Liver specification of human iPSC-derived endothelial cells transplanted into mouse liver.

Background & Aims: Liver sinusoidal endothelial cells (LSECs) are important in liver development, regeneration, and pathophysiology, but the differentiation process underlying their tissue-specific phenotype is poorly understood and difficult to study because primary human cells are scarce. The aim of this study was to use human induced pluripotent stem cell (hiPSC)-derived LSEC-like cells to investigate the differentiation process of LSECs. Methods: hiPSC-derived endothelial cells (iECs) were transplanted into the livers of Fah-/-/Rag2-/-/Il2rg-/- mice and assessed over a 12-week period. Lineage tracing, immunofluorescence, flow cytometry, plasma human factor VIII measurement, and bulk and single cell transcriptomic analysis were used to assess the molecular and functional changes that occurred following transplantation. Results: Progressive and long-term repopulation of the liver vasculature occurred as iECs expanded along the sinusoids between hepatocytes and increasingly produced human factor VIII, indicating differentiation into LSEC-like cells. To chart the developmental profile associated with LSEC specification, the bulk transcriptomes of transplanted cells between 1 and 12 weeks after transplantation were compared against primary human adult LSECs. This demonstrated a chronological increase in LSEC markers, LSEC differentiation pathways, and zonation. Bulk transcriptome analysis suggested that the transcription factors NOTCH1, GATA4, and FOS have a central role in LSEC specification, interacting with a network of 27 transcription factors. Novel markers associated with this process included EMCN and CLEC14A. Additionally, single cell transcriptomic analysis demonstrated that transplanted iECs at 4 weeks contained zonal subpopulations with a region-specific phenotype. Conclusions: Collectively, this study confirms that hiPSCs can adopt LSEC-like features and provides insight into LSEC specification. This humanised xenograft system can be applied to further interrogate LSEC developmental biology and pathophysiology, bypassing current logistical obstacles associated with primary human LSECs. Impact and implications: Liver sinusoidal endothelial cells (LSECs) are important cells for liver biology, but better model systems are required to study them. We present a pluripotent stem cell xenografting model that produces human LSEC-like cells. A detailed and longitudinal transcriptomic analysis of the development of LSEC-like cells is included, which will guide future studies to interrogate LSEC biology and produce LSEC-like cells that could be used for regenerative medicine.

Engineering transplantable human lymphatic and blood capillary networks in a porous scaffold.

Due to a relative paucity of studies on human lymphatic assembly in vitro and subsequent in vivo transplantation, capillary formation and survival of primary human lymphatic (hLEC) and blood endothelial cells (hBEC) ± primary human vascular smooth muscle cells (hvSMC) were evaluated and compared in vitro and in vivo. hLEC ± hvSMC or hBEC ± hvSMC were seeded in a 3D porous scaffold in vitro, and capillary percent vascular volume (PVV) and vascular density (VD)/mm2 assessed. Scaffolds were also transplanted into a sub-cutaneous rat wound with morphology/morphometry assessment. Initially hBEC formed a larger vessel network in vitro than hLEC, with interconnected capillaries evident at 2 days. Interconnected lymphatic capillaries were slower (3 days) to assemble. hLEC capillaries demonstrated a significant overall increase in PVV (p = 0.0083) and VD (p = 0.0039) in vitro when co-cultured with hvSMC. A similar increase did not occur for hBEC + hvSMC in vitro, but hBEC + hvSMC in vivo significantly increased PVV (p = 0.0035) and VD (p = 0.0087). Morphology/morphometry established that hLEC vessels maintained distinct cell markers, and demonstrated significantly increased individual vessel and network size, and longer survival than hBEC capillaries in vivo, and established inosculation with rat lymphatics, with evidence of lymphatic function. The porous polyurethane scaffold provided advantages to capillary network formation due to its large (300-600 µm diameter) interconnected pores, and sufficient stability to ensure successful surgical transplantation in vivo. Given their successful survival and function in vivo within the porous scaffold, in vitro assembled hLEC networks using this method are potentially applicable to clinical scenarios requiring replacement of dysfunctional or absent lymphatic networks.

Liver sinusoidal endothelial cells promote the differentiation and survival of mouse vascularised hepatobiliary organoids.

