Paracrine signals from mesenchymal cell populations govern the expansion and differentiation of human hepatic stem cells to adult liver fates

Authors


  • Potential conflict of interest: Nothing to report.

Abstract

The differentiation of embryonic or determined stem cell populations into adult liver fates under known conditions yields cells with some adult-specific genes but not others, aberrant regulation of one or more genes, and variations in the results from experiment to experiment. We tested the hypothesis that sets of signals produced by freshly isolated, lineage-dependent mesenchymal cell populations would yield greater efficiency and reproducibility in driving the differentiation of human hepatic stem cells (hHpSCs) into adult liver fates. The subpopulations of liver-derived mesenchymal cells, purified by immunoselection technologies, included (1) angioblasts, (2) mature endothelia, (3) hepatic stellate cell precursors, (4) mature stellate cells (pericytes), and (5) myofibroblasts. Freshly immunoselected cells of each of these subpopulations were established in primary cultures under wholly defined (serum-free) conditions that we developed for short-term cultures and were used as feeders with hHpSCs. Feeders of angioblasts yielded self-replication, stellate cell precursors caused lineage restriction to hepatoblasts, mature endothelia produced differentiation into hepatocytes, and mature stellate cells and/or myofibroblasts resulted in differentiation into cholangiocytes. Paracrine signals produced by the different feeders were identified by biochemical, immunohistochemical, and quantitative reverse-transcription polymerase chain reaction analyses, and then those signals were used to replace the feeders in monolayer and three-dimensional cultures to elicit the desired biological responses from hHpSCs. The defined paracrine signals were proved to be able to yield reproducible responses from hHpSCs and to permit differentiation into fully mature and functional parenchymal cells. Conclusion: Paracrine signals from defined mesenchymal cell populations are important for the regulation of stem cell populations into specific adult fates; this finding is important for basic and clinical research as well as industrial investigations. (HEPATOLOGY 2010;)

Human hepatic stem cells (hHpSCs) are uniquely positioned at the foundation of potential liver regeneration therapies because they are the only parenchymal cell subpopulation identified with both the capacity for self-renewal and the capacity to generate numerous progenitors, such as human hepatoblasts (hHBs), committed progenitors and their descendents, mature hepatocytes, and cholangiocytes.1 hHpSCs and hHBs have been found in the livers of donors of all ages and are able to give rise to mature liver tissue in vitro and in vivo.2, 3 In addition to these determined stem cell populations, diverse stem cell populations have been identified and found to be able to be lineage-restricted to a liver fate; these include embryonic stem cells, induced pluripotent stem cells, and multiple forms of mesenchymal stem cells from bone marrow, adipose tissue, and amniotic fluid.4-6 The efficiency of the differentiation of these precursors to a liver fate, whether in vitro or in vivo, results in liver-like cells with overexpression or underexpression of some adult genes and aberrant regulation of genes, and the results are distinct with every preparation. These findings are discussed at length in a recent review.7

We have used hHpSCs as a model system to define the requisite signals for lineage-restricting stem cells to a liver fate. In previous studies, we established methods to isolate hHpSCs and hHBs from fetal, neonatal, pediatric, and adult human livers.2, 8 The hHpSCs are characterized by their uniform morphology, high nucleus-to-cytoplasm ratio, small size (∼7-9 μm in diameter), and tightly packed colony formation. The cells weakly express albumin (ALB), cytokeratin 8 (CK8), CK18, and CK19 but not α-fetoprotein (AFP). The hHBs are larger (∼10-12 μm in diameter), exhibit colonies that are cordlike with bile canaliculi, and express ALB, CK8, CK18, CK19, and AFP. The hHpSCs and hHBs have unique antigenic profiles that include epithelial cell adhesion molecule (EpCAM) for both, neural cell adhesion molecule (NCAM) for hHpSCs, and intercellular cell adhesion molecule (ICAM) for hHBs. More extensive characterizations of these cells and their descendents are provided in previous reports.2 The cells can be clonogenically expanded ex vivo in a serum-free medium tailored for endodermal progenitors [Kubota's medium (KM)]9 and have the potential to differentiate into mature functional hepatocytes and cholangiocytes in vivo.

The microenvironment of stem cell niches modulates stem cell proliferation, influences symmetric division versus asymmetric division, controls differentiation, protects cells from physiological stresses, and helps them to contribute to tissue formation in development and in regeneration in adult life.7 The components of the stem cell microenvironment regulating these processes include distinct cell-cell interactions and paracrine signals, which comprise both soluble and extracellular matrix factors, as well as the three-dimensional (3D) architecture, which shapes and dictates the delivery of these cues.

