Three-Dimensional Cell Culture Scaffold Supports Capillary-Like Network Formation by Endothelial Cells Derived from Porcine-Induced Pluripotent Stem Cells.

INTRODUCTION: Endothelial cells (EC) can be generated from porcine-induced pluripotent stem cells (piPSC), but poor efficiency in driving EC differentiation hampers their application and efficacy. Additionally, the culture of piPSC-derived EC (piPSC-EC) on three-dimensional (3D) scaffolds has not been fully reported yet. Here, we report a method to improve the generation of EC differentiation from piPSC and to facilitate their culture on 3D scaffolds, providing a potential resource for in vitro drug testing and the generation of tissue-engineered vascular grafts. METHODS: We initiated the differentiation of piPSC into EC by seeding them on laminin 411 and employing a three-stage protocol, which involved the use of distinct EC differentiation media supplemented with CHIR99021, BMP4, VEGF, and bFGF. RESULTS: piPSC-EC not only expressed EC markers such as CD31, VE-cadherin, and von Willebrand factor (vWF) but also exhibited an upregulation of EC marker genes, including CD31, CD34, VEGFR2, VE-cadherin, and vWF. They exhibited functional characteristics similar to those of porcine coronary artery endothelial cells (PCAEC), such as tube formation and Dil-Ac-LDL uptake. Furthermore, when cultured on 3D scaffolds, piPSC-EC developed a 3D morphology and were capable of forming an endothelial layer and engineering capillary-like networks, though these lacked lumen structures. CONCLUSION: Our study not only advances the generation of EC from piPSC through an inhibitor and growth factor cocktail but also provides a promising approach for constructing vascular network-like structures. Importantly, these findings open new avenues for drug discovery in vitro and tissue engineering in vivo.

Dopaminergic neurons derived from porcine induced pluripotent stem cell like cells function in the Lanyu pig model of Parkinson's disease.

The induced pluripotent stem cells (iPSCs) are able to differentiate into dopaminergic neurons and execute the therapeutic effects for Parkinson's disease (PD). Here, we established a animal model of PD in Lanyu pigs by injecting 5 mg/kg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP). Next, the porcine iPSC-like cells (piPSC-like cells) were differentiated into D18 neuronal progenitors (D18 NPs) that were transplanted into the striatum to evaluate their therapeutic effects of PD. We showed that after 8 weeks of cell transplantation, the behavior score was significantly ameliorated and fully recovered at the 14th week of cell transplantation. The number of dopaminergic neurons was also significantly improved at the end of the experiment although the number was still about 50% lower than that in the control group. Our findings suggest that piPSC-like cell-derived D18 NPs exhibit a potential for the treatment of PD in the Lanyu pig model.

Transplantation of porcine embryonic stem cells and their derived neuronal progenitors in a spinal cord injury rat model.

BACKGROUND AIMS: The purpose of this study was to investigate therapeutic potential of green fluorescent protein expressing porcine embryonic stem (pES/GFP(+)) cells in A rat model of spinal cord injury (SCI). METHODS: Undifferentiated pES/GFP(+) cells and their neuronal differentiation derivatives were transplanted into the contused spinal cord of the Long Evans rat, and in situ development of the cells was determined by using a live animal fluorescence optical imaging system every 15 days. After pES/GFP(+) cell transplantation, the behavior functional recovery of the SCI rats was assessed with the Basso, Beattie, and Bresnahan Locomotor Rating Scale (BBB scale), and the growth and differentiation of the grafted pES/GFP(+) cells in the SCI rats were analyzed by immunohistochemical staining. RESULTS: The relative green fluorescent protein expression level was decreased for 3 months after transplantation. The pES/GFP(+)-derived cells positively stained with neural specific antibodies of anti-NFL, anti-MBP, anti-SYP and anti-Tuj 1 were detected at the transplanted position. The SCI rats grafted with the D18 neuronal progenitors showed a significant functional recovery of hindlimbs and exhibited the highest BBB scale score of 15.20 ± 1.43 at week 24. The SCI rats treated with pES/GFP(+)-derived neural progenitors demonstrated a better functional recovery. CONCLUSIONS: Transplantation of porcine embryonic stem (pES)-derived D18 neuronal progenitors has treatment potential for SCI, and functional behavior improvement of grafted pES-derived cells in SCI model rats suggests the potential for further application of pES cells in the study of replacement medicine and functionally degenerative pathologies.

