Decellularized human placenta supports hepatic tissue and allows rescue in acute liver failure
KAKABADZE, Zurab, et al.
Tissue engineering with scaffolds to form transplantable organs is of wide interest.
Decellularized tissues have been tested for this purpose, although supplies of healthy donor tissues, vascular recellularization for perfusion, and tissue homeostasis in engineered organs pose challenges. We hypothesized that decellularized human placenta will be suitable for tissue engineering. The universal availability and unique structures of placenta for accommodating tissue, including presence of embedded vessels, were major attractions. We found decellularized placental vessels were reendothelialized by adjacent native cells and bridged vessel defects in rats. In addition, implantation of liver fragments containing all cell types successfully hepatized placenta with maintenance of albumin and urea synthesis, as well as hepatobiliary transport of 99m Tc-mebrofenin, up to 3 days in vitro. After hepatized placenta containing autologous liver was transplanted into sheep, tissue units were well-perfused and self-assembled. Histological examination indicated transplanted tissue retained hepatic cord structures with characteristic hepatic [...]
KAKABADZE, Zurab, et al. Decellularized human placenta supports hepatic tissue and allows rescue in acute liver failure. Hepatology, 2018, vol. 67, no. 5, p. 1956-1969
DOI : 10.1002/hep.29713 PMID : 29211918
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Decellularized Human Placenta Supports Hepatic Tissue and Allows Rescue in
Acute Liver Failure
Zurab Kakabadze,1Ann Kakabadze,1David Chakhunashvili,1Lia Karalashvili,1Ekaterine Berishvili,1Yogeshwar Sharma,2and Sanjeev Gupta2-7
Tissue engineering with scaffolds to form transplantable organs is of wide interest. Decellularized tissues have been tested for this purpose, although supplies of healthy donor tissues, vascular recellularization for perfusion, and tissue homeostasis in engineered organs pose challenges. We hypothesized that decellularized human placenta will be suitable for tissue engineer- ing. The universal availability and unique structures of placenta for accommodating tissue, including presence of embedded vessels, were major attractions. We found decellularized placental vessels were reendothelialized by adjacent native cells and bridged vessel defects in rats. In addition, implantation of liver fragments containing all cell types successfully hepatized pla- centa with maintenance of albumin and urea synthesis, as well as hepatobiliary transport of99mTc-mebrofenin, up to 3 days in vitro. After hepatized placenta containing autologous liver was transplanted into sheep, tissue units were well-perfused and self-assembled. Histological examination indicated transplanted tissue retained hepatic cord structures with characteristic hepatic organelles, such as gap junctions, and hepatic sinusoids lined by endothelial cells, Kupffer cells, and other cell types.
Hepatocytes in this neo-organ expressed albumin and contained glycogen. Moreover, transplantation of hepatized placenta containing autologous tissue rescued sheep in extended partial hepatectomy-induced acute liver failure. This rescue concerned amelioration of injury and induction of regeneration in native liver. The grafted hepatized placenta was intact with healthy tissue that neither proliferated nor was otherwise altered. Conclusion: The unique anatomic structure and matrix of human placenta were effective for hepatic tissue engineering. This will advance applications ranging from biological studies, drug development, and toxicology to patient therapies. (HEPATOLOGY2018;67:1956-1969).
Tissue engineering to generate organs is relevant for transplantation medicine and for basic studies. Despite advances, major challenges remain in bioartiﬁcial organs recapitulating complex structure–function relationships in tissues.(1) Hepatic engineering is of major interest because of its therapeu- tic potential for numerous genetic and metabolic deﬁ- ciencies or acquired conditions.(2) For instance, treatment of serious hepatic injury or acute liver failure (ALF)(3) often requires orthotopic liver transplanta- tion, but simpler alternatives will be helpful to over- come donor organ shortages.
To survive and express functions appropriately, anchorage-dependent cells require suitable scaffolds in engineered organs.(4,5) In the liver, major hepatic func- tions beneﬁt from interactions between hepatocytes and other cell types, including liver sinusoidal endothelial cells (LSECs), Kupffer cells (KCs), and hepatic stellate cells.(6) Hepatic organization of acini with dual systemic and por- tal blood supply is also central in liver biology, since this affects position-speciﬁc gene expression, mass transfer, and biliary transport.(7)Whereas transplantation of hepa- tocytes with or without other cell types with scaffolds allows their survival over the short term,(8) extracellular
Abbreviations: ALF, acute liver failure; ALT, alanine aminotransferase; CT, computed tomography; DAPI, 40,6-diamidino-2-phenylindole; ECM, extracellular matrix; HBSS, Hanks’ balanced salt solution; H&E, hematoxylin and eosin; KC, Kupffer cell; LSEC, liver sinusoidal endothelial cell;
PBS, phosphate buffered saline; PH, partial hepatectomy.
Received July 13, 2017; accepted December 4, 2017.
Additional Supporting Information may be found atonlinelibrary.wiley.com/doi/10.1002/hep.29713/suppinfo.
Supported in part by National Institutes of Health grants R01-DK071111 and R01-DK088561.
CopyrightVC2017 by the American Association for the Study of Liver Diseases.
View this article online at wileyonlinelibrary.com.