The studies reported here are focused on mesenchymal companion cells and their provision of critical paracrine signals regulating the parenchymal lineage stages. Paracrine signals were identified with purified subpopulations of mesenchymal cells cultured under serum-free conditions. A set of these signals was then used to regulate precisely the growth and/or fates of hHpSCs under feeder-free conditions. In a separate report, we are focusing on studies of lineage-dependent soluble signals (J. Uronis and L. Reid, unpublished data, 2010).

Abbreviations

3D, three-dimensional; AFP, α-fetoprotein; ALB, albumin; ASMA, α-smooth muscle actin; BC, bile canaliculus; C1A1, collagen 1A1; C3A1, collagen 3A1, C4A5, collagen 4A5; C5A2, collagen 5A2; CK, cytokeratin; CS-PG, chondroitin sulfate proteoglycan; DAPI, 4′,6-diamidino-2-phenylindole; ELS, elastin; EpCAM, epithelial cell adhesion molecule; ER, endoplasmic reticulum; FN, fibronectin; GFAP, glial fibrillar acidic protein; GAG, glycosaminoglycan; GPYC, glypican; HA, hyaluronan; HDM, hormonally defined medium; hHB, human hepatoblast; hHpSC, human hepatic stem cell; hHpSTC, human hepatic stellate cell; hMSC, human mesenchymal stem cell; HP-PG, heparin proteoglycan; HS-PG, heparan sulfate proteoglycan; HUVEC, human umbilical cord vein endothelial cell; ICAM, intercellular cell adhesion molecule; ICG, indocyanine green; IF, intermediate filament; KDR, kinase insert domain receptor; KM, Kubota's medium; LA4, laminin A4; LB2, laminin B2; LB3, laminin B3; MCM, mesenchymal cell medium; MKM, modified Kubota's medium; MKM-C, modified Kubota's medium for cholangiocytes; MKM-H, modified Kubota's medium for hepatocytes; mRNA, messenger RNA; NCAM, neural cell adhesion molecule; NPC, nonparenchymal cell; qRT-PCR, quantitative reverse transcription polymerase chain reaction; SDC2, syndecan 2; SR, secretin receptor; TEM, transmission electron microscopy; VEGF, vascular endothelial growth factor; vWF, von Willebrand factor.

Materials and Methods

Most of the methods have been presented in detail previously2, 10 or are described in the Supporting Information.

Immunoselection of subpopulations was performed by magnetically activated cell sorting according to the manufacturer's instructions (Miltenyi Biotech) with cell suspensions from human fetal livers or adult human livers. These included the following:

  • Angioblasts: CD133+ or CD117+ cells coexpressing vascular endothelial growth factor receptor 2 [VEGFR2; kinase insert domain receptor (KDR)] from fetal or adult livers.

  • Mature hepatic endothelial cells: CD31++ cells coexpressing KDR from adult livers.

  • Human hepatic stellate cell (hHpSTC) precursors: CD146+ cells from fetal livers.

  • Mature hHpSTCs (pericytes): CD146+ cells from adult livers.

  • hHpSCs: EpCAM+NCAM+ cells from fetal and adult livers.

Results

Isolation of Mesenchymal Subpopulations in Human Livers

Human livers contain two lineages of mesenchymal cell subpopulations that are not hemopoietic cell subpopulations and are CD45-negative. Both are derived from angioblasts: (1) lineage stages of endothelia and (2) hHpSTC precursors and their descendents, mature hHpSTCs (pericytes), and then myofibroblasts. Immunoselection for the different lineage stages of the two subpopulations was performed by magnetically activated cell sorting with specific antigenic profiles, and the cells were used in primary cocultures with hHpSCs. Supporting Information Table 4 provides data for the feeders of both cell lines and primary cultures of mesenchymal cells. Schematic images of the parenchymal and mesenchymal cell lineages are provided in Supporting Information Figs. 5 and 6.

Angioblasts and Endothelia (Fig. 1 and Supporting Information Fig. 2).

Angioblasts were isolated from fetal liver cell suspensions by immunoselection for cells expressing CD117 and VEGFR2 (KDR). The percentage of sorted CD117+KDR+ cells within the fetal liver samples was found to be approximately 0.5%. In culture, they appeared as aggregates demonstrating expression of CD117+ KDR+ (Fig. 1A); other antigens included CD133, NCAM, and von Willebrand factor (vWF) as well as little or no CD31 (platelet/endothelial cell adhesion molecule). They gave rise to mature endothelia that were CD31++, VEGFR+, vWF+, and ICAM1+ and had classic cobblestone-like clusters in monolayer cultures or tubes of cells if they were embedded into hyaluronan (HA) hydrogels or Matrigel.

Figure 1.

