Lineage restriction of human hepatic stem cells to mature fates is made efficient by tissue-specific biomatrix scaffolds§

Authors


  • Potential conflict of interest: Nothing to report.

  • Author contributions: Y.W. and C.C.: Conception and design, collection and assembly of data, data analyses and interpretation, article writing; M.Y. and P.M.: collection and/or assembly of data and analyses on collagen chemistry; M.R. and R.M.: collection and assembly of data on cultures on ground biomatrix scaffolds; M.J.C.: data analysis on the TEM and SEM studies; V.C. and D.A.: collection of data onbiomatrix scaffolds of biliary tree tissue (supplement); E.W. and C.B.: isolation of human hepatic stem cells and assistance with cultures; D.G.: help with some of the editing of article; L.M.R.: conception and design, assembly of data, data analyses and interpretation, article writing and editing, final approval of article and financial suppor

  • §

    Supported by a grant from the North Carolina Biotechnology Center (NCBC), Zenith Biotech (Guilford, CT), Vesta Therapeutics (Bethesda, MD), and from NIH grants (AA014243, IP30-DK065933), NIDDK Grant (DK34987), and an NCI grant (CA016086). A patent application on biomatrix scaffolds has been submitted. V. Cardinale received salary support with a scholarship from Sapienza University of Rome for studies done at UNC. D. Alvaro is supported by MIUR (Italian Minister of University and Research) grants: PRIN #2007, prot. 2007HPT7BA-003.

Abstract

Current protocols for differentiation of stem cells make use of multiple treatments of soluble signals and/or matrix factors and result typically in partial differentiation to mature cells with under- or overexpression of adult tissue-specific genes. We developed a strategy for rapid and efficient differentiation of stem cells using substrata of biomatrix scaffolds, tissue-specific extracts enriched in extracellular matrix, and associated growth factors and cytokines, in combination with a serum-free, hormonally defined medium (HDM) tailored for the adult cell type of interest. Biomatrix scaffolds were prepared by a novel, four-step perfusion decellularization protocol using conditions designed to keep all collagen types insoluble. The scaffolds maintained native histology, patent vasculatures, and ≈1% of the tissue's proteins but >95% of its collagens, most of the tissue's collagen-associated matrix components, and physiological levels of matrix-bound growth factors and cytokines. Collagens increased from almost undetectable levels to >15% of the scaffold's proteins with the remainder including laminins, fibronectins, elastin, nidogen/entactin, proteoglycans, and matrix-bound cytokines and growth factors in patterns that correlate with histology. Human hepatic stem cells (hHpSCs), seeded onto liver biomatrix scaffolds and in an HDM tailored for adult liver cells, lost stem cell markers and differentiated to mature, functional parenchymal cells in ≈1 week, remaining viable and with stable mature cell phenotypes for more than 8 weeks. Conclusion: Biomatrix scaffolds can be used for biological and pharmaceutical studies of lineage-restricted stem cells, for maintenance of mature cells, and, in the future, for implantable, vascularized engineered tissues or organs. (HEPATOLOGY 2011.)

The ongoing revolution in stem cell research has made possible the identification and isolation of stem cell populations including those from fetal and postnatal tissues.1 The potential of human hepatic stem cells (hHpSCs) and other stem/progenitors for pharmaceutical research, cell-based therapies, and tissue engineering relies on being able to isolate them, propagate them in culture and differentiate them to a functional mature cell fate(s).2 Current methods for differentiation of stem cells involve subjecting cells to a mix of soluble signals and/or extracellular matrix components, and the stem cells must be treated with multiple sets of such signals over weeks of time. The adult fate achieved is typical of only partially differentiated cells with over- or underexpression of specific adult genes.3 Here we demonstrate strategies for rapidly differentiating stem cells using matrix scaffolds that elicit more efficient and reproducible responses.

Extracellular matrix is an extraordinarily complex mixture of molecules that are highly regulated, secreted by, and adjacent to cells on one or more of their surfaces, and long understood to be critical for determining the morphology, growth, and differentiation of attached cells.4, 5 Tissue-specific gene expression in cultured cells is improved by culturing the cells on or embedded in matrix extracts or purified matrix components.6, 7 However, individual matrix components, alone or in combination, are unable to recapitulate a tissue's complex matrix chemistry and architecture. This is related to the fact that the matrix components are in patterns associated with natural tissue zones and with histological structures such as blood vessels. This complexity of the tissue matrix is more readily achieved by matrix extracts of decellularized tissue.8-10 Matrix extracts found useful for ex vivo maintenance of cells include amniotic membrane extracts11; Matrigel, an urea extract of a murine embryonal carcinoma12; extracellular matrix (ECM), a detergent- or NaOH-extract of monolayer cell cultures13,14; and biomatrices, an extract of homogenized tissues.10, 15

