Induced Endothelial Cell-Integrated Liver Assembloids Promote Hepatic Maturation and Therapeutic Effect on Cholestatic Liver Fibrosis.

The transplantation of pluripotent stem cell (PSC)-derived liver organoids has been studied to solve the current donor shortage. However, the differentiation of unintended cell populations, difficulty in generating multi-lineage organoids, and tumorigenicity of PSC-derived organoids are challenges. However, direct conversion technology has allowed for the generation lineage-restricted induced stem cells from somatic cells bypassing the pluripotent state, thereby eliminating tumorigenic risks. Here, liver assembloids (iHEAs) were generated by integrating induced endothelial cells (iECs) into the liver organoids (iHLOs) generated with induced hepatic stem cells (iHepSCs). Liver assembloids showed enhanced functional maturity compared to iHLOs in vitro and improved therapeutic effects on cholestatic liver fibrosis animals in vivo. Mechanistically, FN1 expressed from iECs led to the upregulation of Itgα5/ß1 and Hnf4α in iHEAs and were correlated to the decreased expression of genes related to hepatic stellate cell activation such as Lox and Spp1 in the cholestatic liver fibrosis animals. In conclusion, our study demonstrates the possibility of generating transplantable iHEAs with directly converted cells, and our results evidence that integrating iECs allows iHEAs to have enhanced hepatic maturation compared to iHLOs.

Synergistic control of mechanics and microarchitecture of 3D bioactive hydrogel platform to promote the regenerative potential of engineered hepatic tissue.

Culturing autologous cells with therapeutic potential derived from a patient within a bioactive scaffold to induce functioning tissue formation is considered the ideal methodology towards realizing patient-specific regenerative medicine. Hydrogels are often employed as the scaffold material for this purpose mainly for their tunable mechanical and diffusional properties as well as presenting cell-responsive moieties. Herein, a two-fold strategy was employed to control the physicomechanical properties and microarchitecture of hydrogels to maximize the efficacy of engineered hepatic tissues. First, a hydrophilic polymeric crosslinker with a tunable degree of reactive functional groups was employed to control the mechanical properties in a wide range while minimizing the change in diffusional properties. Second, photolithography technique was utilized to introduce microchannels into hydrogels to overcome the critical diffusional limit of bulk hydrogels. Encapsulating hepatic progenitor cells derived via direct reprogramming of tissue-harvested fibroblasts, the application of this strategy to control the mechanics, diffusion, and architecture of hydrogels in a combinatorial manner could allow the optimization of their hepatic functions. The regenerative capacity of this engineered hepatic tissue was further demonstrated using an in vivo acute liver injury model.

Direct monitoring of live human pluripotent stem cells by a highly selective pluripotency sensor.

Human pluripotent stem cells (hPSCs) are a useful cell source for regenerative medicine. Despite having a potential of hPSCs for cell-based therapy, there is a need for a selective human pluripotency sensor for monitoring of live hPSCs. Here, we report the discovery of a novel pluripotency sensor (SHI5) from BODIPY-based library by high-throughput cell-based screening and describe the use of SHI5 to identify and isolate human embryonic stem cells and human induced pluripotent stem cells. We demonstrate that SHI5-based assay can be applied to live cells that gain pluripotency in the reprogramming process without any effect on their viability. We also show that SHI5 is internalized through a clathrin-mediated endocytosis pathway. These findings suggest that SHI5 can be an attractive sensor for pluripotency cells during reprogramming. Taken together, SHI5-based screening for hPSCs opens probably unlimited possibilities of detection probe for hPSC therapy via assures their safety issue.

Etv2- and Fli1-Induced Vascular Progenitor Cells Enhance Functional Recovery in Ischemic Vascular Disease Model-Brief Report.

OBJECTIVE: Vascular progenitor cells (VPCs), which are able to differentiate into both endothelial cells and smooth muscle cells, have the potential for treatment of ischemic diseases. Generated by pluripotent stem cells, VPCs carry the risk of tumorigenicity in clinical application. This issue could be resolved by direct lineage conversion, the induction of functional cells from another lineage by using only lineage-restricted transcription factors. Here, we show that induced VPCs (iVPCs) can be generated from fibroblasts by ETS (E-twenty six) transcription factors, Etv2 and Fli1. Approach and Results: Mouse fibroblasts were infected with lentivirus encoding Etv2 and Fli1. Cell colonies appeared in Fli1- and Etv2/Fli1-infected groups and were mechanically picked. The identity of cell colonies was confirmed by proliferation assay and reverse-transcription polymerase chain reaction with vascular markers. Etv2/Fli1- infected cell colonies were sorted by CD144 (also known as CDH5, VE-cadherin). We defined that CD144-positive iVPCs maintained its own population and expanded stably at multiple passages. iVPCs could differentiate into functional endothelial cells and smooth muscle cells by a defined medium. The functionalities of iVPC-derived endothelial cells and smooth muscle cells were confirmed by analyzing LDL (low-density lipoprotein) uptake, carbachol-induced contraction, and tube formation in vitro. Transplantation of iVPCs into the ischemic hindlimb model enhanced blood flow without tumor formation in vivo. Human iVPCs were generated by human ETS transcription factors ETV2 and FLI1. CONCLUSIONS: We demonstrate that ischemic disease curable iVPCs, which have self-renewal and bipotency, can be generated from mouse fibroblasts by enforced ETS family transcription factors, Etv2 and Fli1 expression. Our simple strategy opens insights into stem cell-based ischemic disease therapy.