Transplantation of embryonic stem cells improves the regeneration of periodontal furcation defects in a porcine model.

OBJECTIVES: Stem cell-based therapy promises to regenerate lost tissue. Embryonic stem (ES) cells are pluripotent and may provide a virtually unlimited source for transplantation. We investigated whether ES cell transplantation improved the regeneration of furcation defects in a porcine model. MATERIAL AND METHODS: Experimental periodontitis was induced in the buccal furcations of the bilateral mandibular 2nd premolars of six minipigs. After 4 weeks, the lesions were surgically debrided and implanted with collagen matrix alone (control site) or collagen matrix overlaid with porcine ES cells expressing green fluorescent protein (pES/GFP(+) ) (test site). After 3 months of healing, the clinical parameters were measured again. The treated teeth with adjacent tissue, and part of the major organs, were processed for GFP immunohistochemistry. RESULTS: We found no obvious teratoma or rejection. The test group had significantly better clinical parameters. Immunohistochemistry (IHC) showed that transplanted pES/GFP(+) cells had differentiated to new periodontal ligament and cementum in the test sites. Surprisingly, GFP(+) cells were also detectable in the repaired control cementum and remote organs. CONCLUSIONS: We conclude that using ES cells to improve the regeneration of periodontal furcation defects is feasible. More studies are required to assess this potential treatment's efficacy and safety.

A protocol for differential staining of cartilages and ossified bones in fetal and adult mouse skeletons using alcian blue and alizarin red S.

The technique for clearing and staining whole specimens consists of many steps. This study discusses the alcian blue/alizarin red S staining method and aims to provide a useful reference and review for users who intend to do this staining. To specifically address the influences of tissue removal on staining results, the mouse fetuses at embryonic stage E18.5 and adult mice at 12 weeks of age were used in this study. The fetuses were divided into three groups: Group 1 skin, muscle, and viscera removed, Group 2 skin and muscle removed, and Group 3 viscera removed. For successful skeletal staining, it was concluded that (1) skin removal from fetuses was necessary for alcian blue staining but unnecessary for alizarin red S staining, (2) removal of muscle surrounding thorax and neck of fetuses could improve transparency effects, (3) retaining fetal viscera would not significantly affect transparency but might avoid the tissue damage, and (4) complete skin, muscle, and viscera removal were essential for good staining of adult mice. The representative images and detailed staining procedures might be good for researchers presently using alcian blue and alizarin red S staining to differentiate cartilages and ossified bones.

Effects of Klf4 and c-Myc Knockdown on Pluripotency Maintenance in Porcine Induced Pluripotent Stem Cell.

OBJECTIVES: The importance of Oct4 and Sox2 in maintaining pluripotency and self-renewal is well-understood, but the functions of Klf4 and c-Myc has not been fully investigated. In the present study, we attempted to determine the roles of Klf4 and c-Myc on pluripotency maintenance of porcine induced pluripotent stem (piPS) cells. MATERIALS AND METHODS: In this experimental study, we performed short hairpin RNA (shRNA) to knock down the Klf4 and c-Myc functions of piPS cells and examined pluripotency markers and teratoma formation to evaluate piPS cell pluripotency. The shRNA-Klf4 and shRNA-c-Myc vectors containing a reporter gene, TagFP635, were transfected into piPS cells by lentivirus infection. The piPS cells fully expressing infrared fluorescence were selected to confirm gene knockdown of Klf4 and c-Myc reverse transcription-polymerase chain reaction (RT-PCR). Next, for pluripotency evaluation, expression of pluripotency markers was detected by immunocytochemical staining, and capability of teratoma formation was investigated by piPS cell transplantation into nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. RESULTS: Our findings indicated that Klf4 and c-Myc functions of piPS cells were knocked down by shRNA transfection, and knockdown of Klf4 and c-Myc functions impaired expression of pluripotency markers such as Oct4, AP, SSEA-3, SSEA-4, TRA-1-6, and TRA-1-81. Furthermore, piPS cells without Klf4 and c-Myc expression failed to form teratomas. CONCLUSIONS: The pluripotency of piPS cells are crucially dependent upon Klf4 and c-Myc expression. These findings, suggesting potential mechanisms of Klf4 and c-Myc contribution to piPS cell formation, have important implications for application, regulation, and tumorigenesis of piPS cells.

Porcine-induced pluripotent stem cell-derived osteoblast-like cells ameliorate trabecular bone mass of osteoporotic rats.

