An acute herb-drug interaction of Magnoliae Officinalis Cortex with methotrexate via inhibiting multidrug resistance-associated protein 2.

Magnoliae Officinalis Cortex (MOC), an herbal drug, contains polyphenolic lignans mainly magnolol (MN) and honokiol (HK). Methotrexate (MTX), a critical drug for cancers and autoimmune deseases, is a substrate of multidrug resistance-associated protein 2 (MRP2) and breast cancer resistance protein (BCRP). This study investigated the effect of coadministration of MOC on the pharmacokinetics of MTX and relevant mechanisms. Sprague-Dawley rats were orally administered MTX alone and with single dose (2.0 and 4.0 g/kg) and repeated seven doses of MOC (2.0 g/kg thrice daily for 2 days, the 7th dose given at 0.5 h before MTX). The serum concentrations of MTX were determined by a fluorescence polarization immunoassay. The results showed that a single dose of MOC at 2.0 g/kg significantly increased the AUC0-t and MRT of MTX by 352% and 308%, and a single dose at 4.0 g/kg significantly enhanced the AUC0-t and MRT by 362% and 291%, respectively. Likewise, repeated seven doses of MOC at 2.0 g/kg significantly increased the AUC0-t and MRT of MTX by 461% and 334%, respectively. Mechanism studies indicated that the function of MRP2 was significantly inhibited by MN, HK and the serum metabolites of MOC (MOCM), whereas BCRP was not inhibited by MOCM. In conclusion, coadministration of MOC markedly enhanced the systemic exposure and mean residence time of MTX through inhibiting the MRP2-mediated excretion of MTX.

Magnolol and Honokiol Inhibited the Function and Expression of BCRP with Mechanism Exploration.

Breast cancer resistance protein (BCRP), one of the ATP-binding cassette (ABC) transporters, was associated with the multidrug resistance (MDR) of chemotherapy. Magnolol (MN) and honokiol (HK) are major bioactive polyphenols of Magnolia officinalis. This study investigated the effects of MN and HK on the function and expression of BCRP for the purpose of developing BCRP inhibitor to overcome MDR. Cell lines including MDCKII-BCRP and MDCKII-WT were used for evaluating the function and expression of BCRP. The results showed that MN (100-12.5 µM) and HK (100-12.5 µM) significantly decreased the function of BCRP by 80~12% and 67~14%, respectively. In addition, MN and HK were verified as substrates of BCRP. Furthermore, MN and HK reduced the protein expression of BCRP, and inhibited the phosphorylation of epidermal growth factor receptor (EGFR) and phosphatidylinositol 3-kinase (PI3K). In conclusion, both MN and HK decreased the function and expression of BCRP via EGFR/PI3K signaling pathway. Therefore, both compounds were promising candidates for reversing the MDR of chemotherapy.

EMP2 induces cytostasis and apoptosis via the TGFß/SMAD/SP1 axis and recruitment of P2RX7 in urinary bladder urothelial carcinoma.

PURPOSE: Urinary bladder urothelial carcinoma (UBUC) is a common malignant disease, and its high recurrence rates impose a heavy clinical burden. The objective of this study was to identify signaling pathways downstream of epithelial membrane protein 2 (EMP2), which induces cytostasis and apoptosis in UBUC. METHODS: A series of in vitro and in vivo assays using different UBUC-derived cell lines and mouse xenograft models were performed, respectively. In addition, primary UBUC specimens were evaluated by immunohistochemistry. RESULTS: Exogenous expression of EMP2 in J82 UBUC cells significantly decreased DNA replication and altered the expression levels of several TGFß signaling-related proteins. EMP2 knockdown in BFTC905 UBUC cells resulted in opposite effects. EMP2-dysregulated cell cycle progression was found to be mediated by the TGFß/TGFBR1/SP1 family member SMAD. EMP2 or purinergic receptor P2X7 (P2RX7) gene expression upregulation induced apoptosis via both intrinsic and extrinsic pathways. In 242 UBUC patient samples, P2RX7 protein levels were found to be significantly and positively correlated with EMP2 protein levels. Low P2RX7 levels conferred poor disease-specific and metastasis-free survival rates, and significantly decreased apoptotic cell rates. EMP2 was found to physically interact with P2RX7. In the presence of a P2RX7 agonist, BzATP, overexpression of both EMP2 and P2RX7 significantly increased apoptotic cell rates compared to overexpression of EMP2 or P2RX7 alone. CONCLUSIONS: EMP2 induces cytostasis via the TGFß/SMAD/SP1 axis and recruits P2RX7 to enhance apoptosis in UBUC. Our data provide new insights that may be employed for the design of UBUC targeting therapies.