More recently, decellularized tissues, prepared by collagenase digestion of a tissue16 or by delipidation followed by distilled water washes,8 have been used to mimic the matrix environment in vivo.17 Even though these protocols result in major losses of some matrix components, the decellularized scaffolds from different tissues or organs, such as small intestinal submucosa (SIS), bladder submucosa matrix (BSM),17, 18 vascular tissue,19 heart,20 airway,21 and liver22 have been used successfully in both preclinical and clinical applications.23

Here we describe a strategy, focused on collagen chemistry, that is ideal for preparing substrata of tissue extracts comprised of tissue-specific matrix components and factors bound to the matrix. The extracts, referred to as biomatrixscaffolds, are potent differentiation substrata especially when used with a serum-free, hormonally defined medium (HDM), tailored for either progenitors, such as Kubota's medium (KM) (24), or for the adult cell type(s) of interest, such as an HDM for mature liver cells.25 In prior studies we established methods for identification and isolation of hHpSCs, human hepatoblasts (hHBs), and committed progenitor subpopulations from livers of all donor ages and identified conditions for their clonogenic expansion.26, 27 In this article we assess the efficacy of biomatrix scaffolds to differentiate hHpSCs to mature fates and to maintain mature parenchymal cells as fully functional for long periods of time.

Abbreviations

AFP, α-fetoprotein; ALB, albumin; ASMA, α-smooth muscle actin; BSM, bladder submucosa matrix; CK, cytokeratin; CS-PG, chondroitin sulfate proteoglycan; CXCR4, chemokine (C-X-C motif) receptor 4; CYP450, cytochrome P450; ECM, extracellular matrix; EpCAM, epithelial cell adhesion molecule; FN, fibronectin; GAGs, glycosaminoglycans; GC, Glisson's capsule; Gly, glycine; HDM, hormonally defined medium; hHB, human hepatoblast; hHpSC, human hepatic stem cell; HS-PG, heparan sulfate proteoglycan; Hyl, hydroxylysine; Hyp, hydroxyproline; KM, Kubota's medium; MACS, magnetically activated cell sorting; MSC, mesenchymal stem cell; PLA2, phospholipase A2; SIS, small intestinal submucosa; SDC, sodium deoxycholate.

Materials and Methods

The details of the methods are given in the Supporting Information online. Here we present only the methods for the preparation of the biomatrix scaffolds.

Procedures for Decellularization.

After anesthesia with ketamine-xylazine, the rat abdominal cavity was opened and a sleevelet with a cannula was inserted into the portal vein to perfuse the entire liver. (1) Perfusion was done with RPMI 1640 for 10 minutes; followed by (2) delipidation with phospholipase A2 (PLA2) combined with a gentle detergent, sodium deoxycholate (SDC) for 30-60 minutes until the tissue becomes transparent, and the effusion becomes clear; (3) perfusion with high salt washes (3.4 M NaCl) until the perfusate is negative for proteins by optical density (OD) at 280 nm; (4) perfusion with nucleases (DNase, RNase) in RPMI 1640 until the perfusate is negative for nucleic acids by OD 260 (see Supporting Fig. S3); (5) Final rinse with RPMI 1640 for 2 hours or more.

The biomatrix scaffolds were quickly frozen on dry ice and frozen sections prepared with a Cryostat, placed onto 24-well cell culture plates, sterilized by gamma irradiation (5000 rads), and rehydrated in medium for 30 minutes before seeding cells. The sections of biomatrix scaffolds covered ≈95% of well surface in the 24-well plate. An alternative method for distributing the biomatrix scaffolds onto culture dishes consisted of pulverizing it to a powder using a freezer mill filled with liquid nitrogen. The powder acquires the consistency of paint at room temperature and can be painted onto any surface, culture dish, or cloth to be used for attaching cells. Details are given in the Supporting Methods.

Results

Biomatrix Scaffolds Are Prepared with a Novel Four-Step Protocol.

Biomatrix scaffolds were prepared using a novel 4-step protocol: (1) gentle delipidation; (2) washes with buffers with salt concentrations at or above 3.4 M, salt concentrations known to maintain the collagens in an insoluble state28; (3) nuclease treatment to eliminate residual nucleic acids; and (4) rinses with a basal medium to eliminate the detergent, salt, and nuclease residues as well as to equilibrate the matrix components with the medium (Fig. 1A).

Figure 1.