AIM: We created rat models of osteoporosis and verified a novel idea to recover bone mass via local cell transplantation. MATERIALS & METHODS: The rats were treated with ovariectomy, 0.1% calcium diet or 3 mg/kg body weight/day of prednisolone and porcine-induced pluripotent stem cell (piPSC)-derived osteoblast-like cells were transplanted into the medullary cavity of the left femurs. RESULTS: The piPSC-derived osteoblast-like cells exerted therapeutic potential on prednisolone treatment group, which confirmed by improvements in trabecular bone volume (15.93 ± 2.20%), bone surface/volume ratio (27.82 ± 1.40 1/mm), thickness (1.40 ± 0.01 mm), separation (0.99 ± 0.10 mm), number (1.13 ± 0.13 1/mm) and total porosity (84.06 ± 2.20%). CONCLUSION: These results first uncovered therapeutic potential of xenotransplantation with piPSCs for glucocorticoid-induced osteoporosis treatment in the rat models.

Porcine induced pluripotent stem cell-derived osteoblast-like cells prevent glucocorticoid-induced bone loss in Lanyu pigs.

The application of appropriate animal models and techniques for the study of osteoporosis is important. Lanyu pigs, a local miniature breed, have been widely used in various biomedical studies in Taiwan. This study aimed to induce bone loss in Lanyu pigs and to examine whether porcine induced pluripotent stem cell (piPSC)-derived osteoblast-like cells could recover bone mass of tibiae via local cell transplantation. piPSCs were directed to differentiate into osteoblast-like cells using osteogenic medium, and differentiated cells expressed osteogenic markers and phenotypes. Twenty mature female Lanyu pigs were divided into four groups, including control (C, 1% calcium diet), treatment 1 (T1, ovariectomy + 1% calcium diet), treatment 2 (T2, ovariectomy + 0.5% calcium diet), and treatment 3 (T3, ovariectomy + 0.5% calcium diet + 1 mg/kg of prednisolone) and were subjected to bone loss induction for twelve months. Micro-CT images revealed that the lowest trabecular bone parameters, such as trabecular bone volume, thickness, separation, number, and total porosity, were detected in the T3 group. The lowest proportions of cortical bone in the proximal metaphysis, proximal diaphysis, and distal diaphysis were also found in the T3 group. These results indicate that ovariectomy, calcium restriction, and prednisolone administration can be applied to induce proper bone loss in Lanyu pigs. After bone loss induction, pigs were subjected to cell transplantation in the left tibiae and were maintained for another six months. Results showed that transplanted piPSC-derived osteoblast-like cells significantly improved trabecular bone structures at transplanted sites and maintained cortical bone structures in the proximal metaphysis. In conclusion, the therapeutic potential of piPSC-derived osteoblast-like cells was confirmed via cell transplantation in the left tibiae of Lanyu pigs. These findings reveal the therapeutic potential of piPSCs for glucocorticoid-induced bone loss in pig models.

In vitro culture period but not the passage number influences the capacity of chimera production of inner cell mass and its deriving cells from porcine embryos.

Mammalian embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass (ICM) of the blastocyst. These cells are able to proliferate continuously without differentiation in vitro under suitable conditions. Their capacity of pluripotency in differentiation will be resumed when they are reintroduced into host embryos, when they will contribute to the embryonic development to form chimeric individuals. Manipulation of ES cells has been mainly established from studies in the mouse, and is powerful in the production of transgenic animals. Porcine ICM-derived cell lines possess the same cellular morphology and in vitro behavior as those of murine ES cells, but have lower efficiency in chimera formation when reintroduced into host embryos. This study was to determine the influences of passage number and the duration of in vitro culture on the capacity of porcine ICM-derived cells in the generation of chimeric embryos. The results showed that when passage number of porcine ICM-derived cells was less than 15, there were no detrimental effects on its integration ability. Extending the culture time up to 6 days in each passage of porcine ICM-derived cells impaired its integration capacity into the host blastocyst. Porcine ICM-derived cells cultured for more than 4 days in each passage should not be used for blastocyst injection if high efficiency of chimera production is to be achieved.

Derivation of porcine pluripotent stem cells for biomedical research.