The structural and physiological complexity of currently available liver organoids is limited, thereby reducing their relevance for drug studies, disease modelling, and regenerative therapy. In this study we combined mouse liver progenitor cells (LPCs) with mouse liver sinusoidal endothelial cells (LSECs) to generate hepatobiliary organoids with liver-specific vasculature. Organoids consisting of 5x103 cells were created from either LPCs, or a 1:1 combination of LPC/LSECs. LPC organoids demonstrated mild hepatobiliary differentiation in vitro with minimal morphological change; in contrast LPC/LSEC organoids developed clusters of polygonal hepatocyte-like cells and biliary ducts over a 7 day period. Hepatic (albumin, CPS1, CYP3A11) and biliary (GGT1) genes were significantly upregulated in LPC/LSEC organoids compared to LPC organoids over 7 days, as was albumin secretion. LPC/LSEC organoids also had significantly higher in vitro viability compared to LPC organoids. LPC and LPC/LSEC organoids were transplanted into vascularised chambers created in Fah-/-/Rag2-/-/Il2rg-/- mice (50 LPC organoids, containing 2.5x105 LPCs, and 100 LPC/LSEC organoids, containing 2.5x105 LPCs). At 2 weeks, minimal LPCs survived in chambers with LPC organoids, but robust hepatobiliary ductular tissue was present in LPC/LSEC organoids. Morphometric analysis demonstrated a 115-fold increase in HNF4α+ cells in LPC/LSEC organoid chambers (17.26 ± 4.34 cells/mm2 vs 0.15 ± 0.15 cells/mm2, p = 0.018), and 42-fold increase in Sox9+ cells in LPC/LSEC organoid chambers (28.29 ± 6.05 cells/mm2 vs 0.67 ± 0.67 cells/mm2, p = 0.011). This study presents a novel method to develop vascularised hepatobiliary organoids, with both in vitro and in vivo results confirming that incorporating LSECs with LPCs into organoids significantly increases the differentiation of hepatobiliary tissue within organoids and their survival post-transplantation.

Bio-engineering a tissue flap utilizing a porous scaffold incorporating a human induced pluripotent stem cell-derived endothelial cell capillary network connected to a vascular pedicle.

Tissue flaps are used to cover large/poorly healing wounds, but involve complex surgery and donor site morbidity. In this study a tissue flap is assembled using the mammalian body as a bioreactor to functionally connect an artery and vein to a human capillary network assembled from induced pluripotent stem cell-derived endothelial cells (hiPSC ECs). In vitro: Porous NovoSorb™ scaffolds (3 mm × 1.35 mm) were seeded with 200,000 hiPSC ECs ±â€¯100,000 human vascular smooth muscle cells (hvSMC), and cultured for 1-3 days, with capillaries formed by 24 h which were CD31+, VE-Cadherin+, EphB4+, VEGFR2+ and Ki67+, whilst hvSMCs (calponin+) attached abluminally. In vivo: In SCID mice, bi-lateral epigastric vascular pedicles were isolated in a silicone chamber for a 3 week 'delay period' for pedicle capillary sprouting, then reopened, and two hiPSC EC ±â€¯hvSMCs seeded scaffolds transplanted over the pedicle. The chamber was either resealed (Group 1), or removed and surrounding tissue secured around the pedicle + scaffolds (Group 2), for 1 or 2 weeks. Human capillaries survived in vivo and were CD31+, VE-Cadherin+ and VEGFR2+. Human vSMCs remained attached, and host mesenchymal cells also attached abluminally. Systemically injected FITC-dextran present in human capillary lumens indicated inosculation to host capillaries. Human iPSC EC capillary morphometric parameters at one week in vivo were equal to or higher than the same parameters measured in human abdominal skin. This 'proof of concept' study has demonstrated that bio-engineering an autologous human tissue flap based on hiPSC EC could minimize the use of donor flaps and has potential applications for complex wound coverage. STATEMENT OF SIGNIFICANCE: Tissue flaps, used for surgical reconstruction of wounds, require complex surgery, often associated with morbidity. Bio-engineering a simpler alternative, we assembled a human induced pluripotent stem cell derived endothelial cell (hiPSC ECs) capillary network in a porous scaffold in vitro, which when transplanted over a mouse vascular pedicle in vivo formed a functional tissue flap with mouse blood flow in the human capillaries. Therefore it is feasible to form an autologous tissue flap derived from a hiPSC EC capillary network assembled in vitro, and functionally connect to a vascular pedicle in vivo that could be utilized in complex wound repair for chronic or acute wounds.

The Vascularised Chamber as an In Vivo Bioreactor.

Vascularisation is key to developing large transplantable tissue constructs capable of providing therapeutic benefits. The vascularised tissue engineering chamber originates from surgical concepts in tissue prefabrication and microsurgery. It serves as an in vivo bioreactor in the form of a closed, protected space surgically created and embedded within the body by fitting a noncollapsible chamber around major blood vessels. This creates a highly angiogenic environment which facilitates the engraftment and survival of transplanted cells and tissue constructs. This article outlines the chamber concept and explores its application in the context of recent advances in biomedical engineering, and how this can play a role in the future of cell therapies and regenerative medicine.

Making cancer visible--Dyes in surgical oncology.