HC-HA/PTX3 from amniotic membrane reverts senescent limbal niche cells to Pax6+ neural crest progenitors to support limbal epithelial progenitors.

Quiescence and self-renewal of human corneal epithelial progenitor/stem cells (LEPC) are regulated by the limbal niche, presumably through close interaction with limbal (stromal) niche cells (LNC). Paired box homeotic gene 6 (Pax6), a conserved transcription factor essential for eye development, is essential for proper differentiation of limbal and corneal epithelial stem cells. Pax6 haploinsufficiency causes limbal stem cell deficiency, which leads to subsequent corneal blindness. We previously reported that serial passage of nuclear Pax6+ LNC resulted in the gradual loss of nuclear Pax6+ and neural crest progenitor status, the latter of which was reverted upon recovery of Pax6. These findings suggest Pax6 plays a pivotal role in supporting the self-renewal of LEPC in limbal niche. Herein, we show that HC-HA/PTX3, a unique matrix purified from amniotic membrane (AM) and consists of heavy chain 1of inter-α-trypsin inhibitor covalently linked to hyaluronic acid and complexed with pentraxin 3, is capable of reverting senescent LNC to nuclear Pax6+ neural crest progenitors that support self-renewal of LEPC. Such reversion is causally linked to early cell aggregation mediated by activation of C-X-C chemokine receptor type 4 (CXCR4)-mediated signaling followed by activation of bone morphogenetic protein (BMP) signaling. Furthermore, CXCR4-mediated signaling, but not BMP signaling, controls recovery of the nuclear Pax6+ neural crest progenitors. These findings not only explain why AM helps in vivo and ex vivo expansion of human LEPC, but they also illuminate the potential role of HC-HA/PTX3 as a surrogate matrix niche that complements stem cell-based therapies in regenerative medicine.

Niche regulation of limbal epithelial stem cells: HC-HA/PTX3 as surrogate matrix niche.

Homeostasis of the corneal epithelium is ultimately maintained by stem cells that reside in a specialized microenvironment within the corneal limbus termed palisades of Vogt. This limbal niche nourishes, protects, and regulates quiescence, self-renewal, and fate decision of limbal epithelial stem/progenitor cells (LEPCs) toward corneal epithelial differentiation. This review focuses on our current understanding of the mechanism by which limbal (stromal) niche cells (LNCs) regulate the aforementioned functions of LEPCs. Based on our discovery and characterization of a unique extracellular matrix termed HC-HA/PTX3 (Heavy chain (HC1)-hyaluronan (HA)/pentraxin 3 (PTX3) complex, "-" denotes covalent linkage; "/" denotes non-covalent binding) in the birth tissue, i.e., amniotic membrane and umbilical cord, we put forth a new paradigm that HC-HA/PTX3 serves as a surrogate matrix niche by maintaining the in vivo nuclear Pax6+ neural crest progenitor phenotype to support quiescence and self-renewal but prevent corneal fate decision of LEPCs. This new paradigm helps explain how limbal stem cell deficiency (LSCD) develops in aniridia due to Pax6-haplotype deficiency and further explains why transplantation of HC-HA/PTX3-containing amniotic membrane prevents LSCD in acute chemical burns and Stevens Johnson syndrome, augments the success of autologous LEPCs transplantation in patients suffering from partial or total LSCD, and assists ex vivo expansion (engineering) of a graft containing LEPCs. We thus envisage that this new paradigm based on regenerative matrix HC-HA/PTX3 as a surrogate niche can set a new standard for regenerative medicine in and beyond ophthalmology.

Macrophage phenotypes and Gas6/Axl signaling in apical lesions.

