A Novel Approach to Orthotopic Hepatocyte Transplantation Engineered With Liver Hydrogel for Fibrotic Livers, Enhancing Cell-Cell Interaction and Angiogenesis.

Hepatocyte transplantation (HCT) is a potential bridging therapy or an alternative to liver transplantation. Conventionally, single-cell hepatocytes are injected via the portal vein. This strategy, however, has yet to overcome poor cell engraftment and function. Therefore, we developed an orthotopic HCT method using a liver-derived extracellular matrix (L-ECM) gel. PXB cells (flesh mature human hepatocytes) were dispersed into the hydrogel solution in vitro, and the gel solution was immediately gelated in 37°C incubators to investigate the affinity between mature human hepatocyte and the L-ECM gel. During the 3-day cultivation in hepatocyte medium, PXB cells formed cell aggregates via cell-cell interactions. Quantitative analysis revealed human albumin production in culture supernatants. For the in vivo assay, PXB cells were encapsulated in the L-ECM gel and transplanted between the liver lobes of normal rats. Pathologically, the L-ECM gel was localized at the transplant site and retained PXB cells. Cell survival and hepatic function marker expression were verified in another rat model wherein thioacetamide was administered to induce liver fibrosis. Moreover, cell-cell interactions and angiogenesis were enhanced in the L-ECM gel compared with that in the collagen gel. Our results indicate that L-ECM gels can help engraft transplanted hepatocytes and express hepatic function as a scaffold for cell transplantation.

Novel approach for reconstruction of the three-dimensional biliary system in decellularized liver scaffold using hepatocyte progenitors.

Reconstruction of the biliary system is indispensable for the regeneration of transplantable liver grafts. Here, we report the establishment of the first continuous three-dimensional biliary system scaffold for bile acid excretion using a novel method. We confirmed the preservation of the liver-derived extracellular matrix distribution in the scaffold. In addition, hepatocyte progenitors decellularized via the bile duct by slow-speed perfusion differentiated into hepatocyte- and cholangiocyte-like cells, mimicking hepatic cords and bile ducts, respectively. Furthermore, qRT-PCR demonstrated increased ALB, BSEP, and AQP8 expression, revealing bile canaliculi- and bile duct-specific genetic patterns. Therefore, we concluded that locally preserved extracellular matrices in the scaffold stimulated hepatic progenitors and provided efficient differentiation, as well as regeneration of a three-dimensional continuous biliary system from hepatic cords through bile ducts. These findings suggest that organ-derived scaffolds can be utilized for the efficient reconstruction of functional biliary systems.

A thioacetamide-induced liver fibrosis model for pre-clinical studies in microminipig.

Drug-induced liver fibrosis models are used in normal and immunosuppressed small animals for transplantation and regenerative medicine to improve liver fibrosis. Although large animal models are needed for pre-clinical studies, they are yet to be established owing to drug sensitivity in animal species and difficulty in setting doses. In this study, we evaluated liver fibrosis by administering thioacetamide (TA) to normal microminipig and thymectomized microminipig; 3 times for 1 week (total duration: 8 weeks). The pigs treated with TA showed elevated blood cytokine levels and a continuous liver injury at 8 weeks. RNA-seq of the liver showed increased expression of fibrosis-related genes after TA treatment. Histopathological examination showed degenerative necrosis of hepatocytes around the central vein, and revealed fibrogenesis and hepatocyte proliferation. TA treatment caused CD3-positive T cells and macrophages scattered within the hepatic lobule to congregate near the center of the lobule and increased αSMA-positive cells. Thymectomized pigs showed liver fibrosis similar to that of normal pigs, although the clinical signs tended to be milder. This model is similar to pathogenesis of liver fibrosis reported in other animal models. Therefore, it is expected to contribute to research as a drug discovery and pre-clinical transplantation models.

Millimeter-thick 3D tissues constructed by densely cellularized core-shell microfluidic bioprinting.

