the Pax6 P0 promoter and lens ectoderm enhancer. Pax6 or Six3 expression vectors were introduced into mES or hES cells by transfection or lentiviral infection and the differentiating ES cells analyzed for lens marker expression. Transfection of mES cells with Pax6 or Six3 but not with other genes induced the expression of lens cell markers and up-regulated GFP reporter expression in Pax6-GFP mES cells by 3 days post-transfection. By 7 days post-transfection, mES cell cultures exhibited a>10-fold increase over controls in the number of colonies expressing γA-crystallin, a lens fiber cell differentiation marker. RT-PCR and immunostaining revealed induction of additional lens epithelial or fiber cell differentiation markers including Foxe3, Prox1, α- and β-crystallins, and Tdrd7. Moreover, γA-crystallin- or Prox1-expressing lentoid bodies formed by 30 days in culture. In hES cells, Pax6 or Six3 lentiviral vectors also induced lens marker expression. mES cells that express lens markers reside close to but are distinct from the Pax6 or Six3 transduced cells, suggesting that the latter induce nearby undifferentiated ES cells to adopt a lens fate by non-cell autonomous mechanisms. In sum, we describe a novel mES cell GFP reporter line that is useful for monitoring induction of lens fate, and demonstrate that Pax6 or Six3 is sufficient to induce ES cells to adopt a lens fate, potentially via non-cell autonomous mechanisms. These findings should facilitate investigations of lens development.
PLoS ONE 12/2014; 9(12). DOI:10.1371/journal.pone.0115106 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Significance: Human-induced pluripotent stem cells (iPSC) can be differentiated into patient-specific cells with a wide spectrum of cellular phenotypes and offer an alternative source of autologous cells for therapeutic use. Recent studies have shown that iPSC-derived fibroblasts display enhanced cellular functions suggesting that iPSC may eventually become an important source of stem cells for regenerative therapies. Recent Advances: The discovery of approaches to reprogram somatic cells into pluripotent cells opens exciting avenues for their use in personalized, regenerative therapies. The controlled differentiation of functional cell types from iPSC provides a replenishing source of fibroblasts. There is intriguing evidence that iPSC reprogramming and subsequent differentiation to fibroblast lineages may improve cellular functional properties. Augmenting the biological potency of iPSC-derived fibroblasts may enable the development of novel, personalized stem cell therapies to treat oral disease. Critical Issues: Numerous questions need to be addressed before iPSC-derived cells can be used as a practical oral therapy. This will include understanding why iPSC-derived cells are predisposed towards differentiation pathways along lineages related to their cell of origin, screening iPSC-derived cells to ensure their safety and phenotypic stability and developing engineered, three-dimensional tissue models to optimize their function and efficacy for future therapeutic transplantation. Future Directions: Future research will need to address how to develop efficient methods to deliver and integrate iPSC-derived fibroblasts into the oral mucosa. This will require an improved understanding of how to harness their biological potency for regenerative therapies that are specifically targeted to the oral mucosa.
[Show abstract][Hide abstract] ABSTRACT: 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 non-diabetic 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 = 0.03). Hierarchical clustering of differentially methylated probes (DMPs, p = 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 = 0.07) and extracellular matrix assembly (p = 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.
Epigenetics: official journal of the DNA Methylation Society 10/2014; 9(10):1339-49. DOI:10.4161/15592294.2014.967584 · 5.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objective: Signaling between fibroblasts and keratinocytes is essential for proper wound healing but is thought to be dysregulated in the diabetic foot ulcer (DFU) environment. An engineered 3D human skin model was used to examine differences in the ability of DFU fibroblasts to support the differentiation and proliferation of human keratinocytes. The long-term goal is to develop improved therapeutic approaches for DFU repair.
Method: Fibroblasts were harvested from diabetic foot ulcers (DFU) and non-ulcerated diabetic feet (DFF) from Type II diabetic patients as well as from site-matched non-diabetic feet (NFF). DFU, DFF or NFF fibroblasts were embedded in collagen gels, normal human keratinocytes (NHKs) were seeded on them and tissues grown at air-liquid interface. Tissues were pulsed with BrdU for 6 hours, formalin-fixed and paraffin embedded. Sections of these tissues were stained by H&E to visualize tissue development and for BrdU to identify proliferating cells. The number of BrdU-positive, basal keratinocytes were counted and expressed as a percentage of the total number of basal keratinocytes per 20x image.
Result: H&E staining showed that all three fibroblast types (DFU, DFF and NFF) were able to support keratinocyte development into a fully differentiated epidermal layer. However, keratinocytes grown on DFU fibroblasts were more proliferative than those grown on DFF fibroblasts (p < .05). The number of proliferating keratinocytes grown on NFF fibroblasts was less than DFUs and greater than DFFs, although not statistically significant.
Conclusion: Our findings indicate that DFU fibroblasts stimulated increased keratinocyte proliferation in 3D tissues when compared with DFF fibroblasts, suggesting that the ulcer environment may direct paracrine signaling between fibroblasts and keratinocytes. This suggests a possible mechanism for the hyperproliferative keratinocytes found in the callus surrounding DFUs. This marks an important step towards understanding mechanisms that will help develop future regenerative therapies to treat oral and cutaneous wounds.
[Show abstract][Hide abstract] ABSTRACT: Objectives: Diabetic foot ulceration impairs quality of life and leads to prolonged hospitalizations, demonstrating the need to develop novel sources of repair-competent, autologous cells. Our lab is developing a novel therapeutic approach, based on induced pluripotent stem cell (iPSC) technologies, whose goal is to reverse chronic wound fibroblasts from a non-healing to a healing phenotype. As a first step towards this goal, we characterized the gene expression profile of fibroblasts cultured from ulcers and normal skin from diabetic patients.