Rat liver biomatrix scaffold preparation. (A) Four-step decellularization process comprised of perfusion wash, delipidation with PLA2 and SDC, high salt washes, and nuclease treatment for nucleic acid removal. (B-D) Four stages in the preparation of rat biomatrix scaffold. (B) After perfusion wash with basal medium for 10 minutes the liver becomes pale; (C) during delipidation, the liver becomes partially transparent under GC; (D) final intact scaffold looks transparent at 40 minutes of perfusion; (E) biomatrix scaffold shown at low magnification. (E1) Visualization of scaffold perfused with rhodamine-labeled dextran particles demonstrates progressive flow from large vessels to the fine blood vessel branches along the channels without leakage, indicating patent vasculature in scaffolds (see also the Supporting Video). Corresponding hematoxylin and eosin (H&E) staining of biomatrix scaffold in different stages demonstrated that the histological structures such as blood vessels and lace-like matrix enveloping the parenchyma are preserved, whereas cells are removed. The normal rat hepatic portal triad structure consisting of the portal vein (PV), hepatic artery (HA), and bile duct (BD) (B1); the matrix fibers becoming apparent as the cells are gradually removed during the decellularization process (C1); decellularized portal triad region, compare (B1) with that in (D1); (D1-3) shows that all of the cells removed from the matrix scaffold but mesh structures are preserved such as the blood vessels, GC, and the lace-like matrix that surrounds muralia of parenchymal cells.

The choices of the rinse media or the buffers for the nucleases can be any of a number of options as long as the salt concentration and ionic strength are such as to maintain the collagens and associated matrix components in an insoluble state. The choice of the delipidation method is also critical to be effective and yet should be gentle. We chose a combination of sodium deoxycholate (SDC) and phospholipase A2 (PLA2) to rapidly degrade the phosphoglyceride located on the cytoplasm membrane and mitochondrial membrane into lysolecithin, a powerful surfactant, which can induce necrosis and cytolysis. The reactive formula is shown in the Supporting Fig. S1.

We avoided prolonged exposure of the scaffolds to the enzymes from the disrupted cells during delipidation and the high salt washes because they can greatly decrease the content of elastin and the content of glycosaminoglycans (GAGs) such as heparan sulfates (HS), chondroitin sulfates (CS), dermatan sulfates (DS), and heparins (HP), sites at which cytokines and growth factors bind.29 We used soybean trypsin inhibitor and careful control of the pH (7.5-8.0) and time (30-60 minutes) to limit the activity of the proteases derived from disrupted cells.

We perfused the whole tissue through relevant vasculature (e.g., portal vein in the liver), enabling us to rapidly isolate (within a few hours) a biomatrix scaffold with minimal loss of matrix components. The rapidity of the isolation is due to the initial step with detergent that delipidates the tissue within ≈30-60 minutes (not hours or days as in protocols used by others, see Supporting Table 5). The resulting biomatrix scaffolds are translucent or white (Fig. 1D). Moreover, using this perfusion method we maintained the primary vasculature channels, portal and hepatic vein, and most of the vascular branches in the liver, which increased the decellularization efficiency (Fig. 1E). Fluorescent rhodamine-labeled dextran particles perfused through the biomatrix scaffolds remained within the remnants of the vasculature, demonstrating that they are patent (Fig. 1E1). There is a progressive flow of the dye from large vessels to the fine blood vessel branches along the channels without leakage (demonstrated even more dramatically in the Supporting Video). This fact will be helpful in the future in revascularization of scaffolds as a means of preparing engineered tissues for either three-dimensional culture and/or for implantation ex vivo.

When sectioned, scaffolds retain the histological structure of the original tissue, including the recognizable remnants of major histological entities such as blood vessels, bile ducts, and Glisson's capsule (GC). Compare Fig. 1B1 and 1D1, in which a section of liver tissue is contrasted with that of a biomatrix scaffold. The matrix remnants of the muralia of parenchymal cells consisted of a lace-like network (Fig. 1D1-1D3).

Collagen, Collagen-Associated Proteins, and Bound Cytokines Are Maintained in the Biomatrix Scaffolds.

The amount of collagens in biomatrix scaffolds was evaluated by amino acid analysis by methods used previously.30 Because hydroxyproline (Hyp) is unique to collagens and collagenous proteins, the collagen composition relative to total protein was expressed as residues of Hyp per 1,000 amino acids. The results demonstrated that collagen content increased from almost undetectable levels, i.e., less than 0.2 residues of Hyp/1,000 in liver, to ≈13 residues of Hyp/1,000 in biomatrix scaffolds. This indicates that delipidation and the high salt washes, described above, did not remove collagens, leaving almost all of the collagens in the biomatrix scaffolds. Detection of significant levels of hydroxylysine (Hyl), another collagen-associated amino acid, and higher levels of glycine (Gly) in biomatrix scaffold supports our conclusion that collagen is markedly enriched in biomatrix scaffolds (Fig. 3A; Supporting Fig. S2, Supporting Table 1).