Dyes share an intricate relationship with oncology. Dyes can cause cancer as chemical carcinogens, but can also be harnessed against cancer when used as diagnostic and therapeutic agents. Histopathology, imaging, and newer molecular diagnostics all rely on dyes, and their use in sentinel lymph node biopsies and intra-operative imaging has helped drive a paradigm shift in cancer surgery towards minimally-invasive and organ sparing approaches with enhanced resection accuracy. As therapeutic agents, the cytotoxicity of specific dyes can be employed in direct chemo-ablation or in photodynamic therapy. The same agent can have dual functionalities in cancer detection and treatment, in a novel field known as theranostics. This is facilitated by newer generation dyes conjugated with tumour-targeting probes such as antibodies, and these bio-conjugate agents can also incorporate nanotechnology or radio-isotopes. Further advances will be closely aligned with our increasing understanding of molecular oncology, and will form a new generation of cancer detection and treatment agents that promote precision medicine for cancer. Dyes and their roles have evolved and been reinvented, but they remain relevant as ever. This review explores the fascinating history of dyes, and their place in the state-of-the-art of oncology.

Enteric neural progenitors are more efficient than brain-derived progenitors at generating neurons in the colon.

Gut motility disorders can result from an absent, damaged, or dysfunctional enteric nervous system (ENS). Cell therapy is an exciting prospect to treat these enteric neuropathies and restore gut motility. Previous studies have examined a variety of sources of stem/progenitor cells, but the ability of different sources of cells to generate enteric neurons has not been directly compared. It is important to identify the source of stem/progenitor cells that is best at colonizing the bowel and generating neurons following transplantation. The aim of this study was to compare the ability of central nervous system (CNS) progenitors and ENS progenitors to colonize the colon and differentiate into neurons. Genetically labeled CNS- and ENS-derived progenitors were cocultured with aneural explants of embryonic mouse colon for 1 or 2.5 wk to assess their migratory, proliferative, and differentiation capacities, and survival, in the embryonic gut environment. Both progenitor cell populations were transplanted in the postnatal colon of mice in vivo for 4 wk before they were analyzed for migration and differentiation using immunohistochemistry. ENS-derived progenitors migrated further than CNS-derived cells in both embryonic and postnatal gut environments. ENS-derived progenitors also gave rise to more neurons than their CNS-derived counterparts. Furthermore, neurons derived from ENS progenitors clustered together in ganglia, whereas CNS-derived neurons were mostly solitary. We conclude that, within the gut environment, ENS-derived progenitors show superior migration, proliferation, and neuronal differentiation compared with CNS progenitors.

Post-operative cognitive dysfunction after knee arthroplasty: a diagnostic dilemma.

Post-operative cognitive dysfunction (POCD) is common in the elderly, and significantly impacts their recovery. We present an unusual diagnostic challenge where a 65-year-old male presented 4-week post-total knee arthroplasty with acute cognitive dysfunction lasting 19 days. Curiously, there were no findings uncovering a specific cause, but during investigation underlying predisposing factors such as depression, mild memory deficits and generalized brain volume loss were identified. The impression after psychogeriatric review was that of an organic brain syndrome with overlay of depression, with a complex presentation as POCD. After escalation of behavioural disturbance, he was commenced on anti-psychotic/depressant, with immediate response. We emphasize the importance of pre-operative evaluation of cognitive function and risk factors in all geriatric patients undergoing elective surgery, and the need for further characterization of POCD, as well as experimental research elucidating the underlying mechanisms to better identify and treat this important post-surgical phenomenon.

Enhanced liver progenitor cell survival and differentiation in vivo by spheroid implantation in a vascularized tissue engineering chamber.

Liver tissue engineering is hampered by poor implanted cell survival due to inadequate vascularization and cell-cell/cell-matrix interactions. Here, we use liver progenitor cell (LPC) spheroids to enhance cell-cell/cell-matrix interactions, with implantation into an angiogenic in vivo mouse chamber. Spheroids were generated in vitro in methylcellulose medium. Day 2 spheroids were optimal for implantation (22,407 +/-645 cells/spheroid), demonstrating maximal proliferation (Ki67 immunolabeling) and minimal apoptosis (caspase-3 immunolabelling). In vivo chambers established bilaterally on epigastric vessels of immunodeficient mice were implanted with equivalent numbers of LPCs as a cell suspension (200,000 cells), or spheroids (9 spheroids). At day 14, a trend of increased LPC survival was observed in spheroid-implanted chambers [pan-cytokeratin (panCK+) cells, p = 0.38, 2.4 fold increase)], with significantly increased differentiation [cytokeratin 18 (CK18+) cells, p < 0.002, 5.1 fold increase)] compared to cell suspension-implanted chambers. At day 45, both measures were significantly increased in spheroid-implanted chambers (panCK, p < 0.006, 16 fold increase) (CK18, p < 0.019, 6 fold increase). Hepatic acini/plates of CK18 + cells expressed hepatocyte nuclear factor 4-α and ß-catenin, indicating ongoing hepatic differentiation. Spheroid cell-delivery significantly increased LPC survival and differentiation compared to conventional cell suspensions. This LPC spheroid/vascularized chamber model has clinical potential to generate three-dimensional vascularized liver tissue for liver replacement.