Methods: Fibroblasts were harvested from non-healing ulcers or normal skin from the feet of diabetic patients (DFU 1, 3, 6) as well as from normal plantar skin and palatal mucosa from non-diabetic patients. Microarray analysis was performed on diabetic fibroblasts and control oral fibroblasts, to identify genes from diabetic patients that were altered. Microarrays were then used to select RNA targets to compare plantar skin vs. oral fibroblasts from non-diabetic patients by RT-PCR to establish how site-specific expression patterns could compare to those seen in diabetic ulcer fibroblasts.
Results: Genes selected from the microarray relevant to wound repair included upregulated LOXL4, SFRP1, and IGF2 and downregulated CDH6. These microarray results were confirmed when DFU 1, 3, 6 were compared to oral fibroblast controls by RT-PCR analysis. However, DFU cells showed different patterns of gene expression when compared to plantar skin control fibroblasts, suggesting the existence of site-specific patterns of gene expression.
Conclusions: Site-matched, plantar fibroblasts displayed different patterns of gene expression compared to non-site matched, oral fibroblasts. Therefore, comparisons between DFU fibroblasts and those from different sites, such as the oral cavity, are of limited use to predict patterns of expression. These studies lay the groundwork for ongoing studies whose goal is to improve the repair potency of DFU fibroblasts following their reprogramming to iPSC and subsequent differentiation to repair-competent fibroblasts.
IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
[Show abstract][Hide abstract] ABSTRACT: Objective: Efforts to better understand interactions between dental epithelium and mesenchyme during odontogenesis have focused on recreating the three dimensional (3D) tissue microenvironments that can mimic this process in vitro. We have developed a 3D co-culture system using epithelial spheroids to study cellular morphology and patterns of gene expression characteristic of epithelial-mesenchymal cross-talk.
Methods: Human epithelial cells (HEP) were cultured on low adhesion plates to generate 3D spheroids that were then plated on confluent monolayers (2D) of dental pulp stem cells (DPSC19 or DPSC23) or human foreskin fibroblasts (HFF) (3D-2D approach). HEP cells (2D) were also seeded as single cells onto confluent layers of DPSCs or HFFs (2D-2D approach). Co-cultures were grown using osteogenic medium for up to three weeks. RNA extracted from these cells were analyzed by RT-PCR or fixed with 4% PFA for immunohistochemical staining.
Results: Cultures grown as spheroids in 3D-2D co-cultures demonstrated epithelial cells with a distinct border while the 2D-2D cultures demonstrated single cell growth when cultured on confluent mesenchymal monolayers. After 3 weeks, calcified structures were detected in 3D-2D cultures when grown on DPSC but not on control HFF cells or in 2D-2D cultures. RT-PCR analysis of genes expressed during early and late odontogenesis are currently underway.
Conclusion: We found that generation of epithelial spheroids with 3D architecture induced differentiation when grown on monolayers of DPSCs, but not when grown as a monolayer in a 2D co-culture system. We expect that ongoing analysis of these 3D co-culture systems will help improve our understanding of factors essential to stem cell commitment to odontogenic pathways that will be broadly applicable to eventual translation for clinical applications in regenerative therapies.
IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
[Show abstract][Hide abstract] ABSTRACT: The human embryo is not a feasible experimental system for the detailed study of implantation and early placentation, so surrogate systems have been sought for investigating the determination of the trophectoderm lineage, its differentiation into trophoblasts of the early implantation site, and subsequently the morphogenesis of the definitive placenta. An alternative to the use of embryos for studying early placental development was revealed by work with human embryonic stem cells (hESC), demonstrating BMP2/4-stimulated trophoblast differentiation, and spontaneous formation from embryoid bodies (EBs). These cells display a trophoblastic transcriptome, as well as a placental protein and steroid hormone secretory profile, and invasive and chemotactic behavior resembling human placental trophoblasts. With EB-derived trophoblasts, two-dimensional and three-dimensional paradigms and other modifications of the culture environment, including extracellular matrix and aggregation with placental fibroblasts, impact on trophoblast differentiation. Recent studies have questioned the identity of the trophoblasts directed by BMP treatment of hESC, and careful attention to culture conditions is needed to interpret different results among research groups. Although the precise placental counterpart of the hESC-derived trophoblast remains unclear, hESC-derived trophoblasts remain an intriguing platform for modeling early implantation.
[Show abstract][Hide abstract] ABSTRACT: 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.
Human Molecular Genetics 03/2011; 20(5):962-74. DOI:10.1093/hmg/ddq542 · 6.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The earliest stages of human development profoundly influence the success of pregnancy and maternal as well as fetal health. One of the most critical developmental processes is the specification of the trophoblast lineage and the formation of the placenta, yet this is the stage that is most difficult to study because of the general inability to do in vitro studies with human embryos or conduct in vivo clinical research during the initial weeks of pregnancy. Embryonic stem cells potentially provide a novel surrogate for the undifferentiated pluripotent cells of the preimplantation embryo, and current research indicates that human embryonic stem cells are capable of differentiation to the trophoblast lineage under the influence of diverse soluble, transcriptional, and environmental cues. This review summarizes current areas of research in these different approaches to the study of placental cell differentiation and suggests avenues of investigation that are both unique and promising in this area.
Seminars in Reproductive Medicine 12/2006; 24(5):314-21. DOI:10.1055/s-2006-952154 · 3.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.