Recently, microfluidic bioprinting methods, which utilize microfluidic devices as printheads to deposit microfilaments, have improved printing resolution. Despite the precise placement of cells, current efforts have not succeeded in forming densely cellularized tissue within the printed constructs, which is highly desired for the biofabrication of solid-organ tissues with firm tissue consistency. In this paper, we presented a microfluidic bioprinting method to fabricate three dimension tissue constructs consisting of core-shell microfibers where extracellular matrices and cells can be encapsulated within the core of the fibers. Using the optimized printhead design and printing parameters, we demonstrated the bioprinting of core-shell microfibers into macroscale constructs and checked the viability of cells after printing. After culturing the printed tissues using the proposed dynamic culture methods, we analyzed the morphology and function of the tissues bothin vitroandin vivo. The confluent tissue morphology in the fiber cores indicates the establishment of intensive cell-cell contacts in the fiber cores, which also leads to the upregulation of the albumin-secretion function compared to the cells cultured in a 2D format. Analysis on the cell density of the confluent fiber cores indicate the formation of densely cellularized tissues with a similar level of cell density ofin-vivosolid organ tissues. In the future, better culture techniques with improved perfusion design are anticipated to enable further the fabrication of thicker tissues, which can be used as thick tissue models or implantation grafts for cell therapy.

Effects of galantamine on social interaction impairments in cholecystokinin receptor-2 overexpression mice.

We examined whether galantamine (GAL), a cholinesterase inhibitor and allosteric potentiating ligand for α7 nicotinic acetylcholine receptor (nAChR), had an impact on emotional abnormalities in forebrain-specific cholecystokinin receptor-2 overexpressed transgenic mice. Treatment with GAL (1 mg/kg, s.c.) attenuated the decrease of social interaction time, but failed to attenuate anxiety-like behavior in the elevated plus-maze test. The effect of GAL was blocked by an α7 nAChR antagonist, methyllycaconitine (3 mg/kg, i.p.). These results suggest that GAL improved social interaction impairments via α7 nAChR and could be useful to treat sociability-related emotional abnormalities.

Lotus-root-shaped cell-encapsulated construct as a retrieval graft for long-term transplantation of human iPSC-derived ß-cells.

Cell therapy using human-stem-cell-derived pancreatic beta cells (hSC-ßs) is a potential treatment method for type 1 diabetes mellitus (T1D). For therapeutic safety, hSC-ßs need encapsulation in grafts that are scalable and retrievable. In this study, we developed a lotus-root-shaped cell-encapsulated construct (LENCON) as a graft that can be retrieved after long-term hSC-ß transplantation. This graft had six multicores encapsulating hSC-ßs located within 1 mm from the edge. It controlled the recipient blood glucose levels for a long-term, following transplantation in immunodeficient diabetic mice. LENCON xenotransplanted into immunocompetent mice exhibited retrievability and maintained the functionality of hSC-ßs for over 1 year after transplantation. We believe that LENCON can contribute to the treatment of T1D through long-term transplantation of hSC-ßs and in many other forms of cell therapy.

Three-dimensional co-culture of blood-brain barrier-composing cells in a culture insert with a collagen vitrigel membrane.

The blood-brain barrier (BBB) is a structure located in brain capillaries that protects the brain from toxic substances in blood due to its high barrier function. The brain capillaries form a layered structure with pericytes, neurons, glial cells, and extracellular matrix proteins that is called neurovascular unit, and the structure is important to express the high barrier function of BBB. Here, we propose a method to construct a three-dimensional BBB tissue using three human BBB-composing cells, including brain endothelial cells, pericytes, and astrocytes, that mimics the in vivo BBB-like layered structure. Primary human brain endothelial cells were plated on the back side (outside) of the collagen vitrigel membrane of a culture insert, pericytes were plated on the upper side (inside), and astrocytes mixed in Matrigel were plated on the pericyte layer. The layered structure was maintained for at least 2 wk. The BBB tissue-loaded collagen vitrigel membrane can be detached from the insert frame using acetone with the tissue fixed intact and used for vertical cryosectioning to analyze the tissue interior. We also measured transendothelial electrical resistance (TEER) in the three-dimensional BBB co-culture to investigate barrier function of the brain endothelial cells. We believe that our co-culture method is useful to study engineered BBB tissues and develop reliable in vitro human BBB models in the future.

Millimeter-thick xenoislet-laden fibers as retrievable transplants mitigate foreign body reactions for long-term glycemic control in diabetic mice.

Transplantation technologies of pancreatic islets as well as stem cell-derived pancreatic beta cells encapsulated in hydrogel for the induction of immunoprotection could advance to treat type 1 diabetes mellitus, if the hydrogel transplants acquire retrievability through mitigating foreign body reactions after transplantation. Here, we demonstrate that the diameter of the fiber-shaped hydrogel transplants determines both in vivo cellular deposition onto themselves and their retrievability. Specifically, we found that the in vivo cellular deposition is significantly mitigated when the diameter is 1.0 mm and larger, and that 1.0 mm-thick xenoislet-laden fiber-shaped hydrogel transplants can be retrieved after being placed in the intraperitoneal cavities of immunocompetent diabetic mice for more than 100 days, during which period the hydrogel transplants can normalize the blood glucose concentrations of the mice. These findings could provide an innovative concept of a transplant that would promote the clinical application of stem cell-derived functional cells through improving their in vivo efficacy and safety.