Through immunohistochemical and ultrastructural studies, we were able to identify in the scaffolds all known collagen types found in liver in situ including fibrillar collagens (collagen types I, III, and V, 10-30 nm in diameter for fibrils and 500-3,000 nm for assembled fibers) and beaded filaments (possibly type VI). Those fibers and filaments are present in the subcapsular connective tissue layer lying beneath the mesothelial layer. Although typical structures of basement membranes were not found along the sinusoids from portal triads to central veins, we found collagen type IV and some bound, small fibrils form net-like, porous 3D lattices, serving as scaffolding for the parenchymal cells (Fig. 2). Collagen type I bundles can be viewed as the principal structure of the scaffolds to which other collagen types, glycoproteins, and proteoglycans are attached. In the Space of Disse, we found small bundles of collagen type I and fibers of collagen types III and VI as well as some type V, which is more abundant near portal triads and central veins. Representative immunohistochemistry data are presented in Fig. 3B, and a summary of matrix components and their locations in normal liver tissue versus those in the biomatrix scaffolds are listed in Fig. 4D. Early studies in the development of the protocols for biomatrix scaffold preparation indicated that the bulk of the cytoskeletal components are lost in the washes (data not shown). Still, we assessed the scaffolds by immunohistochemistry for residues of cytoskeletal components and found no evidence for tubulin, desmin, or actin, trace amounts of cytokeratins 18 and 19, and low levels of vimentin scattered throughout the scaffolds.

Figure 2.

Transmission and scanning electron microscopy images of rat liver biomatrix scaffolds. (A) Low magnification of a blood vessel (BV) probably the portal vein, based on the narrow wall (W) thickness compared to the large diameter of the vessel. The undulations or scalloping of the vessel (sometimes linked to the internal elastic lamina of an artery) is here probably a partial collapse of the vessel wall. Collagen Type I fibers (large arrowhead) are numerous and contains cross-sections of individual fibers that do not take up heavy metal stains (white dots, small arrowheads). (A1) Higher magnification of a vessel wall shows basement membrane (large arrow), amorphous elastin (*), and associated elastic fibers, a rare membrane vesicle remnant (small arrowhead), a collagen Type I banded fiber (arrowhead), and small fibrils (small arrows). The small fibrils are probably fibrillin (Type VI collagen) that associates closely and helps organize Type I collagen. (B) High magnification of Type I collagen with 64 nm banding pattern (arrows). (C) Low magnification of a vessel with a thin wall (BV) and the wall of a larger vessel (W). (D) At higher magnification, the large vessel wall (W) is scalloped, consistent with hepatic artery of a portal triad, see (A). Beneath the wall are numerous Type I collagen bundles (large arrow) linked by long branching thin, reticular (Type III) collagen fibrils (small arrows). (E) A large bundle of Type I collagen has characteristic parallel fibers (large arrow) associated with a variety of smaller fibers (arrow) and nodular or beaded fibers (arrowhead). (F) 3D meshwork of large/small fibers interlinked in a plane that forms a boundary such as to a liver sinusoid. (F1) Higher magnification of the meshwork showing a variety of fibers (arrows): Type III collagen (larger diameter straight), elastic fibers, or Type VI collagen.

Figure 3.

Chemical analyses of collagens and expression of ECM components in biomatrix scaffolds. (A) The content of three amino acids, all found in collagens: Hyp, Hyl, and Gly. The numbers represent the residues of each amino acid / 1,000 amino acids. The data indicate the dramatic increase in the collagen content in the decellularization process going from <0.2% in liver to more than 15% in the biomatrix scaffolds; The Hyp content in the whole liver was too low to be reasonably quantified (less than 0.1 nmol in ≈500 nmol of total amino acids, i.e., <0.2 res/1,000). Thus, in Supporting Table 1 the amount is described as 0. (B) Immunohistochemical staining of matrix molecules in biomatrix scaffolds shows distribution in liver biomatrix scaffolds of laminin (LAM), heparan sulfate (HS), collagen type III (COL3), and fibronectin (FN) and typical basement membrane proteins in association with remnants of blood vessels. At higher magnification one can observe the primary components of basement membrane, including Type IV collagen (COL4), entactin (Ent; also called nidogen), LAM, and perlecan (Per), a form of HS-PG in the portion of the scaffolds near the portal triads. See also the details of the collagen chemistry given in Supporting Table 1 and Supporting Fig. S2.

Figure 4.