Potential conflict of interest: Nothing to report.
matrix (ECM) components in decellularized tissues offer further support.(4)However, major challenges remain; for example, relining with endothelial cells of decellularized vessels to maintain patency has been problematic.(1)Scal- ing up to large animal models for testing clinical applica- tions has also been a hurdle. The source and state of donor organs pose other issues, because disease and post mortem effects might alter cell survival.
We hypothesized that human placenta offers outstanding opportunities for tissue engineering by serving as a suitable receptacle with multiple other advantages. For instance, healthy placentae are available universally. The placenta is endowed with vascular networks for exchanging arterial and venous blood, ample space for transplanted cells or tissues, and ECM containing beneﬁcial factors associated with vas- cular, mesenchymal, parenchymal, and other cell types.(9) This was eminently testable. Moreover, we considered that instead of engineering tissue by implanting one cell type or lineage at a time, which has been the usual approach, using tissue fragments containing all liver cell types at once could work.(10) Here, we developed these principles to generate
“hepatized placenta,” including characterization of various properties in transplanted liver tissue, followed by studies of its therapeutic potential in a large animal model (sheep), where we used autologous liver from donors for rescue in ALF. These studies established the potential of decellular- ized human placenta for tissue engineering.
Materials and Methods
The Institutional Review Board at Tbilisi State Medical University approved tissue procurement.
Informed consent was obtained. Donors were healthy with uncomplicated full-term pregnancies. Placentae were collected immediately after deliveries following standard clinical care.
The Georgian National Medical Research Institute approved animal use (permit #14-48). Animals received unrestricted access to food and water. Wistar rats (n 530) were 8-10 weeks old, weighed 200-250 g, and were obtained from the breeding facility of Tbi- lisi State Medical University. Ether-based anesthesia was used for rat surgery. Female sheep of a Turkish breed weighing 15-20 kg (n527) were obtained from a certiﬁed facility in Georgia with housing in individ- ual pens. For anesthesia, sheep received 0.03-0.05 mg/
kg atropine intramuscularly and 0.5 mg/kg diazepam intravenously followed by 20 mg/kg ketamine intrave- nously and 5 mg/kg xylazine intramuscularly. For anal- gesia, 1 mg/kg ketoprofen was given intramuscularly immediately after surgery and for 3 days subsequently.
To avoid sepsis, 2 mg/kg gentamicin and ceftriaxone each were given intramuscularly twice daily for 6 days.
Animals were monitored for recovery after anesthesia, normal activities, and bleeding or other complications.
The umbilical artery and veins were cannulated and cleared by irrigating normal saline containing heparin.
Placentae were then stored at2808C. For decellulari- zation, after warming to 48C, placentae were perfused through the artery with phosphate-buffered saline (PBS) (pH 7.4) overnight, followed by 0.01%, 0.1%,
From the1Department of Clinical Anatomy, Tbilisi State Medical University, Tbilisi, Georgia;2Department of Medicine, Albert Einstein College of Medicine, Bronx, NY; 3Department of Pathology, Albert Einstein College of Medicine, Bronx, NY; 4Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY;5Diabetes Center, Albert Einstein College of Medicine, Bronx, NY;
6The Irwin S. and Sylvia Chanin Institute for Cancer Research, Albert Einstein College of Medicine, Bronx, NY; and7Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, Bronx, NY.
ADDRESS CORRESPONDENCE AND REPRINT REQUESTS TO:
Sanjeev Gupta, M.D.
Albert Einstein College of Medicine Ullmann Building, Room 625 1300 Morris Park Avenue Bronx, NY 10461
E-mail: firstname.lastname@example.org Tel.: (718) 430-3309
Zurab Kakabadze, M.D., Ph.D.
Department of Clinical Anatomy Tbilisi State Medical University 0177 Tbilisi, Georgia
and 1% sodium dodecyl sulfate in distilled H2O for 3 consecutive 24-hour periods, respectively, at 1 mL min21. To remove sodium dodecyl sulfate, placentae were irrigated with distilled H2O for 30 minutes, 1%
Triton X-100 for 1 hour, and PBS for 3 hours. Placen- tae were stored in PBS containing penicillin and strep- tomycin for up to 7 days at 48C or after draining PBS indeﬁnitely at 2808C. In some instances, placentae were lyophilized and stored at2808C.
Tissue DNA was extracted (G-spin Kit; iNtRON Biotechnology, Seongnam, Korea) and measured at 260 nm (NanoDrop 1000; ThermoFisher Scientiﬁc, Waltham, MA).
ANATOMY OF DECELLULARIZED PLACENTA
For vascular anatomy, the placental artery and vein were cannulated and irrigated with normal saline to clear blood, and casts were obtained with 40 mL 50%
latex in water (NAIRIT L-3, NAIRIT) or Minium Red oxide of lead (Pb3O41PbO [M2]) in turpentine oil. For angiography, radiocontrast was injected into the placental artery. For electron microscopy, samples were ﬁxed in glutaraldehyde and dried in Tousimis Samdri-780 critical point dryer (Tousimis Research, Rockville, MD), followed by gold sputter-coating of ultrathin sections with a scanning electron microscope (Hitachi, Tokyo, Japan).
