Today, liver transplantation is an established and successful procedure that represents the only curative therapy for many primary and secondary liver diseases that lead to liver cirrhosis and consecutive liver failure in end-stage disease. Despite its therapeutic potential, it remains an unspecific approach that is limited by donor organ shortage. In contrast to heart and kidney diseases, no temporary or long-term artificial substitute or procedure is available for liver disorders. This fact raises the necessity to search for alternative approaches toward the treatment of liver failure. Liver cell carcinoma is one of the leading causes of primary liver disease. Among secondary liver diseases, end-stage heart failure and metastasization of other tumors into the liver have an important role. End-stage heart failure also affects the liver and liver dysfunction affects the heart. Chronic and acute heart failure can lead to cardiac cirrhosis and cardiogenic ischemic hepatitis.1 Up to 45% of patients with primary or secondary liver tumors need extended hepatectomy (greater than five segments) to achieve tumor-negative resection margins.2
The liver has a large capacity for regeneration after resection in patients with liver tumors. However, below a critical level of future liver remnant volume (FLRV), partial hepatectomy is accompanied by a significant increase of postoperative liver failure. The FLRV describes that portion of the liver, which is available after the liver resection and so has not been resected during the hepatectomy. This portion is measured clinically by the computerized tomography volumometry. Portovenous application of adult bone marrow stem cells has been shown to enhance hepatic regeneration in a clinical setting.3 The mechanism for homing of cells to the liver remains speculative. Human unrestricted somatic stem cells (USSC) are a newly discovered cell type with relatively low immunogenicity, which can be isolated from cord blood (CB).
Unrestricted somatic stem cells are multipotent and not pluripotent cells, and there is no expression of OCT4A, NANOG, and SOX2 in any of the CB-derived USSC cell subpopulations.4
As already described in the article of Aktas et al.,5 USSC from CB have much longer telomeres as compared to adult bone marrow mesenchymal stem cell (MSC), but do not express hTert, the catalytic subunit of the telomerase activity—that means they are not self-renewing, but still have a much higher proliferative capability as compared to bone marrow cells. Unrestricted somatic stem cells from CB can be differentiated in vitro and in vivo into the mesodermal, endodermal, and ectodermal lineages.4–6 Unrestricted somatic stem cells provide a supportive cell layer for hematopoietic cells, a feature they share with bone marrow MSC. Unrestricted somatic stem cells can be expanded up to 20 passages; however, in routine they are expanded up to passage 14–16 corresponding to 55-60 population doublings.4,5 Notably, USSCs release a multitude of cytokines.5 These features make USSC suitable candidates for regenerative therapy. When transplanted into an acutely ischemic heart, USSCs significantly improved left ventricular (LV) function and prevented scar formation as well as LV dilation.6 Differentiation, apoptosis, and macrophage mobilization at infarct site were excluded as underlying mechanisms and paracrine effects are most likely to account for the observed effects of USSC treatment in the heart. Nygren et al.7 showed that adult stem cell-derived cardiomyocytes were derived exclusively through cell fusion and not transdifferentiation.
The aim of the current study was to evaluate the impact of transplanted human multipotent CB-derived USSC on liver regeneration in acute ischemia and identify the underlying mechanisms in an ovine model.
Preparation of USSC
Cord blood was collected from human umbilical cord vein. Unrestricted somatic stem cells were isolated and characterized as previously described.4 Collection, processing, and initial characterization of CB units obtained by the José Carreras CB bank in Düsseldorf from its 80 participating collection sites/maternity hospitals (8,000 units per year) is performed routinely. Only approximately 30% of these units are suitable for hematopoietic banking, mainly due to cell number limitations. Therefore, sufficient CB units are available for research purposes, i.e. the donor mother’s consent to use for research if donations are not suitable for banking. In USSC cultures initiated from 772 CB samples, so far only in 43% were USSC colonies observed. On average, there were between 1 and 11 USSC colonies/CB. For the subsequent generation of USSC colonies, 30% good manufacturing practice (GMP)-grade fetal calf serum (FCS), low-glucose Dulbecco’s Modified Eagle Medium (DMEM)/10−7 M dexamethasone was used for the initiation of the cultures. Expansion of USSC was performed in 30% GMP-grade FCS, low-glucose DMEM in a closed system applying cell stacks (Costar Corning). Serum was identified as the most critical factor for generation and expansion, and FCS needs to be carefully preselected to allow for extensive amplification of multipotent USSC. The combination of the SEPAX (SEPAX Cell Processing Device, Sepax GenesisBPS, Hackensack, NJ) procedure together with the cell stack system (1, 2, 5, and 10 layers) allows cell numbers of 1 × 109 USSC to be obtained within 4–5 passages. These USSC products could be cryopreserved and thawed expanded further in clinical grade quality. Unrestricted somatic stem cells used in the experiment here were in passage seven corresponding to 10 population doublings. The quality of the cells was verified by histology (Figure 1). Previous experiments showed that the USSC can be an efficient cell source—after the mentioned population doublings, as far as their ability as progenitor cells and their number is concerned.4–6
Immunophenotyping of USSC
Flow cytometric assessment was performed on a BD FACSCanto flow cytometer using the FACSDIVA software (version 5.0.3., BD Biosciences Pharmingen San Diego, California) for recording and WinMDI 2.8 for analysis. All samples were incubated for 30 min and washed with phosphate-buffered saline for each staining step; for fixation, paraformaldehyde (2% end concentration) was used.