BACKGROUND/PURPOSE: Macrophages participate in the periapical inflammation with pro-inflammatory M1 cells and anti-inflammatory M2 cells. Gas6/Axl signal is the responsible pathway for the activation of M1 and polarization of M2. The aim of this study was to compare the number of CD16+ M1 cells, CD206+ M2 cells, and Gas6/Axl expression between apical granulomas and radicular cysts. MATERIALS AND METHODS: Twenty-four cases of granuloma and twenty of cysts were submitted to immunohistochemistry using anti-CD16 and anti-CD206 antibodies for determining M1 and M2 macrophages and investigating the cells with positive Gas6 and Axl expression. RESULTS: There were more numerous of M1 macrophages in radicular cysts (175.9 ±â€¯87.7) compared to apical granuloma (116.6 ±â€¯55.8), and M2 macrophages was higher in cysts (204.0 ±â€¯97.6) than granuloma (152.9 ±â€¯64.6). The level of Gas6/Axl expression were similar. There was a significant different in M1 macrophage (P = 0.014) between two diagnosis. In patients with or without root resorption, the number of M1 were 194.6 ±â€¯57.2 compared with 139.1 ±â€¯79.6. The number of M2 were 241.7 ±â€¯81.4 and 164.6 ±â€¯77.1. The expression of Axl was stronger in root resorption patients (191.1 ±â€¯43.6), but the tendency in Gas6 expression was similar. Significant differences were noted in high M2 infiltration and Axl positive lesions. CONCLUSION: It appears that macrophages associated with significantly higher numbers in radicular cysts than apical granuloma. Meanwhile, macrophages and Axl receptor was intensively expressed in patients with root resorption, related to severe inflammation.

Laparoendoscopic single-site myomectomy using conventional laparoscopic instruments and glove port technique: Four years experience in 109 cases.

OBJECTIVE: To report a single surgeon's experience with 109 laparoendoscopic single-site myomectomy (LESS-M) using conventional laparoscopic instruments and a homemade glove port system. MATERIALS AND METHODS: A total of 109 consecutive women who underwent LESS-M between March 2011 and April 2015 were reviewed. RESULTS: The mean age and body mass index were 38.3 ± 6.5 years and 22.1 ± 3.0 kg/m2. The mean diameter of the largest myoma and the mean number of myomas were 8.1 ± 2.4 cm and 1.6 ± 0.7. The mean weight of the myomas was 223.2 ± 159.7 g. The most common type of myoma was intramural (61%), followed by subserosal (23%), submucosal (9%), and intraligamental (7%). The most common site of the myomas was anterior (39%), followed by posterior (38%), lateral (15%), and fundal (9%). The mean operative time and estimated blood loss were 138.5 ± 43.8 min and 104.9 ± 270.1 mL. Two patients (1.8%) required intraoperative transfusion. The mean hospital stay was 2.5 ± 0.6days. There were no conversions to laparotomy, but three patients(2.8%) were converted to two-port laparoscopic myomectomy. No patient experienced any major complication, including bowel, ureter, bladder injuries, or incisional hernia. Six women became pregnant after the operation, and five of these patients delivered their babies at full term by cesarean section. One patient delivered her baby at a gestational age at 32 weeks due to idiopathic polyhydramnios by cesarean section. One patient had the second pregnancy and delivery after LESS-M. Fourteen patients (12.8%) had small recurrent myomas that did not require treatment. CONCLUSION: LESS-M is a feasible alternative for patients with symptomatic myomas, and this technique can provide cosmetic advantages compared to conventional laparoscopic surgery.

Senescence Mediated by p16INK4a Impedes Reprogramming of Human Corneal Endothelial Cells into Neural Crest Progenitors.

Human corneal endothelial cells (HCECs) have limited proliferative capacity due to "contact-inhibition" at G1 phase. Such contact-inhibition can be delayed from Day 21 to Day 42 by switching EGF-containing SHEM to LIF/bFGF-containing MESCM through transient activation of LIF-JAK1-STAT3 signaling that delays eventual nuclear translocation of p16INK4a. Using the latter system, we have reported a novel tissue engineering technique by implementing 5 weekly knockdowns with p120 catenin (p120) and Kaiso siRNAs since Day 7 to achieve effective expansion of HCEC monolayers to a transplantable size with a normal HCEC density, through reprogramming of HCECs into neural crest progenitors by activating p120-Kaiso-RhoA-ROCK-canonical BMP signaling. Herein, we noted that a single knockdown with p120-Kaiso siRNAs at Day 42 failed to achieve such reprogramming when contact inhibition transitioned to senescence with nuclear translocation of p16INK4a. In contrast, 5 weekly knockdowns with p120-Kaiso siRNAs since Day 7 precluded senescence mediated by p16INK4a by inducing nuclear translocation of Bmi1 because of sustained activation of JAK2-STAT3 signaling downstream of p120-Kaiso-RhoA-ROCK signaling. STAT3 or Bmi1 siRNA impeded nuclear exclusion of p16INK4a and suppressed the reprogramming induced by p120-Kaiso siRNAs, suggesting that another important engineering strategy of HCEC lies in prevention of senescence mediated by nuclear translocation of p16INK4a.