Pattern of ECM components from portal triad to central vein in biomatrix scaffolds. Histological comparison from portal triad (zone 1) to central vein (zone 3) of normal liver (A) and liver biomatrix scaffold (B); both are hematoxylin/eosin stained sections. (C) The model illustrating a stem cell and maturational lineage system in the liver with representative matrix components shown that they form patterns associated with the liver zonation. The components are listed in order of abundance from the findings of immunohistochemistry. The known lineage stages within human livers begin periportally in zone 1 (around portal triads) and progress in maturation ending with apoptotic cells in zone 3. The known matrix chemistry identified in the liver's stem cell niche is comprised of hyaluronans, type III collagen, a form of laminin that binds to α6β4 integrin, and a weakly sulfated form of CS-PG.49, 50 Just outside the stem cell niche are found Type IV collagen, normally sulfated CS-PGs and HS-PGs, and forms of laminin binding to αβ1 integrin. HP-PGs have been documented to be located uniquely pericentrally.51, 52 (D) The survey of matrix components and their location in liver versus those in biomatrix scaffolds, data summarized from immunohistochemistry findings (N/D = not tested. *Found by others to be exclusively near central veins). Most components of the cytoskeleton are lost during the washes (data not shown), residues of some, but not all, cytoskeletal proteins are present. The scaffolds are devoid of tubulin, desmin, and actin (phalloidin assays). However, there are trace amounts of cytokeratins scattered randomly in the scaffolds; trace amounts of α-smooth muscle actin around remnants of blood vessels at the portal triads; and low levels of vimentin throughout.

The matrix associated with the bile ducts and portions of the hepatic vascular systems (arterial and venous vessels) consists of typical basement membrane structures and so is quite distinctive from the thin layers of the matrix associated with the vascular structures found in the sinusoids. Laminin, entactin/nidogen, perlecan, and collagen type IV are found in the portal triad, whereas only perlecan and some collagen type IV are found in the Space of Disse (data not shown). Enormous amounts of hydrophobic, wavy elastin are present; it crosslinks together and forms sheets and fibers restricted primarily to the subcapsular connective tissue, portal regions, and arterial walls. Fibronectins are ubiquitous and prevalent throughout the scaffolds and are especially abundant in the Space of Disse, where they form either fine filaments or granular deposits (Figs. 2, 3B).

Immunohistochemistry indicates that known proteoglycans in the tissue are preserved in the biomatrix scaffolds (Figs. 3B, 4D). Among heterogeneous proteoglycans identified, syndecan was found intercalated and continuously along sinusoids, and perlecan is more punctuate in the Space of Disse. The forms of HS-PGs and CS-PGs are present throughout the remnants of the sinusoids in the biomatrix scaffolds and in patterns correlating with known zonation of liver tissue.

Proteoglycans and other matrix components are important reservoirs for cytokines and growth factors that bind tightly to their GAGs.31 Most growth factors and hormones are found in biomatrix scaffolds at physiological concentrations. In Table 1 the data are given from the lysates of rat livers versus rat liver biomatrix scaffolds, and in Supporting Table 2 parallel data are from human bile duct tissue versus bile duct biomatrix scaffolds. Interestingly, there were a few examples (e.g., bFGF) that were strongly enriched in liver biomatrix scaffolds over that found in liver lysates. The growth factors and cytokines bound are distinct qualitatively and quantitatively between the scaffolds of liver versus bile duct tissue, implicating either tissue-specificity or species-specificity, a conclusion that awaits further analyses on multiple tissues. Alternatively, it may be due, in part, to the fact that bile duct scaffolds were prepared, from necessity, by shaking the tissue in buffers on a rocker and not by perfusion through vasculature.

Table 1. Analyses of Growth Factors Bound to Liver Biomatrix Scaffolds
NameCytokine Full NameRat LiversRat Biomatrix ScaffoldsPercent
bFGFBasic fibroblast growth factor100.06394.14394
EGFEpidermal growth factor74.8176.02102
EGF REpidermal growth factor receptor92.8181.6488
FGF-4Fibroblast growth factor-415.0613.2188
FGF-6Fibroblast growth factor-64.813.7778
FGF-7Fibroblast growth factor-710.066.3263
GCSFGranulocyte-colony stimulating factor348.06338.2097
GDNFGlial-derived neurotrophic factor81.3143.5954
GM-CSFGranulocyte macrophage-colony stimulating factor133.56105.3879
HB-EGFHeparin-binding epidermal growth factor44.5638.2386
IGFBP-1Insulin-like growth factor binding proteins 167.8170.40104
IGFBP-3Insulin-like growth factor binding proteins 3140.81201.90143
IGFBP-4Insulin-like growth factor binding proteins 483.5658.9271
IGFBP-6Insulin-like growth factor binding proteins 691.8172.1979
IGF-IInsulin-like growth factor-I1.561.98127
IGF-I SRInsulin-like growth factor-I7.313.5148
IGF-IIInsulin-like growth factor-23749.063482.5293
M-CSFMacrophage-colony stimulating factor170.31134.6879
M-CSF RMacrophage colony stimulating factor receptor70.5650.4772
NT-3Neurotrophin-325.565.0320
NT-4Neurotrophin-455.0643.5979
PDGF R aPlatelet- derived growth factor receptor alpha10.5621.11200
PDGF R bPlatelet- derived growth factor receptor beta113.8185.4675
PDGF-AAPlatelet- derived growth factor AA62.06106.40171
PDGF-ABPlatelet- derived growth factor AB19.3119.34100
PDGF-BBPlatelet- derived growth factor BB9.5614.23149
PlGFPhosphatidylinositol glycan anchor biosynthesis, class F4.818.36174
SCFStromal cell-derived factor-12.0642.562064
SCF RStromal cell-derived factor receptor17.0617.80104
TGF-aTransforming growth factor alpha21.3121.63102
TGF-bTransforming growth factor-beta330.31342.77104
TGF-b 2transforming growth factor-beta 2134.06152.34114
TGF-b 3Transforming growth factor-beta 31.060.1817
VEGFVascular endothelial growth factor70.5694.14133
VEGF R2Vascular endothelial growth factor receptor 213.5611.9388
VEGF R3Vascular endothelial growth factor receptor 3459.5646.9110