Establishment of a Large Animal Surgery Model for Portovenous Access, Partial Liver Embolization, and Cell Transplantation
During all experiments, the “Principles of laboratory animal care” (NIH Publication No. 86–23, revised 1985) as well as federal guidelines for the use of human tissue and the Animal Welfare Law of Baden Württemberg were followed. All recipient animals were female, 2 years of age, and had a body weight of 65–82 kg.
A preclinical surgical animal model for portovenous embolization was established at our department. In a sheep model (n = 8), animals were put into a left lateral recumbency position. Portal vein cannulation was performed after resection of the 12th rib and accessing the portal vein. Under systemic administration of Heparin, a port access sheath (5 Fr, Cordis Corporation, Miami, FL) was placed into the portal vein. Subsequently the portal venous system was visualized by contrast medium using fluoroscopy (Figure 2A). After identification of the right liver lobe, an angiography catheter was placed into the right portal vein and acute hepatic ischemia was induced by selective injection of microbeads (Contour SE 300–500, Boston Scientific, Ratingen, Germany) until contrast medium stasis was reached (Figure 2B). Subsequently, 40 million USSC were injected into the left portal vein (Figure 2C). The control group (n = 5) received only medium.
Detection of In Vivo Differentiation into Liver
Livers of the sheep were fixed in buffered formalin and embedded in paraffin. Liver sections (2 µm) were dewaxed and incubated for 10 min at 90°C for target retrieval. After blocking endogenous peroxidase by EnVision Blocking Reagent (Dako, Hamburg, Germany), sections were incubated in serum-free protein block (Dako) for 10 min and for 2 hours in Tris-buffered saline containing 0.1% gelatin and the primary Ab antihuman serum albumin (clone HSA-11; 1:100; Sigma, Munich, Germany) or monoclonal antihuman hepatocyte Ab (clone OCH1E5; Dako). After washing, sections were incubated for 30 min with labeled polymer (EnVision System; Dako) and immunoreactivity was visualized by incubation with diaminobenzidine tetrahydrochloride. For microdissection, applying the PALM MicroBeam System immunohistochemistry was performed as described previously with the monoclonal antihuman hepatocyte, clone OCH1E5 Ab except that sections were mounted on foil-laminated glass slides.
Single-Cell Polymerase Chain Reaction Analysis of Fusion/Cell Hybrids
Isolation of single cells of human origin from human and sheep chimera liver tissue sections as well as single cells of ovine origin from chimera liver tissue sections were performed using the PALM MicroBeam System (P.A.L.M. Microlaser Technologies, Munich, Germany). After target cell identification (the chimeric sheep liver slides were previously stained with the human hepatocyte-specific Ab) and dissection from the surrounding tissue by a nitrogen laser beam (laser-manipulated microdissection), another strong laser was used to catapult (laser pressure catapult) the microdissected material directly into a PALM AdhesiveCap (P.A.L.M. Microlaser Technologies). Before polymerase chain reaction (PCR) amplifications, cells were digested by 200 ng/µl proteinase K (Sigma) for 12–16 hours at 56°C before proteinase K was inactivated for 10 min at 99°C. To analyze individual micromanipulated parenchymal liver cells from human liver and human (stained) and ovine (unstained) cells from chimeric sheep liver tissue, genomic fragments of the human VH1, ovine VH7 as well as human TCRV7.2 and ovine T-cell receptor C-DNA genes were amplified with all external primer pairs in a first PCR round. One microliters of aliquots from the first amplification round were then subjected to a second round of fully nested PCR amplification using internal PCR primers. The sequences of the primers used for PCR amplification are given in Table 1. Single-cell PCR amplifications were performed in a 50 µl reaction mix volume containing 1.5 mM MgCl2 (Qiagen, Hilden Germany), 200 µM of each dNTP, 2.5 µM of each primer, 1 × PCR buffer (Qiagen), and 2.5U HotStar Taq Polymerase (Qiagen). Cycling conditions for the first round included a single 2-min denaturation step at 95°C, followed by 34 cycles at 95°C for 1 min, 56°C for 30 seconds, and 72°C for 1 min and a 5-min incubation at 72°C. For the second PCR round, 45 cycles of amplification were used. To rule out interspecies cross-reactivity of the primer pairs used, we also amplified DNA from ovine cells with human-specific primers and vice versa. Amplification products were analyzed by agarose gel electrophoresis.