DECELLULARIZED VESSELS AND GRAFTING
Placental artery segments were irrigated with water for 12 hours and treated with heparin as described pre- viously.(11) Decellularized arteries were ﬁlled with 1%
protamine sulfate under 120 mm Hg pressure, irri- gated with water for 30 minutes, and soaked in 1000 U/mL heparin for 48 hours. Vessels were sterilized in PBS containing 0.1% peracetic acid (Sigma Chemical Co., St. Louis, MO) for 3 hours. For mechanical strength, 2.5- to 3-cm-long vessel segments of 1.5-2.5 mm diameter were cannulated at either end with poly- propylene catheters secured by 4-0 proline (Ethicon Inc., Somerville, NJ). These segments were immersed in a normal saline bath at 378C, and saline was pumped with increasing pressure into one end until bursting occurred. For the ability of vessels to bridge
arterial defects, the common carotid artery was divided between ligatures in rats. Polyethylene cuffs with han- dles and a 0.7-mm outer diameter and 0.6-mm inner diameter were applied to the two ends of artery accord- ing to the method described by Zou et al.(11) The edges of artery ends were everted over cuffs, and decel- lularized placental vessels were ﬁxed with 9-0 sutures to the cuffs. Rats were euthanized 5, 10, 15, 30, and 40 days after surgery. Angiography used radiocontrast or ﬂuorescein. Formalin-ﬁxed graft sections were examined by immunostaining for desmin, a-smooth muscle actin and von Willebrand factor (Abcam, Cambridge, UK).
HARVESTING AND PROCESSING OF SHEEP LIVER AND
The left lobe of liver (30%, typically 110-120 g) was removed after ligating its pedicle with midline laparot- omy. The resected liver was irrigated through the por- tal vein branch with Hanks’ balanced salt solution (pH 7.4). The liver was fragmented with a mechanical tis- sue chopper for 5-6 minutes at room temperature.
This time was determined in earlier studies. Fragments
>1-2 mm in size were removed by passage through two layers of surgical gauze and ﬁltrate collected in 30 mL of Hanks’ balanced salt solution. Fragment viabil- ity was determined using Trypan blue dye exclusion.
Fragments were ﬁxed in formalin for histology. For hepatization, the placenta was trimmed to size and the tissue integrity was conﬁrmed by irrigating saline. Up to 150-mL liver fragments, 10-15 mL per cotyledon, were injected through the placenta capsule with an 18- to 20-gauge needle. Typically, the time from decellu- larized placenta preparation to use was <30 minutes.
The hepatized placenta was tested by injecting colored latex into the vessels. For albumin secretion, RPMI 1640 medium was infused at 10 mL/min for 3 days.
Aliquots of perfusate were stored daily for albumin content. For urea synthesis, 5 mM ammonium chlo- ride was added to infusate for 1 hour, and perfusate was collected daily over this period. For hepatic trans- port, 3.8 mBq of 99mTc–Bromo-Billaron (Medi- Radiopharma, Budapest, Hungary) was injected into placental artery with dynamic imaging (Gamma- camera Siemens E-cam) using 20% window centered on 140 keV at 5 seconds per frame for 1 minute and 1 minute per frame for 59 minutes. Multiple regions of interest were analyzed.
HEPATIZED PLACENTA IN SHEEP
Sheep were given 100 U heparin intravenously to decrease thrombosis. The right femoral artery and vein were partially side-clamped. The hepatized placenta was implanted subcutaneously in the ilio-inguinal space. After end-to-side anastomoses of placental and femoral arteries and veins with 6-0 proline, the graft was perfused. Vascular ﬂow was examined using Doppler ultrasonography (Logiq7 Ultrasound Machine; GE Electronics, Tbilisi, Georgia) and com- puted tomography (CT) angiography (Innova-3100 Digital Cath & Angio, GE Electronics) with ULTRAVIST-300 intravenously at 3-5 mL/s to 50 mL. Arterial, venous, and renal phases were identiﬁed by established protocols.
PARTIAL HEPATECTOMY MODEL OF ALF IN SHEEP
After midline laparotomy, hepatic lobes were iso- lated and resected, except for portions of the anterior lobes and entire caudate lobe, to constitute 85% partial hepatectomy (PH). Hemostasis and bile leaks were secured by 5-0 sutures or ﬁbrin (Tisseel; Baxter, Hochstadt, Germany). Hepatized placenta was trans- planted after PH was completed while sheep were under anesthesia. Blood samples were collected at intervals. Animals were euthanized with intravenous pentobarbital sodium. At necropsy, liver and graft dimensions were recorded for estimating organ vol- ume, which was converted to weight by reference liver samples containing blood from healthy sheep. Simi- larly, placental graft dimensions were recorded for comparisons in animal groups.