Chemistry of the Biomatrix Scaffolds Correlates with Histology.

A significant feature of this new protocol is the retention of the matrix chemistry in patterns correlating with hepatic acinar zones 1-3 from portal triad to central vein and with histological entities such as vascular channels and GC, as shown in Fig. 4A-C. The matrix chemistry periportally in zone 1 is similar to that found in fetal livers and consists of type I and type III collagens, laminin, and forms of CS-PGs. It transitions to a different matrix chemistry in the mid-acinar (zone 2) and pericentral zones (zone 3), ending with a very stable matrix with high levels of type IV collagen and HP-PGs.32

Myriad proteins (e.g., growth factors and hormones, coagulation proteins, various enzymes) are known to bind to the matrix and to be held stably by way of binding to the discrete and specific sulfation patterns in the GAGs or to other matrix components.29 Thus, the matrix chemistry transitions from its start point in the stem cell niche having labile matrix chemistry associated with high turnover and minimal sulfation to stable matrix chemistries and having increasing amounts of sulfation with progression towards the pericentral zone. We hypothesize that maintenance of the natural architecture and matrix chemistry correlating with histology will facilitate recellularization in tissue engineering processes by guiding cells to specific sites on the biomatrix scaffolds and/or providing the proper mix of signals to drive differentiation into mature cells.

Biomatrix Scaffold Can Be Prepared from Different Tissues and Species.

The biomatrix scaffolds can be prepared from any tissue, normal or diseased, and from any species. In the supplement we show biomatrix scaffolds from human pancreas, biliary tree, and duodenum and from rat pancreas (Supporting Figs. S6-S9). Figures 5–7 and Supporting Fig. S5 show effects of bovine or rat liver biomatrix scaffolds on hepatic cells. In addition, biomatrix scaffolds have been prepared from human abdominal aorta, iliac vein, and from rat and pig intestine (data not shown). Histological, ultrastructural, and immunohistochemical studies on the biomatrix scaffolds suggested a marked tissue specificity, but not species specificity, in their structure, chemical composition, and functions (data not shown).

Figure 5.

Characterization of hHpSCs on liver biomatrix scaffolds versus on type I collagen. Phase-contrast images (A-D) show the morphologic changes of hHpSC colonies derived from the same liver and cultured in serum-free Kubota's medium and on tissue culture plastic (A), one of the conditions for self-replication, versus in the differentiation conditions of the serum-free differentiation medium for liver, and on Type I collagen (B) versus on bovine liver biomatrix scaffolds (C,C1,D). The cultures transitioned to cells by days 7-12 with increased cytoplasmic/nuclear ratio and marked glycogen expression (C) and then to ones with classic polygonal hepatocyte morphology interspersed by clear bile canaliculi (C1), a culture morphology that persisted thereafter, as indicated in the representative culture at day 24 (D). Reverse-transcription polymerase chain reaction (RT-PCR) assays show gene expression changes of hHpSCs under self-replication conditions on culture plastic versus on rat liver biomatrix scaffolds on day 7 (E). We compared expression of hHpSC markers, including CXCR4 and EpCAM; early hepatocytic genes including CK19, HNF 6, FOXA2, AFP, and low levels of albumin; mature hepatocytic markers including high levels of albumin, transferrin, CYP3A4, tyrosine aminotransferase (TAT), and glucose-6-phoshatase (G6PC), and cholangiocytic genes, including CFTR, gamma glutamyl transpeptidase (GGT1), anion exchange 2 (AE2), and apical sodium-dependent bile acid transporter (ASBT). Biochemical assays measuring urea synthesized in cultures on type I collagen versus on rat liver biomatrix scaffolds and P450 3A4 activity (F) in cultures on type I collagen versus on biomatrix scaffolds prepared from either rat or bovine livers at day 12.

Figure 6.