Unrestricted somatic stem cells express high levels of CD13, CD29, CD44, CD71, CD73, CD105, CD146, CD166, and HLA-ABC. All lines tested were negative for CD31, CD34, CD45, CD56, CD106, AC133 (CD133/1), CD271, and HLA-DR. Fluorescence-activated cell sorting analysis did not reveal any significant differences between the different USSC lines; the immunophenotype is independent of the passages used.4
In Vivo Differentiation of USSC into Hepatic Cells
Sheep livers were taken 1 month after USSC transplantation to examine the human hepatocye development (Figure 3). The specific staining of the human hepatocyte Ab OCH1E5 in a human liver is depicted in Figure 4. In Figure 4, D and E, a different human-specific Ab that recognizes human albumin, monoclonal antibody HAS-11, was applied showing a specific staining for the human liver and no reactivity with the sheep liver. By contrast, the sheep liver transplanted with USSC showed a strong staining with human albumin (Figure 4). Because human albumin is a secreted protein, the majority of cells stained positive, some showed a very strong pattern of albumin distribution, and the ones in the periphery showed a weaker staining.
Liver Cell-Specific Differentiation of USSC Not Caused by Cell Fusion
Single microdissected liver parenchymal cells from chimeric liver tissue were tested for the coexistence of human and ovine genomes in these cells to determine whether the human cells that integrated into the sheep liver parenchyma had acquired an organ-specific differentiated phenotype through cell fusion with indigenous ovine liver parenchymal cells or by liver cell differentiation. This was performed by micromanipulating single liver parenchymal cells from chimeric liver sections, in which either expression of human proteins could be detected or were devoid of human proteins, and analyzing them separately by single-cell PCR. Each cell was transferred into a PALM AdhesiveCap, digested by proteinase K, and subjected to two rounds of nested PCR with 80 pg human DNA as control. A human PCR product was obtained in all control reactions, yet not a single PCR product of sheep origin. Human DNA fragments originating from the human immunoglobulin heavy chain (IGH) or T-cell receptor beta chain (TCRB) loci amd sheep DNA fragments from ovine IGH and TCRD loci were coamplified during the analysis of 80 cells from chimeric tissue prestained with the human hepatocyte-specific Ab OCH1E5. In 35 of these 80 cells derived from paraffin sections, amplification gave rise to a PCR product for human DNA, whereas not a single sheep-specific PCR product could be detected (Table 1). As shown in Table 1, only PCR products of ovine and not of human DNA origin were obtained after coamplification of human and ovine DNA fragments from cells micromanipulated from chimeric tissue that did not stain for human proteins. Neither human nor ovine DNA could be amplified in 16 PCR reactions to which only PCR buffer, but no cells, was added as negative control. At best, if fusion events occur at all under physiologic noninjury conditions they might account only for a very low frequency of the differentiated liver cell-specific phenotype of the USSC-derived hepatocytes that integrated into the chimeric liver tissue.
The formation of new hepatocytes well integrated in an ischemic liver is a promising development in future treatment of liver diseases. The damaged liver is known to express cytokines and chemoattractants, such as stroma-derived factor-1, that are thought to participate in stem cell homing from extrahepatic sources to the liver.8 Wang et al.9 also demonstrated that cell fusion is the main source of bone-marrow-derived hepatocytes and not differentiation. Hepatic engraftment from extrahepatic progenitor cells is accelerated in cases of liver damage when contrasted with noninjured liver tissue.9,10 The data shown here in our study using USSC to analyze fusion versus nonfusion events document a substantial degree of
USSC-differentiated human parenchymal liver cells per slide in the liver. No indication of cell fusion was detectable in the chimeric sheep liver. Our conclusion is that the main mechanism leading to the presence of new hepatocytes with a human phenotype in the ovine liver is in fact the de novo generation of liver cells. We could demonstrate that compared to bone-marrow-derived stem cells USSC are better suited for liver regeneration, because the actual number of healthy hepatocytes can be increased by USSC transplantation. Based on the work of Kluth et al.,4 we could show that USSC naturally, in contrast to BM MSC, do not differentiate toward the adipogenic lineage. Delta-like 1/preadipocyte factor 1 (DLK-1/PREF-1) was identified as the distinguishing transcription factor. Delta-like 1 can play at least two different roles during development.
First, it is an established player for adipogenesis. Second several groups demonstrated DLK-1 expression in the embryo, which marks the growing branches of organs that develop through the process of branching morphogenesis. Delta-like 1 expression can be detected in the cardiac mesoderm during early embryogenesis. Second, with regard to the endodermal differentiation, the fact that DLK-1 is specifically expressed in fetal liver suggests that DLK-1 is implicated in proliferation or differentiation of hepatocytes as shown by Kluth et al.4 In addition to DLK-1, USSC are similar to fetal and adult liver HOX negative; in contrast BMM SC are HOX positive. This in vitro differentiation data already implicate a different function based on the ontology of the cells applied. Moreover, despite the fact that no immunosuppressive regimen was used in our preclinical model, no sign of cellular infiltration as a consequence of possible cellular rejection of the xenografts was visible. To clarify how long cellular rejection can be avoided, further experiments over a longer period of time will be required. In addition, additional tests to evaluate humoral rejection must be performed.
Ghodsizad and Fahy helped with the study concept and provided technical support; Waclawczyk, Liedtke, Berjon, and Barrios helped in acquiring data; Mehrabi helped in interpretation of data; and Karck and Kögler in design.
The authors thank Dirk Mahnkopf, Antje Mittag, and A. Lefort for technical assistance.
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