Tissues were ﬁxed in formalin and parafﬁn- embedded sections stained with hematoxylin and eosin or Masson’s trichrome. Glycogen was stained as described previously,(12) except no counterstain was used. Lysosomal acid phosphatase staining was per- formed as described previously.(13) For immunostain- ing, antigen retrieval used citrate buffer for 15 minutes and endogenous peroxidase activity was quenched by 3% hydrogen peroxide in methanol. Sections were blocked with 3% bovine serum albumin in Tris- buffered saline for 45 minutes and incubated with
anti-human antibodies fora-smooth muscle actin, des- min, or von Willebrand factor (Abcam, Cambridge, UK) for 18 hours at 48C followed by EnVision1Sys- tem labeled horseradish peroxidase–conjugated sec- ondary antibody (DAKO) for 30 minutes at room temperature. Tissue sections were developed in 3,30- diaminobenzidine-chromogen substrate mix (DAKO) and hematoxylin counterstain. For DNA damage, sec- tions were treated with 250 ng/mL RNAse for 1 hour at 378C, denatured by 4 M HCl for 7 minutes with neutralization in 50 mM Tris base for 2 minutes, fol- lowed by blocking in 10% goat serum for 1 hour and incubated in 8-oxo-dG antibody overnight at room temperature (Trevigen, Gaithersburg, MD).
Connexin-32 was stained by anti-mouse immunoglob- ulin G (Sigma) for 1 hour at room temperature. Albu- min was stained with polyclonal anti-rat immunoglobulin G (US Biochemical Corp, Cleveland, OH). For Ki-67, tissue sections were blocked in 5%
goat serum, permeabilized by 0.2% Triton X-100 (Sigma) in PBS for 1 hour, and incubated with anti- Ki67 (BD Biosciences, San Jose, CA) overnight at 48C. Detection used Alexa Fluor 647-conjugated goat anti-mouse or Alexa Fluor 488-conjugated donkey anti-sheep immunoglobulin G (Invitrogen, Eugene, OR) for 1 hour at room temperature with 40,6-diami- dino-2-phenylindole (DAPI) counterstain.
Samples were stored for total serum bilirubin, ala- nine aminotransferase (ALT), albumin, and urea. Pro- thrombin time was determined in fresh plasma samples. These were measured in a clinical laboratory.
Data are expressed as the mean 6 standard devia- tion or median (range) as appropriate. Statistical sig- niﬁcances were identiﬁed using the Studentttest, chi- squared test, or log-rank test. P values <0.05 were considered signiﬁcant.
DECELLULARIZED HUMAN PLACENTA ALLOWED
Anatomic studies, including corrosion casts (Sup- porting Fig. S1), revealed ample arterial and venous
networks in placenta and cotyledons. This was impor- tant for tissue perfusion and mass transfer within coty- ledons with implanted liver. After decellularization, vascular structures were intact without gross bulging or weakening (Fig. 1A). The DNA content after decellu- larization was <1% of intact placenta (4.6 lg DNA/
mg dry weight). Nucleated cells were removed and stroma was well-preserved (Fig. 1B, 1C). Electron microscopy veriﬁed empty vascular and luminal spaces with ECM protrusions (Fig. 1D). Moreover, angiog- raphy revealed major and branching placental vessels leading into cotyledons were intact and ﬁlled promptly
(Fig. 1E). This entry and exit of contrast from cotyle- dons occurred within seconds (Supporting Video 1).
These parameters were naturally signiﬁcant for tissue engineering. We also examined decellularized placenta after storage in saline at 48C for days or lyophilization and cryopreservation at2808C for months and found these were intact.
To ensure suitability of decellularized vessels, we tested tensile strength to bursting (Supporting Fig.
S2). In matched arterial segments with or without decellularization (n 5 10 each), including storage at 2808C, the mean burst point was 250 kPa versus 210
FIG. 1. Decellularization of human placenta with ECM and vascular structures. (A) Gross appearance of decellularized placenta. (B, C) H&E staining showing removal of cells under 310 magniﬁcation (B). A vessel with adjacent stroma in decellularized placenta under 3100 magniﬁcation (C). (D) Scanning electron microscopy of decellularized placenta indicating empty lumens and spaces (white arrows) along with projections of ECM (black arrows, right panel). Scale bars: left to right, 1 mm, 50lm, and 20lm. (E) Angiography of decellularized placenta with time-lapse images after radiocontrast in placental artery showing progressive ﬁlling of major and minor vessels and capillaries, including cotyledons. Filling of vessels required only seconds with simultaneous exit (Support- ing Video S1).
kPa, respectively (P>0.05). These decellularized ves- sels bridged arterial defects in rats (n515) (Support- ing Fig. S3), where all grafts showed excellent color and blood ﬂow after 0, 3, 5, or 20 days (Supporting Fig. S4). Angiography conﬁrmed graft patency. Simi- larly, decellularized veins bridged jugular vein defects (data not shown). Histology conﬁrmed graft lumens were patent over up to 40 days. Remarkably, grafted vessels contained endothelial cells expressing von Wil- lebrand factor and stromal cells expressing a-smooth muscle actin and desmin migrating from native tissues.
For transplantation of hepatized placenta, we trimmed excess tissue and injected donor liver through the capsule (Supporting Fig. S5). Histology veriﬁed liver fragments contained multiple cell types and
tissues were intact despite passage through needles.