Niche Regulation of Limbal Epithelial Stem Cells: Relationship between Inflammation and Regeneration.

Human limbal palisades of Vogt are the ideal site for studying and practicing regenerative medicine due to their accessibility. Nonresolving inflammation in limbal stroma is common manifestation of limbal stem cell (SC) deficiency and presents as a threat to the success of transplanted limbal epithelial SCs. This pathologic process can be overcome by transplantation of cryopreserved human amniotic membrane (AM), which exerts anti-inflammatory, antiscarring and anti-angiogenic action to promote wound healing. To determine how AM might exert anti-inflammation and promote regeneration, we have purified a novel matrix, HC-HA/PTX3, responsible for the efficacy of AM efficacy. HC-HA complex is covalently formed by hyaluronan (HA) and heavy chain 1 (HC1) of inter-α-trypsin inhibitor by the catalytic action of tumor necrosis factor-stimulated gene-6 (TSG-6) and are tightly associated with pentraxin 3 (PTX3) to form HC-HA/PTX3. In vitro reconstitution of the limbal niche can be established by reunion between limbal epithelial progenitors and limbal niche cells on different substrates. In 3-dimensional Matrigel, clonal expansion indicative of SC renewal is correlated with activation of canonical Wnt signaling and suppression of canonical bone morphogenetic protein (BMP) signaling. In contrast, SC quiescence can be achieved in HC-HA/PTX3 by activation of canonical BMP signaling and non-canonical planar cell polarity (PCP) Wnt signaling, but suppression of canonical Wnt signaling. HC-HA/PTX3 is a novel matrix mitigating nonresolving inflammation and restoring SC quiescence in the niche for various applications in regenerative medicine.

Hypoglycemic Activity through a Novel Combination of Fruiting Body and Mycelia of Cordyceps militaris in High-Fat Diet-Induced Type 2 Diabetes Mellitus Mice.

Diabetes mellitus (DM) is currently ranked among leading causes of death worldwide in which type 2 DM is reaching an epidemic proportion. Hypoglycemic medications for type 2 DM have either proven inadequate or posed adverse effects; therefore, the Chinese herbal products are under investigation as an alternative treatment. In this study, a novel combination of fruiting body and mycelia powder of herbal Cordyceps militaris number 1 (CmNo1) was administered to evaluate their potential hypoglycemic effects in high-fat diet- (HFD-) induced type 2 DM in C57BL/6J mice. Body weight, fasting blood glucose (FBG), oral glucose tolerance test (OGTT), and blood biochemistry indexes were measured. Results indicated that CmNo1 lowered the blood glucose level by increasing insulin sensitivity, while no change in body weight was observed. Increased protein expression of IRS-1, pIRS-1, AKT, pAKT, and GLUT-4 in skeletal muscle and adipose tissue was found indicating restoration of insulin signaling. Additionally, PPAR-γ expression in adipose tissue restored the triglyceride and cholesterol levels. Finally, our results suggest that CmNo1 possesses strong hypoglycemic, anticholesterolemic, and antihypertriglyceridemic actions and is more economical alternate for DM treatment.

HC-HA/PTX3 Purified From Amniotic Membrane Promotes BMP Signaling in Limbal Niche Cells to Maintain Quiescence of Limbal Epithelial Progenitor/Stem Cells.

