Behzad Gerami-Naini

Amniocytes can serve a dual function as a source of iPS cells and feeder layers

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.

Genome-wide DNA methylation analysis identifies a metabolic memory profile in patient-derived diabetic foot ulcer fibroblasts

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.

Functional Maintenance of Differentiated Embryoid Bodies in Microfluidic Systems: A Platform for Personalized Medicine

Hormone replacement therapies have become important for treating diseases such as premature ovarian failure or menopausal complications. The clinical use of bioidentical hormones might significantly reduce some of the potential risks reportedly associated with the use of synthetic hormones. In the present study, we demonstrate the utility and advantage of a microfluidic chip culture system to enhance the development of personalized, on‐demand, treatment modules using embryoid bodies (EBs). Functional EBs cultured on microfluidic chips represent a platform for personalized, patient‐specific treatment cassettes that can be cryopreserved until required for treatment. We assessed the viability, differentiation, and functionality of EBs cultured and cryopreserved in this system. During extended microfluidic culture, estradiol, progesterone, testosterone, and anti‐müllerian hormone levels were measured, and the expression of differentiated steroidogenic cells was confirmed by immunocytochemistry assay for the ovarian tissue markers anti‐müllerian hormone receptor type II, follicle‐stimulating hormone receptor, and inhibin β‐A and the estrogen biosynthesis enzyme aromatase. Our studies showed that under microfluidic conditions, differentiated steroidogenic EBs continued to secrete estradiol and progesterone at physiologically relevant concentrations (30–120 pg/ml and 150–450 pg/ml, respectively) for up to 21 days. Collectively, we have demonstrated for the first time the feasibility of using a microfluidic chip system with continuous flow for the differentiation and extended culture of functional steroidogenic stem cell‐derived EBs, the differentiation of EBs into cells expressing ovarian antigens in a microfluidic system, and the ability to cryopreserve this system with restoration of growth and functionality on thawing. These results present a platform for the development of a new therapeutic system for personalized medicine.

Efficient Differentiation of Steroidogenic and Germ-Like Cells from Epigenetically-Related iPSCs Derived from Ovarian Granulosa Cells

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.

Generation of Induced Pluripotent Stem Cells from Diabetic Foot Ulcer Fibroblasts Using a Nonintegrative Sendai Virus

Diabetic foot ulcers (DFUs) are nonhealing chronic wounds that are a serious complication of diabetes. Since induced pluripotent stem cells (iPSCs) may offer a potent source of autologous cells to heal these wounds, we studied if repair-deficient fibroblasts, derived from DFU patients and age- and site-matched control fibroblasts, could be reprogrammed to iPSCs. To establish this, we used Sendai virus to successfully reprogram six primary fibroblast cell lines derived from ulcerated skin of two DFU patients (DFU8, DFU25), nonulcerated foot skin from two diabetic patients (DFF24, DFF9), and healthy foot skin from two nondiabetic patients (NFF12, NFF14). We confirmed reprogramming to a pluripotent state through three independent criteria: immunofluorescent staining for SSEA-4 and TRA-1-81, formation of embryoid bodies with differentiation potential to all three embryonic germ layers in vitro, and formation of teratomas in vivo. All iPSC lines showed normal karyotypes and typical, nonmethylated CpG sites for OCT4 and NANOG. iPSCs derived from DFUs were similar to those derived from site-matched nonulcerated skin from both diabetic and nondiabetic patients. These results have established for the first time that multiple, DFU-derived fibroblast cell lines can be reprogrammed with efficiencies similar to control fibroblasts, thus demonstrating their utility for future regenerative therapy of DFUs.

Pax6- and Six3-Mediated Induction of Lens Cell Fate in Mouse and Human ES Cells

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.