Pluripotent stem cells including embryonic stem cells (ESCs), embryonic germ cells (EGCs), and induced pluripotent stem cells (iPSCs) are capable of self-renew and limitlessly proliferating in vitro with undifferentiated characteristics. They are able to differentiate in vitro, spontaneously or responding to suitable signals, into cells of all three primary germ layers. Consequently, these pluripotent stem cells will be valuable sources for cell replacement therapy in numerous disorders. However, the promise of human ESCs and EGCs is cramped by the ethical argument about destroying embryos and fetuses for cell line creation. Moreover, there are still carcinogenic risks existing toward the goal of clinical application for human ESCs, EGCs, and iPSCs. Therefore, a suitable animal model for stem cell research will benefit the further development of human stem cell technology. The pigs, on the basis of their similarity in anatomy, immunology, physiology, and biochemical properties, have been wide used as model animals in the study of various human diseases. The development of porcine pluripotent stem cell lines will hold the opportunity to provide an excellent material for human counterpart to the transplantation in biomedical research and further development of cell-based therapeutic strategy.

Evaluation of decellularized extracellular matrix of skeletal muscle for tissue engineering.

OBJECTIVE: We evaluated the effectiveness of enzyme-detergent methods on cell removal of mouse skeletal muscle tissue and assessed the biocompatibility of the decellularized tissues by an animal model. METHODS: The mouse latissimus dorsi (LD) muscles underwent decellularization with different enzyme-detergent mixtures (trypsin-Triton X-100, trypsin-sodium dodecyl sulfate (SDS), trypsin-Triton X-100-SDS). The effectiveness of decellularization was assessed by histology and DNA assay. The content in collagen and glycosaminoglycan was measured. The biomechanical property was evaluated in uniaxial tensile tests. For biocompatibility, the decellularized muscle specimens were implanted in situ and the tissue samples were retrieved at day 10, 20, and 30, to evaluate the host-graft inflammatory reaction. RESULTS: Extensive washing of the mouse LD muscles with an enzyme-detergent mixture (trypsin and Triton X-100) can yield an intact matrix devoid of cells, depleted of more than 93% nuclear component and exhibiting comparable biomechanical properties with native tissue. In addition, we observed increased infiltration of inflammatory cells into the scaffold initially, and the presence of M1 (CD68)-phenotype mononuclear cells 10 days after implantation, which decreased gradually until day 30. CONCLUSIONS: The enzyme-detergent method can serve as an effective method for cell removal of mouse skeletal muscle. In short-term follow-up, the implanted scaffolds revealed mild inflammation with fibrotic tissue formation. The decellularized extracelluar matrix developed herein is shown to be feasible for further long-term study for detailed information about muscle regeneration, innervation, and angiogenesis in vivo.

Inhibition of fumonisin B1 cytotoxicity by nanosilicate platelets during mouse embryo development.

Nanosilicate platelets (NSP), the form of natural silicate clay that was exfoliated from montmorillonite (MMT), is widely used as a feed additive for its high non-specific binding capacity with mycotoxins such as fumonisin B1 (FB1), and has been evaluated its safety for biomedical use including cytotoxicity, genotoxicity, and lethal dosage (LD). In the study, we further examined its toxicity on the development of CD1 mouse embryos and its capacity to prevent teratogenesis-induced by FB1. In vitro cultures, NSP did not disturb the development and the quality of intact pre-implantation mouse embryos. Further, newborn mice from females consumed with NSP showed no abnormalities. NSP had an unexpected high adsorption capacity in vitro. In contrast to female mice consumed with FB1 only, a very low residual level of FB1 in the circulation, reduced incidence of neutral tube defects and significantly increased fetal weight were observed in the females consumed with FB1 and NSP, suggesting a high alleviation effect of NSP on FB1 in vivo. Furthermore, FB1 treatment disturbed the gene expression of sphingolipid metabolism enzymes (longevity assurance homolog 5, LASS 5; sphingosine kinase 1, Sphk1; sphingosine kinase 2, Sphk2; sphingosine 1- phosphate lyase, Sgpl1; sphingosine 1-phosphate phosphatase, Sgpp1) in the maternal liver, uterus, fetus, and placenta, but NSP administration reversed the perturbations. Based on these findings, we conclude that NSP is a feasible and effective agent for supplementary use in reducing the toxicity of FB1 to animals.

Directed differentiation into neural lineages and therapeutic potential of porcine embryonic stem cells in rat Parkinson's disease model.