To explore how limbal niche cells (LNCs) may control quiescence, self-renewal, and corneal epithelial lineage commitment/differentiation of limbal epithelial progenitor/stem cells (LEPCs), we have established an in vitro sphere assay by reunion between the two cell types in three-dimensional Matrigel. The resultant sphere exhibits inhibition of corneal epithelial lineage commitment/differentiation and marked clonal growth of LEPCs, of which the latter is correlated with activation of canonical Wnt signaling. Herein, we have created a similar reunion assay in immobilized heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3), which is purified from amniotic membrane (AM) and consists of a complex formed by hyaluronic covalently linked to heavy chain 1 of inter-α-inhibitor and noncovalently linked to pentraxin 3. The resultant spheres exhibited similar suppression of corneal epithelial lineage commitment/differentiation but upregulation of quiescence markers including nuclear translocation of Bmi-1, and negligible clonal growth of LEPCs. This outcome was correlated with the suppression of canonical Wnt but activation of noncanonical (Planar cell polarity) Wnt signaling as well as BMP signaling in both LEPCs and LNCs. The activation of BMP signaling in LNCs was pivotal because nuclear translocation of pSmad1/5/8 was prohibited in hLEPCs when reunioned with mLNCs of conditionally deleted Bmpr1a;Acvr1(DCKO) mice. Furthermore, ablation of BMP signaling in LEPCs led to upregulation of cell cycle genes, downregulation of Bmi-1, nuclear exclusion of phosphorylated Bmi-1, and marked promotion of the clonal growth of LEPCs. Hence, HC-HA/PTX3 uniquely upregulates BMP signaling in LNCs which leads to BMP signaling in LEPCs to achieve quiescence, helping explain how AM transplantation is clinically useful to be used as a matrix for ex vivo expansion of LEPCs and to treat corneal blindness caused by limbal stem cells deficiency.

Random-Coil Behavior of Chemically Denatured Topologically Knotted Proteins Revealed by Small-Angle X-ray Scattering.

Recent studies on the mechanisms by which topologically knotted proteins attain their natively knotted structures have intrigued theoretical and experimental biophysicists. Of particular interest is the finding that YibK and YbeA, two small trefoil knotted proteins, remain topologically knotted in their chemically denatured states. Using small-angle X-ray scattering (SAXS), we examine whether these chemically denatured knotted proteins are different from typical random coils. By revisiting the scaling law of radius of gyration (Rg) as a function of polypeptide chain length for chemically denatured proteins and natively folded proteins, we find that the chemically denatured knotted proteins in fact follow the same random coil-like behavior, suggesting that the formation of topological protein knots do not necessarily require global compaction while the loosely knotted polypeptide chains are capable of maintaining the correct chirality without defined secondary or tertiary structures.

Unraveling the folding mechanism of the smallest knotted protein, MJ0366.

Understanding the mechanism by which polypeptide chains thread themselves into topologically knotted structures has emerged to be a challenging subject not least because of the additional complexity associated with the spontaneous and efficient knotting and folding events. While recent theoretical calculations have made significant progress in establishing the atomistic folding pathways for a number of knotted proteins, experimental data on the folding stabilities and kinetic pathways of knotted proteins has been sparse. Using MJ0366 from Methanocaldococcus jannaschii, the smallest knotted protein known to date, as a model system, we set out to systematically investigate its folding equilibrium, kinetics, and internal dynamics under native and chemically denatured states. NMR hydrogen-deuterium exchange analysis indicates that the knotted region is the most stable structural element within the novel fold. Additionally, (15)N spin relaxation analysis reveals the presence of residual structures in urea-denatured MJ0366. Despite the apparent two-state equilibrium unfolding behavior during chemical denaturation, the kinetic unfolding pathway of MJ0366 involves the dissociation of the homodimeric native state into a native-like monomeric intermediate followed by unfolding into a denatured state. Our results provide comprehensive structural information regarding the folding dynamics and kinetic pathways of MJ0366, whose small size is ideal for converging experimental and theoretical findings to better understand the underlying principles of the folding of knotted proteins.

LIF-JAK1-STAT3 signaling delays contact inhibition of human corneal endothelial cells.