3D culture of functional human iPSC-derived hepatocytes using a core-shell microfiber.

Human iPSC-derived hepatocytes hold great promise as a cell source for cell therapy and drug screening. However, the culture method for highly-quantified hepatocytes has not yet been established. Herein, we have developed an encapsulation and 3D cultivation method for iPSC-hepatocytes in core-shell hydrogel microfibers (a.k.a. cell fiber). In the fiber-shaped 3D microenvironment consisting of abundant extracellular matrix (ECM), the iPSC-hepatocytes exhibited many hepatic characteristics, including the albumin secretion, and the expression of the hepatic marker genes (ALB, HNF4α, ASGPR1, CYP2C19, and CYP3A4). Furthermore, we found that the fibers were mechanically stable and can be applicable to hepatocyte transplantation. Three days after transplantation of the microfibers into the abdominal cavity of immunodeficient mice, human albumin was detected in the peripheral blood of the transplanted mice. These results indicate that the iPSC-hepatocyte fibers are promising either as in vitro models for drug screening or as implantation grafts to treat liver failure.

Cell-laden microfibers fabricated using µl cell-suspension.

Current microfluidic methods for cell-laden microfiber fabrication generally require larger than 100 µl of cell-suspensions. Since some 'rare' cells can be only acquired in small amounts, the preparation of >100 µl cell-suspensions with high-cell density can be both expensive and time consuming. Here, we present a facile method capable of fabricating cell-laden microfibers using small-volume cell-suspensions. The method utilizes a 3D-printed coaxial microfluidic device featured with a 'luer-lock inlet' to effectively load cell-suspensions in a deterministic volume (down to 5 µl) with a low sample-loss. In experiments, we demonstrate the formation of fibrous tissues consisting of various kinds of cells. Investigations on the morphology and function of the encapsulated cells show the viability of the cells is not significantly affected by the fabrication process, and also indicate the potential of using our method to perform quantitative assays on fiber-shaped tissues, while reducing the overall material and time consumption.

Visualization of sequential conversion of human intermediately reprogrammed stem cells into iPS cells.

Analysis of gene expression in single cells is required to understand somatic cell reprogramming into human induced pluripotent stem cells (iPSCs). To facilitate this, we established intermediately reprogrammed stem cells (iRSCs), pre-iPSC lines. The iRSC-iPSC conversion system enables the reproducible monitoring of reprogramming events and the analysis of progressive gene expression profiles using single-cell microarray analysis and genome editing. Here, single-cell microarray analysis showed the stage-specific sequential gene activation during the conversion of iRSCs into iPSCs, using OCT4, TDGF1 and E-CADHERIN as marker genes. Out of 75 OCT4-related genes, which were significantly up-regulated after the activation of OCT4, and entry into the mesenchymal-to-epithelial transition (MET), LIN28 (LIN28A) and FOXO1 were selected for applying to gene expression visualization. Multicolored visualization was achieved by the genome editing of LIN28 or FOXO1 with mCherry into OCT4-GFP iRSCs. Fluorescent analysis of gene activity in individual cells showed that OCT4 was dispensable for maintenance, but required for activation, of the LIN28 and FOXO1 expression in reprogramming.

Cell fiber-based three-dimensional culture system for highly efficient expansion of human induced pluripotent stem cells.

Human pluripotent stem cells are a potentially powerful cellular resource for application in regenerative medicine. Because such applications require large numbers of human pluripotent stem cell-derived cells, a scalable culture system of human pluripotent stem cell needs to be developed. Several suspension culture systems for human pluripotent stem cell expansion exist; however, it is difficult to control the thickness of cell aggregations in these systems, leading to increased cell death likely caused by limited diffusion of gases and nutrients into the aggregations. Here, we describe a scalable culture system using the cell fiber technology for the expansion of human induced pluripotent stem (iPS) cells. The cells were encapsulated and cultured within the core region of core-shell hydrogel microfibers, resulting in the formation of rod-shaped or fiber-shaped cell aggregations with sustained thickness and high viability. By encapsulating the cells with type I collagen, we demonstrated a long-term culture of the cells by serial passaging at a high expansion rate (14-fold in four days) while retaining its pluripotency. Therefore, our culture system could be used for large-scale expansion of human pluripotent stem cells for use in regenerative medicine.