This study was conducted to direct porcine embryonic stem (pES) cells differentiating into neural lineages and to investigate therapeutic potential of GFP-expressing pES (pES/GFP(+)) in the rat model of Parkinson's disease (PD). Directed differentiation of pES into neural lineages was induced by suspension culture in medium containing RA, SHH, and FGF combinations without going through embryoid body formation. A high yield of nestin-expressing neural precursors was found in all treatments on day 2 after the 12-day induction. On day 6 after replating, more than 86.2 and 83.4% of the differentiated cells stained positively for NFL and MAP2, respectively. The expression of TH, ChAT, and GABA specific markers were also observed in these NFL-positive neural cells. The undifferentiated pES/GFP(+) cells and their neuronal differentiation derivatives were transplanted into the Sprague-Dawley (SD) rat's brain, and their survival and development was determined by using live animal fluorescence optical imaging system every 15 days. The results showed that fluorescent signals from the injection site of SD rats' brain could be detected through the experimental period of 3 months. The level of fluorescent signal detected in the treatment group was twofold that of the control group. The results of behavior analysis showed that PD rats exhibited stably decreased asymmetric rotations after transplantation with pES/GFP(+)-derived D18 neuronal progenitors. The dopaminergic differentiation of grafted cells in the brain was further confirmed by immunohistochemical staining with anti-TH, anti-DA, and anti-DAT antibodies. These results suggested that the differentiation approach we developed would direct pES cells to differentiate into neural lineages and benefit the development of novel therapeutics involving stem cell transplantation.

Identification of early transcripts related to male development in chicken embryos.

Early transcripts related to male development in chicken embryos and their expression profiles were examined. A total of 89 and 127 candidate male development transcripts that represented 83 known and 119 unknown non-redundant sequences, respectively, were characterized in an embryonic day 3 (E3; Hamburger and Hamilton Stage 20: HH20) male-subtract-female complementary DNA library. Of 35 selected transcripts, quantitative reverse transcription-polymerase chain reaction validated that the expression levels of 25 transcripts were higher in male E3 whole embryos than in females (P < 0.05). Twelve of these transcripts mapped to the Z chromosome. At 72 wk of age, 20 and 4 transcripts were expressed at higher levels in the testes and brains of male than in the ovaries and brains of female chickens (P < 0.05), respectively. Whole mount and frozen cross-section in situ hybridization, as well as Western blotting analysis further corroborated that riboflavin kinase (RFK), WD repeat domain 36 (WDR36), and EY505808 transcripts; RFK and WDR36 protein products were predominantly expressed in E7 male gonads. Treatment with an aromatase inhibitor formestane at E4 affected the expression levels at E7 of the coatomer protein complex (subunit beta 1), solute carrier family 35 member F1, LOC427316 and EY505812 transcripts across both sexes (P < 0.05), similar to what was observed for the doublesex and mab-3 related transcription factor 1 gene. The interaction effects of sex by formestane treatment were observed in 15 candidate male development transcripts (P < 0.05). Taken together, we identified a panel of potentially candidate male development transcripts during early chicken embryogenesis; some might be regulated by sex hormones.

Establishment and characterization of novel porcine embryonic stem cell lines expressing hrGFP.

The purpose of this study was to establish transgenic porcine embryonic stem (pES) cell lines that can stably express report gene. Established pES cell line at passage 44 was transfected with pAAV-hrGFP Control Plasmid by electroporation-mediated, viral vector-mediated, and liposome-mediated strategies. Although there were several pES colonies expressing green fluorescent protein (GFP) obtained from the retrovirus-mediated and liposome-mediated transfection methods, no stable GFP-expressing pES cell line was then derived. A total of 28 GFP-expressing pES cell colonies were obtained following electroporation with two DC pulses of 150 V/cm for 10 msec and three GFP-expressing pES (pES/GFP(+)) cell lines were established. These pES/GFP(+) cell lines stably expressed exogenous GFP and continuously proliferated in vitro for more than 90 passages in 20 months. They maintained normal karyotype of 36 + XX and typical characteristics of pluripotent stem cells, including expression of pluripotent markers Oct-4, AP, SSEA-4, TRA-1-60, and TRA-1-81, formation of embryoid bodies under suspension culture. They were able to differentiate in vitro into neural and cardiomyocytic lineage, respectively, under suitable induction. To our knowledge, there has been no report of establishing GFP-expressing pES cell lines. These novel pES/GFP(+) cell lines established in this study might serve as a nonrodent model and would benefit to the studies involving ES cell transplantation, cell replacement therapy, and tissue regeneration due to their traceable capacity.