Mesenchymal cell subpopulations. (A) Aggregates of angioblasts were positive for VEGFR2 (KDR) and were rimmed by individual stellate cell precursors positive for CD146 (pink; arrowheads) and other markers not shown (desmin, ASMA, and β-3 integrin).2, 21 All the assessed mesenchymal cell populations were negative for CD45 (data not shown). (B) Mature endothelia, especially form adult livers, formed swirling aggregates of cells in monolayer cultures and formed tubelike structures if they were embedded into HAs or Matrigel. The endothelia strongly expressed CD31 (red) and vWF (green). The nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). (C) The stellate cell precursors that were separated from the aggregates of angioblasts were short (<10 μm) and had their nucleus on one end of a cell resembling a comma. (D) Mature stellate cells were found in abundance in cell suspensions of adult human livers when they were freshly plated; they were bipolar cells at least 15 to 20 μm long, and the nucleus was centrally located. With time in culture and especially after exposure to serum, they grew even longer (they could be 50 μm long and express a high level of ASMA). The CD146 expression in the precursors can be compared to the expression in the activated stellate cells. (E) Quantitative RT-PCR analyses of matrix components in freshly isolated liver mesenchymal cells [CD117 = angioblasts (CD117+/VEGFR2+); CD31 = endothelia; CD146 = stellate cells]. The RNAs from different fetal (F) or adult (A) cell subpopulations were subjected to qRT-PCR analyses for matrix molecules. The assayed proteoglycans included HS-PG2 (perlecan), GPYC, and SDC2; the assayed collagens included C1A1, C3A1, C4A5, and C5A2. The assayed basal adhesion molecules were laminin isoforms (LA4, LB2, and LB3) and FN; ELS was also assayed. The data from the maximal values among the six cellular subpopulations for each matrix component were given a value of 100% and were used to standardize the data for the other cellular subpopulations. Thus, the amount of the matrix component produced by the other cell populations was given as a fraction of the maximum one. This allowed us to see that forms of HS-PGs, laminins, ELS, and type III and IV collagens dominated in the endothelial cell lineages, whereas all the collagens, proteoglycans, FN, and ELS dominated in the stellate ones. Bars indicate standard deviations (n = 6). Abbreviations: C1A1, collagen 1A1; C3A1, collagen 3A1, C4A5, collagen 4A5; C5A2, collagen 5A2; ELS, elastin; FN, fibronectin; GPYC, glypican; LA4, laminin A4; LB2, laminin B2; LB3, laminin B3; mRNA, messenger RNA; SDC2, syndecan 2.

hHpSTC Lineage (Fig. 1 and Supporting Information Fig. 3).

The hHpSTC precursors were recognizable by their morphology as short (<10 μm), bipolar cells with their nucleus on one end, and they expressed CD146. They had very low levels of desmin, α-smooth muscle actin (ASMA), vitamin A, and lipids. They were negative for glial fibrillar acidic protein, were found at the edges of aggregates of angioblasts (arrowheads, Fig. 1A), and were found separately from these clusters. They gave give rise to mature hHpSTCs (also called hepatic-specific pericytes) strongly expressing CD146.11, 12 Freshly isolated hHpSTCs from adult liver cell suspensions were longer (∼15-20 μm), and their nuclei were more centrally located than those found in the precursors. The pericytes expressed elevated levels of ASMA, desmin, glial fibrillar acidic protein, and vitamin A and had high lipid contents (not shown). When hHpSTCs were plated onto culture plastic and in KM supplemented with 5% fetal bovine serum, they were activated into the cells with a myofibroblast phenotype emerging within 3 to 5 days of culture (Supporting Information Fig. 3). The cells were even longer (up to 50 μm or longer), had a centrally located nucleus, and expressed the highest levels observed for ICAM1, ASMA, and desmin.

Expression of Matrix Components in the Mesenchymal Cell Lineages

We surveyed the biological activities of numerous mesenchymal cell lines and primary cultures of mesenchymal cells as feeders (Supporting Information Table 4). We eventually realized that even transient exposure to serum resulted in muting of the distinctions in paracrine signals produced by the different mesenchymal cell subpopulations and their skewing toward biological activity typical for fibrosis or cirrhosis. Therefore, a serum-free medium for mesenchymal cells (MCM) was developed (Supporting Information Fig. 1) that enabled us to define feeder effects and the paracrine signals produced with freshly immunoselected mesenchymal cell subpopulations from fetal human livers versus adult human livers under serum-free conditions in short-term cultures (up to 2 weeks).