OCT4 activity during conversion of human intermediately reprogrammed stem cells to iPSCs through mesenchymal-epithelial transition.

To facilitate understanding the mechanisms of somatic reprogramming to human induced pluripotent stem cells (iPSCs), we have established intermediately reprogrammed stem cells (iRSCs), human mesenchymal cells that express exogenous Oct4, Sox2, Klf4 and c-Myc (OSKM) and endogenous SOX2 and NANOG. iRSCs can be stably maintained at low density. At high density, however, they are induced to enter mesenchymal-epithelial transition (MET), resulting in reprogramming to an iPSC state. Morphological changes through MET correlate with silencing of exogenous OSKM, and upregulation of endogenous OCT4. A CRISPR/Cas9-mediated GFP knock-in visualized the temporal regulation of endogenous OCT4 in cells converting from iRSC to iPSC state. OCT4 activation coincident with silencing of OSKM occurred prior to entering MET. Notably, OCT4 instability was frequently observed in cells of developing post-MET colonies until a late stage (>200 cells), demonstrating that OCT4-activated post-MET cells switched from asymmetric to symmetric cell division in late stage reprogramming.

Self-renewal and pluripotency acquired through somatic reprogramming to human cancer stem cells.

Human induced pluripotent stem cells (iPSCs) are reprogrammed by transient expression of transcription factors in somatic cells. Approximately 1% of somatic cells can be reprogrammed into iPSCs, while the remaining somatic cells are differentially reprogrammed. Here, we established induced pluripotent cancer stem-like cells (iCSCs) as self-renewing pluripotent cell clones. Stable iCSC lines were established from unstable induced epithelial stem cell (iESC) lines through re-plating followed by embryoid body formation and serial transplantation. iCSCs shared the expression of pluripotent marker genes with iPSCs, except for REX1 and LIN28, while exhibited the expression of somatic marker genes EMP1 and PPARγ. iESCs and iCSCs could generate teratomas with high efficiency by implantation into immunodeficient mice. The second iCSCs isolated from dissociated cells of teratoma from the first iCSCs were stably maintained, showing a gene expression profile similar to the first iCSCs. In the first and second iCSCs, transgene-derived Oct4, Sox2, Klf4, and c-Myc were expressed. Comparative global gene expression analyses demonstrated that the first iCSCs were similar to iESCs, and clearly different from human iPSCs and somatic cells. In iCSCs, gene expression kinetics of the core pluripotency factor and the Myc-related factor were pluripotent type, whereas the polycomb complex factor was somatic type. These findings indicate that pluripotent tumorigenicity can be conferred on somatic cells through up-regulation of the core pluripotency and Myc-related factors, prior to establishment of the iPSC molecular network by full reprogramming through down-regulation of the polycomb complex factor.

Cure of ADPKD by selection for spontaneous genetic repair events in Pkd1-mutated iPS cells.

Induced pluripotent stem cells (iPSCs) generated by epigenetic reprogramming of personal somatic cells have limited therapeutic capacity for patients suffering from genetic disorders. Here we demonstrate restoration of a genomic mutation heterozygous for Pkd1 (polycystic kidney disease 1) deletion (Pkd1(+/-) to Pkd1(+/R+)) by spontaneous mitotic recombination. Notably, recombination between homologous chromosomes occurred at a frequency of 1~2 per 10,000 iPSCs. Southern blot hybridization and genomic PCR analyses demonstrated that the genotype of the mutation-restored iPSCs was indistinguishable from that of the wild-type cells. Importantly, the frequency of cyst generation in kidneys of adult chimeric mice containing Pkd1(+/R+) iPSCs was significantly lower than that of adult chimeric mice with parental Pkd1(+/-) iPSCs, and indistinguishable from that of wild-type mice. This repair step could be directly incorporated into iPSC development programmes prior to cell transplantation, offering an invaluable step forward for patients carrying a wide range of genetic disorders.

Human and mouse induced pluripotent stem cells are differentially reprogrammed in response to kinase inhibitors.