Immunofluorescence staining of cells lineage restricted from hHpSCs on biomatrix scaffolds. (A) Stained with hepatic specific marker: albumin (Alb, red) and hHpSC cell surface marker: EpCAM (green). Note that cells plated on biomatrix scaffold do not express EpCAM. Scale bar = 200 μm. (B) Stained with early hepatic marker α-fetoprotein (AFP, red) and with an antibody to human CK19 (green) that at this level of expression is indicative of mature cholangiocytes. This antibody to CK19 assays is human-specific and did not stain the residue of rat CK19 in the scaffolds not seeded with cells (data not shown). The AFP expression is low but still evident at day 7. Scale bar = 200 μm. (C) Stained with Alb (red) and hepatic stellate cell marker, α-smooth muscle actin (ASMA, green). The expression of albumin and ASMA is a strong indication that both maturing hepatocytes and stellate cells are present. Scale bar = 100 μm. (D) Stained with functional hepatic protein CYP450 (red) and cholangiocytes-specific marker, secretin receptor (SR, green) showing that the maturing hepatocytes and cholangiocytes are functional and express classic markers for these two cell types. Scale bar = 200 μm.

Figure 7.

Stability of fully functional, mature human hepatocytes on biomatrix scaffolds. Adult human hepatocytes plated in the differentiation medium and onto type I collagen (A,B) versus on bovine liver biomatrix scaffolds (C) that were cryogenically pulverized, dispersed in medium, and allowed to sediment onto the plates. Cells on Type I collagen are fully viable and at their peak of differentiation from 7-12 days (A shown at 7 days); they begin to deteriorate after ≈2 weeks, and by 20 days (B) they are dead, dying, and nonfunctional. By contrast, those plated onto liver biomatrix scaffolds (C) are functional for at least 8 weeks (we have not assessed them at longer times yet); here shown after 21 days in culture on pulverized liver biomatrix scaffolds. P450 3A4 assays on cultures of two separate preparations of cryopreserved adult human liver cells plated onto biomatrix scaffolds versus on type I collagen and assayed on day 12 (D). The sample ZHep-007 is representative of cryopreserved samples with good attachment after thawing; the sample ZL-013 is representative of those lots that have poor or no attachment after thawing. Thus, even these poorer quality samples are able to attach to biomatrix scaffolds and remain viable long term. In both samples assayed the levels of P450s are higher when cultured on liver biomatrix scaffolds. With time on the biomatrix scaffolds, the lots of poorer quality cryopreserved cells will improve; studies are ongoing to assess the extent of this improvement and the time required for it to occur (data not shown).

Biomatrix Scaffolds Induced and/or Maintained Differentiation of Cells.

Plating hHpSCs onto dishes with sections of liver biomatrix scaffolds and in HDM tailored for adult liver cells resulted in essentially 100% of the viable cells attached within a few hours onto biomatrix scaffolds, whether intact or after cryogenic pulverization. The colonies of cells that initially formed on the sections of scaffolds retained some of their stem cell phenotype, as the cells in the center of the colonies were able to resist staining with dyes (Supporting Fig. S4) and expressed classic hepatic progenitor markers, such as chemokine (C-X-C motif) receptor 4 (CXCR4) and epithelial cell adhesion molecule (EpCAM) (Fig. 5E). They divided once or twice and then transitioned into cell cycle arrest and into 3D cord-like morphologies typical for cultures of mature parenchymal cells (Figs. 5, 6 for stem cells differentiation; compare with Fig. 7 and Supporting Fig. S5 for maintenance of mature hepatocytes). The HDM used did not require all the usual cytokines or growth factors because these are present bound to the biomatrix scaffolds. The transition to growth arrest correlated with staining throughout the colonies with viability dyes (Supporting Fig. S4), with loss of expression of EpCAM and CXCR4 (Fig. 5E) and with a steady increase in the expression of adult-specific hepatocytic and cholangiocytic genes such as urea and cytochrome P450 3A4 (Fig. 5F).

Normal adult rat and human hepatocytes were plated onto type I collagen versus on biomatrix scaffolds from rat or bovine livers and into HDM for adult cells. The adult parenchymal cells were able to attach to scaffolds within a few minutes (even in serum-free medium) versus within hours on type I collagen; remained in growth arrest from the point of attachment; and remained viable and fully functional for more than 8 weeks on scaffolds, versus only about ≈2 weeks on type I collagen (Fig. 7 and Supporting Fig. S5). The levels of functions of the mature liver cells on biomatrix scaffolds for weeks proved to be the same or similar to the findings of others of freshly isolated, adult hepatocytes.33 The dramatic distinctions are that the cultures on type I collagen deteriorated rapidly after 2 weeks, whereas those on biomatrix scaffolds remained stable morphologically and functionally for as long as the cultures were maintained (Fig. 7 and Supporting Fig. S5).