Human corneal endothelial cells (HCECs) responsible for corneal transparency have limited proliferative capacity in vivo because of "contact-inhibition." This feature has hampered the ability to engineer HCECs for transplantation. Previously we have reported an in vitro model of HCECs in which contact inhibition was re-established at Day 21, even though cell junction and cell matrix interaction were not perturbed during isolation. Herein, we observe that such HCEC monolayers continue to expand and retain a normal phenotype for 2 more weeks if cultured in a leukemia inhibitory factor (LIF)-containing serum-free medium. Such expansion is accompanied initially by upregulation of Cyclin E2 colocalized with nuclear translocation of phosphorylated retinoblastoma tumor suppressor (p-Rb) at Day 21 followed by a delay in contact inhibition through activation of LIF-Janus kinase1 (JAK1)-signal transducer and activator of transcription 3 (STAT3) signaling at Day 35. The LIF-JAK1-STAT3 signaling is coupled with upregulation of E2F2 colocalized with nuclear p-Rb and with concomitant downregulation of p16(INK4a), of which upregulation is linked to senescence. Hence, activation of LIF-JAK1-STAT3 signaling to delay contact inhibition can be used as another strategy to facilitate engineering of HCEC grafts to solve the unmet global shortage of corneal grafts.

Corneal neovascularization and contemporary antiangiogenic therapeutics.

Corneal neovascularization (NV), the excessive ingrowth of blood vessels from conjunctiva into the cornea, is a common sequela of disease insult that can lead to visual impairment. Clinically, topical steroid, argon laser photocoagulation, and subconjunctival injection of bevacizumab have been used to treat corneal NV. Sometimes, the therapies are ineffective, especially when the vessels are large. Large vessels are difficult to occlude and easily recanalized. Scientists and physicians are now dedicated to overcoming this problem. In this article, we briefly introduce the pathogenesis of corneal NV, and then highlight the existing animal models used in corneal NV research-the alkali-induced model and the suture-induced model. Most of all, we review the potential therapeutic targets (i.e., vascular endothelial growth factor and platelet-derived growth factor) and their corresponding inhibitors, as well as the immunosuppressants that have been discovered in recent years by corneal NV studies.

Using induced pluripotent stem cell-derived conditional medium to attenuate the light-induced photodamaged retina of rats.

BACKGROUND: Light injury to photoreceptor cells and retinal pigment epithelium may lead to oxidative stress and irreversible degeneration of retina, especially degeneration of the high energy-demanded macula. The model of retinal photodamage could be applied to age-related macular degeneration and other degenerative retinal diseases for exploring new treatments. Based on broadly investigated induced pluripotent stem cells (iPSC) in the field of retinal degeneration, we aimed to clarify further how the interaction progresses between iPSC-conditional medium (CM) and light-damaged retina. METHODS: iPSCs were generated from murine embryonic fibroblasts of C57/B6 mice by retroviral transfection of three factors: Oct4, Sox2, and Klf4. Cytokine array was performed to analyze the components of CM. Sprague-Dawley rats receiving white light exposure to retina were viewed as an animal model of light injury. The rats were divided into four subgroups: light-injured rats receiving intravitreal injection of iPSC-CM, apoptotic iPSC-CM, or sodium phosphate buffer (PBS); and a control group without light damage. The electroretinography and thickness of outer nuclear layer were measured to document the therapeutic effects in each condition. Apoptosis arrays for detecting annexin V and caspase 3 were performed in the retinal tissues from each group. RESULTS: Murine embryonic fibroblasts were induced into iPSCs and expressed the marker genes similar to embryonic stem cells. These iPSCs can differentiate into Embryoid bodies (EBs), three germ layers in vitro and develop teratoma in severe combined immunodeficiency mice. The quantitative polymerase chain reaction of our iPSC-CM showed significantly elevated fibroblast growth factor-2, glial cell-derived neurotrophic factor, and insulin-like growth factor-binding proteins-1, -2, and -3. Compared to rats without photodamage, the light-injured rats receiving iPSC-CM had less reduction of outer nuclear layer thickness on Day 21 than other groups treated with either PBS or apoptotic iPSC-CM. In the same animal model, both a- and b-waves of electroretinography measurement in the group treated with iPSC-CM were significantly maintained compared to the control group and others with apoptotic iPSC-CM or PBS treatment. The apoptosis assay also demonstrated lower levels of annexin V and caspase 3 in the group with iPSC-CM treatment than in other groups presenting increasing apoptotic markers. CONCLUSION: The conditional medium of iPSCs contains plenty of cytoprotective, immune-modulative and rescue chemicals, contributing to the maintenance of neuronal function and retinal layers in light-damaged retina compared with apoptotic iPSC-CM and PBS. The antiapoptotic effect of iPSC-CM also shows promise in restoring damaged neurons. This result demonstrates that iPSC-CM may serve as an alternative to cell therapy alone to treat retinal light damage and maintain functional and structural integrity of the retina.