Conventional human induced pluripotent stem cells (hiPSCs), reprogrammed from somatic cells by induced expression of Oct4, Sox2, Klf4, and c-Myc, are phenotypically different from mouse embryonic stem cells (ESCs). In mice, culture in N2B27 serum-free 2i media (mitogen-activated protein kinase/extracellular signal-regulated kinase and glycogen synthase kinase 3 inhibitors; PD0325901 and CHIR99021) plus leukemia inhibitory factor (LIF) (2i+LIF medium) enriches for germline competent ESCs. Here, we demonstrate that flat-shaped hiPSC colonies can be reprogrammed into bowl-shaped multi-potent stem cells (2i-hiPSCs) by using 2i+LIF medium. Mechanical dissociation of 2i-hiPSC colonies enables stable maintenance for >20 passages. Importantly, gene expression profiling demonstrated that 2i-hiPSCs more closely resemble primitive neural stem cells (PNSCs). Notably, this 2i-induced phenotype was generated from conventional hiPSCs, but not human ESCs (hESCs), thus correlating with the observation of neuroectodermal SOX1-positive colonies in conventional hiPSCs, but not hESCs in 2i+LIF medium. Thus, 2i-hiPSCs, which are nonteratoma forming PNSCs, may represent a safe source of cells for neural research and regenerative medicine.

Sox2 expression effects on direct reprogramming efficiency as determined by alternative somatic cell fate.

Induced pluripotent stem cells (iPSCs) are generated by directly reprogramming somatic cells by forcing them to express the exogenous transcription factors, Oct4, Sox2, Klf4 and c-Myc (OSKM). These cells could potentially be used in clinical applications and basic research. Here, we explored the molecular role of Sox2 by generating iPSCs that expressed Sox2 at various levels. Low Sox2 (LS) expression increased the efficiency of generating partially reprogrammed iPSCs in combination with OKM. Notably, we detected a significant increase in the number of fully reprogrammed iPSCs with three factors of OK and LS. LS expression was linked with the reduced expression of ectoderm and mesoderm marker genes. This indicates that cell differentiation into the ectoderm and mesoderm lineages was impeded during reprogramming. The quality of the iPSCs that was generated by using OK and LS was comparable to that of iPSCs that were produced via conventional OSK as seen by pluripotent marker gene expression and chimera formation. We conclude that Sox2 plays a crucial role in a dose-dependent manner in direct reprogramming of somatic cells to iPSCs.

Efficient reprogramming of human and mouse primary extra-embryonic cells to pluripotent stem cells.

Practical clinical applications for current induced pluripotent stem cell (iPSC) technologies are hindered by very low generation efficiencies. Here, we demonstrate that newborn human (h) and mouse (m) extra-embryonic amnion (AM) and yolk-sac (YS) cells, in which endogenous KLF4/Klf4, c-MYC/c-Myc and RONIN/Ronin are expressed, can be reprogrammed to hiPSCs and miPSCs with efficiencies for AM cells of 0.02% and 0.1%, respectively. Both hiPSC and miPSCs are indistinguishable from embryonic stem cells in colony morphology, expression of pluripotency markers, global gene expression profile, DNA methylation status of OCT4 and NANOG, teratoma formation and, in the case of miPSCs, generation of germline transmissible chimeric mice. As copious amounts of human AM cells can be collected without invasion, and stored long term by conventional means without requirement for in vitro culture, they represent an ideal source for cell banking and subsequent 'on demand' generation of hiPSCs for personal regenerative and pharmaceutical applications.

Molecular evidence for the presence of new Babesia species in feral raccoons (Procyon lotor) in Hokkaido, Japan.

We recently reported that feral raccoons (Procyon lotor) with splenomegaly native to Japan were carriers of a Babesia microti-like parasite identical to that found in the United States, which was likely introduced to Japan from North America via raccoons imported as pets. Thus, we attempted extensive molecular survey for piroplasma infections of feral raccoon with normal spleen in Hokkaido, Japan using nested PCR that target broadly to 18S ribosomal RNA gene (SSU-rDNA) of all the parasites in the genus Babesia, Theileria, Cytauxzoon and B. microti group. Of the 348 raccoon samples analyzed, 9 gave positive signals. Cloning and phylogenetic analysis on SSU-rDNA sequences revealed that six of nine positives were found to be infected with Babesia and the remaining three with previously unreported Sarcocystis. Babesia sequences were further separated into two distantly related groups, those that reside in a novel phylogenetic group were consisted solely of four parasites found in this study, while those which included one identical sequence found in the three of our specimens were assembled together with both Babesia parasites of tick's in Japan and of raccoon's in U.S. These results may indicate that not only a B. microti-like parasite but also at least two yet undescribed Babesia species are being established in their new life cycles in the feral raccoon populations in Japan.