Vessel casts and angiography conﬁrmed transplanted tissue was well perfused (data not shown). After implantation in placenta and perfusion over 3 days in vitro, tissue maintained cord structure with sinusoids and was healthy (Supporting Fig. S5), To determine mass transfer, we perfused hepatized placenta with
99mTc-mebrofenin, which speciﬁcally enters and exits hepatocytes by way of deﬁned transporters—members of solute carrier family and Abcc2, respectively.(14)The tracer was incorporated uniformly over 5-10 minutes in hepatized placenta and cleared progressively over 60 minutes (Supporting Fig. S6). This99mTc-mebrofenin uptake and excretion kinetics reproduced that in ani- mals and people.(10,14) Moreover, it reemphasized
FIG. 2. Hepatized placenta in sheep. (A) Hepatization of pla- centa with autologous sheep liver. The images show the decellularized placenta after trimming. The hepatized pla- centa is shown immediately after injection of tissue frag- ments. The vessel ﬁlling with colored latex highlights the pat- ent arteries (red) and vein (blue) and intact capsule after hepati- zation. The sagittal section of the graft shows the liver tissue along with latex dye inside the graft, indicating perfusion. (B) Histology of grafts after 1, 10, 15, and 45 days in sheep indi- cating transplanted tissue and blood-ﬁlled spaces at early times, followed by tissue reorga- nization at later times. The transplanted liver was within the placental cotyledons contained on the outside by the placental capsule. The transplanted liver was perfused by vessels ordinar- ily supplying blood to cotyle- dons (H&E staining; original magniﬁcation:340).
hepatic tissue perfusion. In placenta hepatized with 10%-20% sheep liver, albumin was secreted in perfus- ate to 2.0 6 0.2 g/L, 3.4 6 0.3 g/L, and 3.0 6 0.8 g/L on days 1, 2, and 3, respectively. In 60-minute samples with ammonium chloride infusion, urea was produced to 3.162.2 mg/L, 3.461.9 mg/L, and 4.8 6 1.4 mg/L urea on days 1, 2, and 3, respectively.
This proof of hepatic transport and synthetic functions coupled with angiographic and radiotracer evidences of graft perfusion set the stage for studies of hepatized placenta in vivo.
HEPATIZED PLACENTA IN SHEEP
To avoid rejection, we used autologous sheep liver.
Decellularized placentae were banked prospectively.
To accommodate grafts in the inguinal region and to avoid the vascular “steal” phenomenon, excess placental tissue was trimmed before anastomosis with femoral vessels (Supporting Fig. S7). We examined hepatized placentae 1, 5-7, 14-15, 20, 30, and 45 days after transplantation (n51-2 each; total510) (Supporting Table S1). No sheep was lost during PH for liver har- vest. In some instances, perfusion of hepatized placen- tae was veriﬁed by casts (Fig. 2A). Latex entered hepatized placentae and appeared in hepatic tissue.
Typically, we transplanted hepatized placenta within 25-60 minutes after PH in sheep. Immediately after
vascular anastomoses, grafts were well-perfused with- out excessive distension or rupture. Subsequently, the graft produced visible inguinal protuberance. Trans- plantation of hepatized placenta caused no morbidity or mortality. Evaluation of grafts after 1, 10, 15, and 45 days showed healthy placental and femoral vessel anastomoses. Histology at 1 day after grafting showed that placental cotyledons and vessels were ﬁlled with blood (Fig. 2B). Subsequently, tissue coalesced to form conﬂuent hepatic sheets with hepatocytes and sinus- oids containing blood and other cells within enclosures of connective tissue stroma from cotyledons and other cell types populated the placental capsule.
Ultrasound imaging and Doppler ultrasonography 1, 5, 10, 15, and 20 days after transplantation veriﬁed blood ﬂow in graft vessels (Supporting Fig. S8). Pla- cental grafts were also evaluated by total body CT and angiography as indicated in images from 20 days after transplantation (Fig. 3). Blood ﬂowed in both arterial and venous phases indicating grafts contained active arterial and venous channels.
More detailed analysis indicated additional vessels from subcutaneous tissue in graft capsule (Fig. 4A).
The graft interior was typically ﬁlled with healthy tis- sue interspersed with blood-ﬁlled vascular spaces (Fig.
4B). Tissue sections showed graft contained conﬂuent hepatic tissue (Fig. 4C). On occasion, less viable areas of transplanted tissue were also noted. Transplanted
FIG. 3. CT angiography of transplanted placental graft in sheep. (A) Hepatized placenta visualized in the inguinal region 20 days after transplantation with ﬁlling of vessels by radio- contrast (arrow). (B) Sequential images in sagittal sections indi- cating vessels ﬁlled in the arterial and venous phases (arrows).
liver in grafts included hepatocytes with lobular organi- zation in portal-like areas such as ductal structures and sinusoids containing blood and other cell types, leading to structures corresponding to central veins (Fig. 4D).