Delayed animal aging through the recovery of stem cell senescence by platelet rich plasma.

Aging is related to loss of functional stem cell accompanying loss of tissue and organ regeneration potentials. Previously, we demonstrated that the life span of ovariectomy-senescence accelerated mice (OVX-SAMP8) was significantly prolonged and similar to that of the congenic senescence-resistant strain of mice after platelet rich plasma (PRP)/embryonic fibroblast transplantation. The aim of this study is to investigate the potential of PRP for recovering cellular potential from senescence and then delaying animal aging. We first examined whether stem cells would be senescent in aged mice compared to young mice. Primary adipose derived stem cells (ADSCs) and bone marrow derived stem cells (BMSCs) were harvested from young and aged mice, and found that cell senescence was strongly correlated to animal aging. Subsequently, we demonstrated that PRP could recover cell potential from senescence, such as promote cell growth (cell proliferation and colony formation), increase osteogenesis, decrease adipogenesis, restore cell senescence related markers and resist the oxidative stress in stem cells from aged mice. The results also showed that PRP treatment in aged mice could delay mice aging as indicated by survival, body weight and aging phenotypes (behavior and gross morphology) in term of recovering the cellular potential of their stem cells compared to the results on aged control mice. In conclusion these findings showed that PRP has potential to delay aging through the recovery of stem cell senescence and could be used as an alternative medicine for tissue regeneration and future rejuvenation.

Activation of RhoA-ROCK-BMP signaling reprograms adult human corneal endothelial cells.

Currently there are limited treatment options for corneal blindness caused by dysfunctional corneal endothelial cells. The primary treatment involves transplantation of healthy donor human corneal endothelial cells, but a global shortage of donor corneas necessitates other options. Conventional tissue approaches for corneal endothelial cells are based on EDTA-trypsin treatment and run the risk of irreversible endothelial mesenchymal transition by activating canonical Wingless-related integration site (Wnt) and TGF-ß signaling. Herein, we demonstrate an alternative strategy that avoids disruption of cell-cell junctions and instead activates Ras homologue gene family A (RhoA)-Rho-associated protein kinase (ROCK)-canonical bone morphogenic protein signaling to reprogram adult human corneal endothelial cells to neural crest-like progenitors via activation of the miR302b-Oct4-Sox2-Nanog network. This approach allowed us to engineer eight human corneal endothelial monolayers of transplantable size, with a normal density and phenotype from one corneoscleral rim. Given that a similar signal network also exists in the retinal pigment epithelium, this partial reprogramming approach may have widespread relevance and potential for treating degenerative diseases.

Isolation and characterization of mesenchymal progenitor cells from human orbital adipose tissue.

PURPOSE: Adipose-derived stem cells (ASCs) have gained importance due to their myriad potential clinical applications. We hypothesize that progenitor cells also exist besides those conventionally isolated from the stromal vascular fraction (SVF). METHOD: Central and medial orbital adipose tissues obtained from patients during eyelid surgery were digested with collagenase for 3 or 16 hours at 37°C with or without shaking. After centrifugation, the remaining cell pellet was resuspended and filtered to yield flow through in SVF and retained cells (RC) on the filter. Single cells from RC and SVF were cultured on 5% coated Matrigel in serum-free modified embryonic stem cells medium (MESCM) for 10 passages. The progenitor status was evaluated by the expression of a number of markers by qPCR and immunofluorescence staining as well as their plasticity for endothelial and tri-lineage differentiation. RESULTS: Type I collagenase digestion for 3 hours under shaking was significantly less effective in releasing progenitor cells than collagenase A digestion for 16 hours without shaking. Following filtration, cells in SVF and RC, of which the latter were tangled in collagen IV-containing matrix, expressed different markers of progenitor cells. Cells from SVF and RC could be expanded for 10 passages on coated Matrigel in MESCM and exhibited similar or better potential to differentiate into vascular endothelial cells, chondrocytes, osteocytes, and adipocytes than SVF cells expanded on plastic in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). CONCLUSIONS: Different progenitor cells can be isolated and expanded from orbital adipose tissues. Further characterization of their mesodermal or neuroectodermal origin might enhance clinical outcome when used as a source of autologous stem cells for ocular surface regeneration.