Oct4 and Hnf4α-induced hepatic stem cells ameliorate chronic liver injury in liver fibrosis model.

Direct conversion from fibroblasts to generate hepatocyte like-cells (iHeps) bypassing the pluripotent state has been described in previous reports as an attractive method acquiring hepatocytes for cell-based therapy. The limited proliferation of iHeps, however, has hampered it uses in cell-based therapy. Since hepatic stem cells (HepSCs) possess self-renewal and bipotency with the capacity to differentiate into both hepatocytes and cholangiocytes, they have therapeutic potential for treating liver disease. Here, we investigated the therapeutic effects of induced HepSCs (iHepSCs) on a carbon tetrachloride (CCl4)-induced liver fibrosis model. We demonstrate that Oct4 and Hnf4a are sufficient to convert fibroblasts into expandable iHepSCs. Hepatocyte-like cells derived from iHepSCs (iHepSC-HEPs) exhibit the typical morphology of hepatocytes and hepatic functions, including glycogen storage, low-density lipoprotein (LDL) uptake, Indocyanine green (ICG) detoxification, drug metabolism, urea production, and albumin secretion. iHepSCs-derived cholangiocyte-like cells (iHepSC-CLCs) expressed cholangiocyte-specific markers and formed cysts and tubule-like structures with apical-basal polarity and secretory function in three-dimensional culture condition. Furthermore, iHepSCs showed anti-inflammatory and anti-fibrotic effects in CCl4-induced liver fibrosis. This study demonstrates that Oct4 and Hnf4α-induced HepSCs show typical hepatic and biliary functionality in vitro. It also presents the therapeutic effect of iHepSCs in liver fibrosis. Therefore, directly converting iHepSCs from somatic cells may facilitate the development of patient-specific cell-based therapy for chronic liver damage.

Oct4-induced oligodendrocyte progenitor cells enhance functional recovery in spinal cord injury model.

The generation of patient-specific oligodendrocyte progenitor cells (OPCs) holds great potential as an expandable cell source for cell replacement therapy as well as drug screening in spinal cord injury or demyelinating diseases. Here, we demonstrate that induced OPCs (iOPCs) can be directly derived from adult mouse fibroblasts by Oct4-mediated direct reprogramming, using anchorage-independent growth to ensure high purity. Homogeneous iOPCs exhibit typical small-bipolar morphology, maintain their self-renewal capacity and OPC marker expression for more than 31 passages, share high similarity in the global gene expression profile to wild-type OPCs, and give rise to mature oligodendrocytes and astrocytes in vitro and in vivo. Notably, transplanted iOPCs contribute to functional recovery in a spinal cord injury (SCI) model without tumor formation. This study provides a simple strategy to generate functional self-renewing iOPCs and yields insights for the in-depth study of demyelination and regenerative medicine.

Possible involvement of Class III phosphatidylinositol-3-kinase in meiotic progression of porcine oocytes beyond germinal vesicle stage.

Phosphatidylinositol-3-kinases (PI3Ks) play pivotal roles in meiotic progression of oocytes from metaphase I to metaphase II stage. Using a Class III-specific inhibitor of PI3K, 3-methyladenine (3MA), this study shows that Class III PI3K may be essential for meiotic progression of porcine oocytes beyond germinal vesicle (GV) stage. Treatment of immature porcine oocytes with 3MA for 22-42 h arrested them at the GV stage, irrespective of the presence or absence of cumulus cells. Furthermore, a significantly high proportion (60.9 ± 13.8%) of 3MA-treated oocytes acquired a nucleolus completely surrounded by a rim of highly condensed chromatin (GV-II stage). The GV-arresting effect of 3MA was, however, completely reversible upon their further culture in the absence of 3MA for 22 h. When cumulus-oophorus-complexes (COCs), arrested at the GV stage for 22 h by 3MA, were further cultured for 22 h in the absence of 3MA, 96.1 ± 1.5% of oocytes reached the MII stage at 42 h of IVM and did not differ from non-treated control oocytes with respect to their ability to fertilize, cleave and form blastocyst (P > 0.05) upon in vitro fertilization (IVF) or parthenogenetic activation (PA). These data suggest that 3MA efficiently blocks and synchronizes the meiotic progression of porcine oocytes at the GV stage without affecting their ooplasmic maturation in terms of post-fertilization/activation in vitro embryonic development. Our data also provide indirect evidence for the likely participation of Class III PI3K in meiotic maturation of porcine oocyte beyond the GV stage.