We observed endothelial cells and additional cell types in hepatic vessels and sinusoids. To conﬁrm the
presence of LSECs and KCs, we stained lysosomal acid phosphatase activity, which is a hallmark of these cell types.(13) Such LSECs and KCs with lysosomal acid phosphatase were abundant in transplanted tissue (Fig. 4E). To demonstrate whether transplanted hepa- tocytes maintained cell-to-cell communication for
FIG. 4. Integrity of hepatized placenta in sheep. (A) Transplanted placenta explanted from sheep after 1 month with healthy struc- tures. (B) Sagittal section of explanted placenta with transplanted liver and interspersed vessels. (C, D) H&E staining showing that the placenta wall was reorganized (C, arrow) around the transplanted liver, which contained ductal as well as venous structures lined by endothelial cells (arrowheads) and other cell types (open arrows) (D). Original magniﬁcations: panel C,340; panel D, left, 340;
panel D, middle and right,3400. (E) Staining for lysosomal acid phosphatase as a hallmark of LSECs (top left, arrowheads) and KCs (top left, open arrows), gap junctions in hepatocytes (top right, arrows), albumin in hepatocytes (bottom left, arrow), and glyco- gen in hepatocytes (bottom right, arrows). Nuclei in panels showing gap junctions and albumin were counterstained with DAPI. Orig- inal magniﬁcation:3400.
homeostasis, we localized gap junctions in adjacent cells. Immunostaining for connexin-32, which is spe- ciﬁc to hepatocytes,(15) indicated their presence in
transplanted tissue (Fig. 4E). Moreover, hepatocytes in grafts expressed albumin (Fig. 4E). Also, hepatocytes contained glycogen, which was at higher levels in cells
FIG. 5. Rescue of sheep in ALF with hepatized placenta. (A-D) Mobilization, ligation, and removal of left and right lobes, left and right median lobes, and quadrate lobes in sheep for 85% PH. (E) Survival analysis with no mortality in controls and loss of all animals in group 1 between 2 and 5 days after PH. In group 2, 2 of 7 animals died after 2 or 4 days due to thrombosis of graft vessels (P<
0.05). (F) Total serum bilirubin levels (top left) increased signiﬁcantly in groups 1 and 2, peaking on days 2-3. Serum ALT levels (top right) increased in all groups, peaking on days 2-3. Serum albumin levels (bottom left) decreased in group 1 and group 2. Pro- thrombin time (bottom right) increased by up to 3.5- and 2.6-fold above controls on days 2-3 in groups 1 and 2. In surviving animals, test abnormalities resolved gradually. All data points included 3-7 animals. *P<0.05.
adjacent to vascular spaces compared with ductal areas to suggest position-speciﬁc hepatic function (Fig. 4E).
(7) This unique hepatic organization is not readily found in hepatocytes transplanted into ectopic sites.(8)
Neither bile plugs nor other evidence for cholestasis were observed, suggesting that bile produced in trans- planted tissue was secreted by bile canaliculi into adja- cent sinusoidal blood followed by drainage into
FIG. 6. Tissue studies in sheep with ALF and transplantation of hepatized placenta. (A) Liver morphology with H&E staining was normal in controls after 20 days (inset). In groups 1 and 2 with ALF, hepatic necrosis (inset, long arrow), inﬂammation (inset, closed arrowhead), and apoptosis (inset, open arrowheads) were noted at death. In group 2 with recovery after 20 days, only occasional areas with less healthy hepatocytes were found (inset). 8-oxo-dG adducts were infrequent after 20 days in controls (top), but were wide- spread after death with ALF in groups 1 and 2 (middle), and were again infrequent in group 2 with recovery (bottom) (insets, arrows mark nuclei with adducts in pink). Ki-67 staining (pink) showed cells were rarely positive in healthy controls or group 1 with ALF (arrow in middle panel). By contrast, livers in group 2 contained areas with multiple Ki-67–positive cells (arrow, inset). (B) Placental graft from surviving sheep in group 2. The liver was intact with no evidence of ﬁbrosis on Masson’s trichrome staining or of necrosis, inﬂammation, or apoptosis on H&E staining. Hepatocytes in grafts were healthy, and transplanted tissue was perfused as indicated by presence of blood cells, including in sinusoidal spaces (inset, middle panel). Ki-67–positive cells (arrows) were infrequent. Nuclei were counterstained with DAPI. Original magniﬁcation:3400.
systemic circulation. This does not imply that bile ducts had been formed. Taken together, we concluded hepatized placenta engrafted and functioned in sheep over the long-term.
THERAPEUTIC POTENTIAL OF HEPATIZED PLACENTA AND RESCUE OF SHEEP IN ALF
To determine whether hepatized placenta would replace deﬁcient functions, we induced ALF in sheep by 85% PH, leaving behind only the caudate lobe plus portions of anterior lobes (Fig. 5A-5D). The hepatic loss caused progressive physical decline, lethargy, and death after 2-5 days. At necropsy, livers were small, indicating lack of liver regeneration. Then we hepat- ized banked placentae with liver from sheep subjected to 85% PH followed by transplantation into the same sheep within 1-2 hours. Placental grafts contained 20% by volume of liver. Animal groups were as fol- lows: sham controls undergoing laparotomy without PH (n 5 3), test group 1 with 85% PH to induce ALF (n57), and test group 2 with 85% PH plus hep- atized placenta (n57). The placentae were grafted in the inguinal region as described above. All sham- treated controls recovered from surgery uneventfully (Fig. 5E). Group 1 with ALF worsened with lethargy and death in 7 of 7 (100%) animals. However, in group 2 with ALF plus hepatized placenta, 2 of 7 animals were less active and died on days 2 and 4 after PH, but 5 (71%) animals survived, which was superior to the outcome in group 1 (P<0.05). Mortality in two group 2 animals was due to thrombosis of femoral vessel anastomoses and graft necrosis. Blood tests indicated higher serum ALT levels, peaking 2-3 days after sur- gery to 4-, 7-, and 6-fold above normal in controls, group 1 and group 2, respectively (Fig. 5F). Total serum bilirubin was unchanged in controls but increased in groups 1 and 2, peaking to 23- and 19- fold above normal 2-3 days after surgery, respectively.