Discussion

Biomatrix scaffolds contain most of the tissue's extracellular matrix components and matrix-bound cytokines and growth factors, providing a composite set of chemical signals that can be used as an insoluble, stable scaffolding with an extraordinary ability to induce hHpSCs to adult liver fates as well as maintain adult cells fully differentiated for weeks. In comparing the extant types of matrix extracts from decellularized tissues with that of biomatrix scaffolds (Supporting Table 5), it is clear that physical, enzymatic, and chemical treatments have substantial effects on the composition, mechanical behavior, and host responses to biological scaffolds derived from the decellularization of native tissues and organs and, accordingly, have important implications for their in vitro and in vivo applications. All other existing methods for preparation of substrata or scaffolds remove a large portion of matrix components either through use of matrix-degrading enzymes16 or using buffers that dissolve portions of the matrix.9 Physical methods (e.g., snap freezing and agitation) can work to prepare matrix extracts from tissues with a layered structure such as dermis (e.g., SIS, BSM)34 but are not useful for organs with complex tissue structures such as liver. By contrast, the method for biomatrix scaffolds resulted in loss of most cellular proteins but preserved essentially all of the collagens and collagen-associated components including the matrix-bound cytokines and growth factors.

Extracellular matrix is embedded in a mosaic lipid bilayer, which in even the simplest organism is a complex, heterogeneous, and dynamic environment. The delipidation method is a critical facet of the protocol. The commonly used methods for decellularization of tissues involve ionic detergents such as SDC and sodium dodecyl sulfate (SDS). SDC is relatively milder than SDS, tends to cause less disruption to the native tissue architecture, and is less effective at solubilizing both cytoplasmic and nuclear cellular membranes.35 There are no reports of tissue decellularization using SDC alone. Many studies have made use of a harsh nonionic detergent (e.g., Triton X-100)36 or zwitterionic detergents (e.g., 3-(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, CHAPS).37 By contrast, our method of using a combination of SDC and PLA2 delipidated the tissue rapidly and gently.

At least 29 types of collagens (I-XXIX) have been identified with functional roles in cell adhesion, differentiation, growth, tissue development, and structural integrity.38, 39 The major structural component in the matrix, collagens, are known to remain insoluble in high salt concentrations and at neutral pH,28, 40-42 a finding that is the basis of our strategy in preparation of biomatrix scaffolds. The strategy has added advantages that collagens enable preservation of matrix components bound to them, such as laminins and fibronectins (FNs), small leucine-rich proteoglycans (PGs), and GAGs that in turn preserve cytokines, growth factors, or cell surface receptors bound to them.

Biomatrix scaffolds are unique in their profound ability to induce rapid and consistent differentiation of stem/progenitor cells, such as hHpSCs, to adult fates and to maintain those lineage-restricted cells, or to maintain adult cells plated onto the scaffolds, as viable and fully functional cells for many weeks (>8 weeks).

Differentiation of stem cells, such as embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, or varying forms of mesenchymal stem cells (MSCs) into fully mature liver cell types requires multiple sets of signals (soluble and matrix) presented in stages, with induction by one set requiring priming to respond to a different set, and takes many weeks, up to 6 weeks of culture, to generate cells having the adult liver fate.43 Moreover, lineage restriction of MSCs to liver fates gives inconsistent results with adult cells having mixed hepatocyte and MSC phenotypes.3, 44, 45 The hepatocyte-like cells from any of these precursors express some, but never all, of the major liver-specific genes, with variability in which genes are observed, and with the protein levels for hepatic genes being usually low46 or high for one hepatic gene and negligible for others.3, 47, 48 For reasons unknown, the results are different from preparation to preparation. In contrast, differentiation of hHpSCs on biomatrix scaffolds resulted in essentially all cells expressing a classic adult phenotype with urea, albumin, and CYP450 activities at near normal levels within 1 to 2 weeks in culture and with stability of that phenotype for many weeks. We assume that the biomatrix scaffolds can greatly facilitate differentiation of other stem cell populations, such as ES, iPS, and MSCs to an adult liver phenotype, a hypothesis now being tested.

The ability to differentiate stem cells on biomatrix scaffolds and in an HDM to achieve mature and functional cells and tissues offers considerable opportunities for academic, industrial, and clinical programs enabling the use of well-differentiated cell types for every type of analytical study, and, excitingly, enabling an improved way to generate implantable, revascularized tissues or organs that might be used for basic research and clinical programs.

Preliminary studies, still ongoing, suggest that scaffolds are both efficient inducers of differentiation and also can dictate fate. If true, it implicates the exciting potential of being able to define variables dictating fate and deriving them from the microenvironment versus those entirely within the stem cells.

Acknowledgements

We thank Dr. V. Madden for TEM and SEM processing; Dr. Y. Rong for the rat hepatocyte preparations, and Lucendia English for glassware washing and lab management.

Ancillary

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