Differentiation of diabetic foot ulcer–derived induced pluripotent stem cells reveals distinct cellular and tissue phenotypes

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. FASEB J. 33, 1262–1277 (2019). www.fasebj.org

Induced pluripotent stem cells to generate skin tissue models

Abstract Human-induced pluripotent stem cells (iPSCs) can be differentiated into a wide spectrum of multiple, isogenic, patient-specific cell types that offer an important new source of cells for the construction of 3-D skin tissue models. iPSCs have been differentiated to specific cell lineages, including fibroblasts, keratinocytes, melanocytes, and endothelial cells that can be incorporated into skin tissue models that support the development of a tissue microenvironment with a well-differentiated, stratified squamous epithelium. When incorporated into 3-D skin tissues, these iPSC-derived cell types display cellular behaviors that mimic their in vivo functions. Further development of skin tissues constructed with iPSC-derived cells will generate versatile models to study the pathogenesis of skin disease processes and to test the safety and efficacy of new products and drugs designed to treat these diseases.

An Embryonic and Induced Pluripotent Stem Cell Model for Ovarian Granulosa Cell Development and Steroidogenesis

Embryoid bodies (EBs) can serve as a system for evaluating pluripotency, cellular differentiation, and tissue morphogenesis. In this study, we use EBs derived from mouse embryonic stem cells (mESCs) and human amniocyte–derived induced pluripotent stem cells (hAdiPSCs) as a model for ovarian granulosa cell (GC) development and steroidogenic cell commitment. We demonstrated that spontaneously differentiated murine EBs (mEBs) and human EBs (hEBs) displayed ovarian GC markers, such as aromatase (CYP19A1), FOXL2, AMHR2, FSHR, and GJA1. Comparative microarray analysis identified both shared and unique gene expression between mEBs and the maturing mouse ovary. Gene sets related to gonadogenesis, lipid metabolism, and ovarian development were significantly overrepresented in EBs. Of the 29 genes, 15 that were differentially regulated in steroidogenic mEBs displayed temporal expression changes between embryonic, postnatal, and mature ovarian tissues by polymerase chain reaction. Importantly, both mEBs and hEBs were capable of gonadotropin-responsive estradiol (E2) synthesis in vitro (217-759 pg/mL). Live fluorescence-activated cell sorting–sorted AMHR2+ granulosa-like cells from mEBs continued to produce E2 after purification (15.3 pg/mL) and secreted significantly more E2 than AMHR2− cells (8.6 pg/mL, P < .05). We conclude that spontaneously differentiated EBs of both mESC and hAdiPSC origin can serve as a biologically relevant model for ovarian GC differentiation and steroidogenic cell commitment. These cells should be further investigated for therapeutic uses, such as stem cell–based hormone replacement therapy and in vitro maturation of oocytes.

Trophoblast differentiation, invasion and hormone secretion in a three-dimensional in vitro implantation model with rhesus monkey embryos

BackgroundThe initiation of primate embryo invasion into the endometrium and the formation of the placenta from trophoblasts, fetal mesenchyme, and vascular components are essential for the establishment of a successful pregnancy. The mechanisms which direct morphogenesis of the chorionic villi, and the interactions between trophectoderm-derived trophoblasts and the fetal mesenchyme to direct these processes during placentation are not well understood due to a dearth of systems to examine and manipulate real-time primate implantation. Here we describe an in vitro three-dimensional (3-D) model to study implantation which utilized IVF-generated rhesus monkey embryos cultured in a Matrigel explant system.MethodsBlastocyst stage embryos were embedded in a 3-D microenvironment of a Matrigel carrier and co-cultured with a feeder layer of cells generating conditioned medium. Throughout the course of embryo co-culture embryo growth and secretions were monitored. Embedded embryos were then sectioned and stained for markers of trophoblast function and differentiation.ResultsSigns of implantation were observed including enlargement of the embryo mass, and invasion and proliferation of trophoblast outgrowths. Expression of chorionic gonadotropin defined by immunohistochemical staining, and secretion of chorionic gonadotropin and progesterone coincident with the appearance of trophoblast outgrowths, supported the conclusion that a trophoblast cell lineage formed from implanted embryos. Positive staining for selected markers including Ki67, MHC class I, NeuN, CD31, vonWillebrand Factor and Vimentin, suggest growth and differentiation of the embryo following embedding.ConclusionsThis 3-D in vitro system will facilitate further study of primate embryo biology, with potential to provide a platform for study of genes related to implantation defects and trophoblast differentiation.