Serum albumin decreased in group 1 and group 2 to 18-20 g/L versus 30-35 g/L in controls 2-3 days after surgery. Similarly, group 1 and group 2 animals showed coagulation abnormalities peaking on days 2-3 after surgery. In surviving animals, liver tests normal- ized over time.
We euthanized surviving animals 20 days after PH.
For histological evidences of liver injury, we examined nonsurvivors (groups 1 and 2), survivors with hepatized placenta (group 2), and sham-treated controls eutha- nized after 20 days. Liver morphology was normal in
sham-treated controls (Fig. 6A). In groups 1 and 2 with ALF, interspersed hepatic necrosis or apoptosis and ductular reactions with mild biliary hyperplasia were noted, consistent with liver injury. In group 2 sur- vivors, livers were healthy, although with greater KC abundances. We found widespread oxidative adducts on guanosine residues (8-oxo-dG) in group 1 with ALF (Fig. 6A,Supporting Fig. S9). This is a compo- nent of PH-induced DNA damage and ALF.(16) By contrast, 8-oxo-dG adducts were infrequent in con- trols or group 2 following recovery over 20 days. More- over, tissue analysis for regenerative activity showed extremely rare Ki-67–positive cells, 1 per 10-20 micro- scopic ﬁelds (3200) in healthy controls or group 1 with ALF. By contrast, we found 1-10 Ki-67–positive hepatocytes per ﬁeld in group 2 sheep. Also, ductal cells with Ki-67 were present. Additional controls for Ki-67 staining were included (Supporting Fig. S9).
Examination of placental grafts from group 2 veriﬁed that transplanted tissue was intact (Fig. 6B). These liver grafts were nonﬁbrotic with Masson’s trichrome and were without inﬂammation, necrosis, or apoptosis in hematoxylin and eosin (H&E)-stained sections.
Transplanted hepatocytes were healthy and hepatic sinusoids contained blood to indicate perfusion. In grafts, Ki-67 was expressed rarely (1-2 cells per 10-20 ﬁelds). This ﬁnding suggests that the native liver but not tissue in placental graft was regenerating in ALF.
Based on liver dimensions, we estimated a healthy sheep liver to be 877 6 216 g (n 5 3). The livers of group 1 sheep at death were 114655 g (n57), rep- resenting 13% 6 6% of healthy controls (P < 0.05).
By contrast, after 20 days, the livers of group 2 sheep had recovered to 370678 g (n55), which was 3.2- fold greater than those in group 1, but was still only 42%68% of healthy controls (P<0.05). The placen- tal grafts in group 2 sheep weighed 163 6 12 g and 19368 g at transplantation and after sacriﬁce, respec- tively, which was 18%-19% of healthy liver mass and increased minimally compared with native livers in group 2.
These studies established that human placenta offers suitable anatomical structure, vascular access, and ECM components for tissue engineering. Mechani- cally strong vessels and capillary networks in placenta ensured excellent tissue perfusion. Blood entered pla- cental cotyledons rapidly via arterioles and exited
equally rapidly from venules, which was veriﬁed by angiography. Latex casting indicated that after hepati- zation, vascular ﬂow into cotyledons was maintained.
Histology also veriﬁed the presence of blood within cotyledons and sinusoids of hepatic tissue in placenta.
Radiotracer studies with 99m Tc-mebrofenin veriﬁed entry and exit of the tracer from cotyledons containing hepatic tissue. Radiological studies, including Doppler ultrasonography and CT veriﬁed blood ﬂow in sheep.
Moreover, ECM in vessels allowed migration of native endothelial and stromal cells during graft engraftment, which was critical for graft perfusion. These properties in hepatized placenta were highly signiﬁcant for treat- ing ALF. Because applications of decellularized organs have been restricted by major challenges, including thrombosis of damaged vessels with platelet aggrega- tion, leading to only short-term tissue survival over hours or days,(17) these large animal studies will be quite important.
The universal availability of human placenta would allow use of this otherwise discarded material for trans- lational and clinical applications. Successful determina- tion of metabolic and synthetic functions in placenta grafts will be useful as tools for drug testing or toxico- logical studies in vitro. This should beneﬁt from the availability of multiple liver cell types from the outset.
Moreover, individual cotyledons could be dissected for obtaining multiple units for such applications. These possibilities will be best realized by further studies with human tissues.
As indicated by our experience, decellularized pla- centae may be banked, including after lyophilization, and prepared with tissue grafts rapidly for use at short notice. The decellularization procedure itself is simple, requiring only common chemicals and resources. The high mechanical strength of decellularized placental vessels and their capacity to bridge arterial and venous defects indicates additional potential for vascular reconstruction or bypasses (e.g., in coronary or periph- eral arterial diseases), which may be of further interest for tissue engineering.(18)The migration of endothelial and other cell types in decellularized placental vessels suggested that necessary factors were immobilized in ECM. Similarly, the capsule of decellularized placenta, as well as its interior containing transplanted liver tis- sue, was revascularized by migrating cells. Previously, placental ECM was found to contain collagen, elastin and glycosaminoglycans, along with numerous cyto- kines, chemokines and growth factors, including con- tributors to chemotaxis (e.g., GCP, SDF-1), and angiogenesis or vasculogenesis (i.e., VEGF, HGF,
EGF, FGFs, PDGF, TGF-beta).(19) Interestingly, some of these substances also exert hepatotrophic effects, as in cases of VEGF, HGF, FGFs, EGF, IGF-1, IGFBPs, and so forth. Therefore, such proper- ties in placental ECM likely beneﬁted hepatic tissue engineering. Moreover, the placenta serves unique roles in other major biological processes, such as immunological mechanisms,(9)which should have fur- ther beneﬁts for tissue engineering.
We considered self-assembly of transplanted liver fragments in decellularized placenta to be important because this simultaneously provided multiple cell types in the graft. Introducing one cell type at a time into decellularized organs has been a general limitation for graft vascularization and survival.(1)Here, reorgani- zation of transplanted liver tissue was likely fostered by various cell types, including biliary cells, LSECs, and KCs. The multidimensional structure of liver frag- ments should have conferred advantages for tissue homeostasis through superior cell-to-cell interactions and access to autocrine or paracrine signals.(6)Another aspect of tissue homeostasis involves metabolic inter- mediates that contribute in gene expression regula- tion.(7) Similarly, gene expression is regulated by local cytokine levels; for example, networks of inﬂammation-associated cytokines, chemokines, and receptors under control of tumor necrosis factora had major effects on survival of transplanted hepato- cytes.(20)The transplanted liver tissue was morphologi- cally intact in placenta with hepatocytes expressing metabolic functions ex vivo or in vivo (i.e., glycogen storage, albumin secretion, urea synthesis, and hepato- biliary transport). Although we observed interspersed bile duct structures in hepatized placenta, bile was excreted in blood for disposal by the native liver as determined in other ectopic sites with99mTc-mebrofe- nin,(10)as also found here. These aspects will be help- ful for drug testing or toxicology models where multiple liver cell types will be particularly valuable.
Our studies in sheep yielded a large animal model of ALF, where autologous tissue was convenient for dem- onstrating rescue with hepatized placenta by avoiding complexities due to rejection in allogeneic transplants.
The capacity of transplanted tissue for ammonia- ﬁxation, as shown by urea synthesis in our studies, would have contributed in treating metabolic encepha- lopathy, whereas provision of hepatic support should have allowed liver regeneration in sheep with ALF.
The substantial hepatic DNA damage in sheep with ALF and regression of this damage during recovery in recipients of hepatized placenta were in agreement
with such beneﬁts for liver regeneration. Moreover, native liver regenerated after placental grafts, as indi- cated by Ki-67–positive cells, which were rare in grafted liver tissue. The native liver was also larger in animals receiving placental grafts and approached nearly 40%-50% of the size of healthy control liver. On the other hand, placental grafts enlarged only slightly during this period. Taken together, we concluded that native liver regeneration was advanced by grafted liver possibly by hepatic support and also paracrine signals from transplanted tissue. Although the nature of puta- tive paracrine factors supporting liver regeneration is unknown, it should be noteworthy that transplantation of mature hepatocytes or of pluripotent stem cell- derived hepatocytes in the peritoneal cavity also pro- moted liver regeneration in animals with ALF through metabolic support plus paracrine factor release, includ- ing with similar regression of hepatic DNA damage.(8,21)
The successful application of hepatized placenta in ALF will be appropriate because this may allow bridg- ing to orthotopic liver transplantation or time for regeneration of the native liver to avoid orthotopic liver transplantation altogether. The incorporation of multi- ple liver cell types in hepatized placenta should be of interest for studies of pathophysiological mechanisms or development of therapies in other conditions. For instance, LSECs constitute the major source of FVIII, which is deﬁcient in hemophilia A, and transplantation of healthy LSECs was sufﬁcient for curing hemo- philia.(22) Moreover, KCs also contributed in FVIII expression.(23) It should be noteworthy that sheep modeling hemophilia A have been available for testing the therapeutic beneﬁts of alternative approaches.(24) Also, transplantation of healthy tissue in placental grafts could be useful for enzymatic deﬁciency states (e.g., Crigler-Najjar syndrome).(2) Although these and other enzyme deﬁciency states are amenable to in vivo gene therapy, inadvertent systemic effects of gene transfer vectors could potentially be avoided by this approach. The procedure could be reversed and trans- planted tissue removed for further safety, of course.
Similarly, transplantation of pancreatic islets into the portal vein is of value for type-1 diabetes mellitus, but superior locations for transplanting islets where these could be revascularized are needed.(25) Whether pan- creatic islets could be combined with hepatization of placenta could be another possibility. The potential of cells derived from pluripotent or other stem cells for repopulating decellularized placenta(26) should also be of interest.
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Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep.29713/suppinfo.