Using this strategy, we determined that all the mesenchymal subpopulations produced multiple types of collagens, basal adhesion molecules, proteoglycans, and elastin but at quite different levels (Fig. 1E and Table 1). The angioblasts (CD117+/KDR+ or CD133+/KDR+) from fetal livers produced less matrix than any of the tested mesenchymal cell subpopulations, produced low levels of type III, IV, and V collagens (only type III was detectable by immunohistochemistry), laminin A4 but not the other laminins or fibronectin, chondroitin sulfate proteoglycans (CS-PGs) and low levels of syndecan (only CS-PGs were detected by immunohistochemistry), and HAs. Those from adult livers produced higher levels of syndecan, laminin A4, fibronectin, and type IV collagen. Fetal liver–derived endothelial (CD-31+) cells made all of the forms of heparan sulfate proteoglycans (HS-PGs), type I, III, and V collagens (but not type IV collagen), low levels of laminin B2 and fibronectin, and elastin. Adult liver–derived endothelial cells (CD-31++) made high levels of HS-PG2 and syndecan, type I and IV collagens, laminins A4 and B3, fibronectin, and elastin. In summary, the matrix chemistry in angioblast and endothelial subpopulations was dominated by HS-PGs and some but not all laminin forms, and there was a significant increase in elastin with development.

Table 1. Extracellular Matrix Components Found in Feeder Cells
 Matrix Components
Angioblasts*HUVECshMSCsSTO FeedersNPCs Minus FibroblastsCD31+ or VEGFR+ cellsCD146+ (ASMA+) cells
  • The data were taken from immunohistochemistry or biochemistry examinations. Bolding indicates the matrix components found in particular abundance. The amount of staining for matrix components was least with the angioblasts and maximum with the CD146+ cells. ASMA was used as on the marker for stellate cells. Others have shown that bone marrow–derived mesenchymal stem cells in their undifferentiated state produce type I and X collagens and CS-PG forms that include decorin and versican.30

  • Abbreviations: hMSC, human mesenchymal stem cell; HUVEC, human umbilical cord vein endothelial cell; NPC, nonparenchymal cell.

  • *

    CD117+KDR+ or CD133+KDR+.

  • The NPCs were depleted of human fibroblasts with magnetic immunoselection.

Source of the cellsHuman fetal liverHuman umbilical veinBone marrowMurine embryosHuman fetal liverHuman fetal and adult livers
Collagen types producedSmall amounts of type IIIType IVType I and types III and IVTypes III and IVType I and type IVType III in fetal livers shifting to type IV in adult liversType I in fetal livers shifting to types I and IV in adult livers
Proteoglycans/GAGsCS-PGs and HAsCS-PGsCS-PGs, HS-PGs, and HAsCS-PGsHS-PGsCS-PGs and HS-PGs
Adhesion proteins and other proteinsNegligible levels of those assayedFibronectinFibronectin and lamininsFibronectin and lamininsLow levels of fibronectin and high levels of laminin and elastin increasing in adultsLow levels of fibronectin and elastin in fetal livers increasing to very high levels of these plus laminin in adult livers
Response of hHpSCs when cocultured with these mesenchymal cellsSelf-replicationSlow lineage restriction (>36-48 hours)Lineage restriction to hHBs and committed progenitors within ∼24 hoursDifferentiation into mature parenchymal cells

The stellate cell subpopulations produced the highest amounts of most of the analyzed matrix components, expressed low or negligible levels of laminins, strongly expressed fibronectin and elastin, and were high producers of all the collagens (especially type I) and the CS-PGs. As for the angioblast and endothelial cell subpopulations, the levels were highest in those from adult livers.

Supporting Information Fig. 7 summarizes the findings from changes in the matrix components at various stages of the lineage up to mature hepatocytes and cholangiocytes, and it also provides information from other reports. In addition, the composition of serum-free, hormonally defined medium (HDM) for the different stages is shown, and it is the same composition established in previous studies.9

Strong Dependence on Mesenchymal Companion Cells

Rigorous purification of parenchymal cells away from their native mesenchymal cell partners resulted in a loss of viability of the parenchymal cells (especially the stem cells and progenitors), as shown previously.2, 9 Cocultures of hHpSCs with different subpopulations of mesenchymal feeder cells elicited distinct biological responses. Those with angioblasts remained stem cells, and those with precursors to hepatic stellate cells and endothelia became hepatoblasts; this provided distinctive antigenic, biochemical, and ultrastructural features for both parenchymal and mesenchymal cell populations (Figs. 2 and 3). The hHpSC/angioblast partnership resulted in cells that were tightly bound to one another on their lateral borders through large numbers of tight junctions, desmosomes, and interdigitated microvilli. Efforts to disperse the angioblasts and hHpSCs into single cells were not successful with the customary enzymes (e.g., trypsin, chymotrypsin, dispase, and collagenases), and they resulted in a rapid loss of cell viability. Mechanical passaging, as used for human embryonic stem cells in culture, resulted in reasonably efficient passaging of hHpSCs13 and was used for the studies reported here.

Figure 2.

Colonies of (A-C) hHpSCs and (D,E) hHBs. (A) hHpSCs expressed EpCAM (green) and NCAM (red), whereas (D) hHBs expressed EpCAM (green) and AFP (red). Nuclei were stained blue with 4′,6-diamidino-2-phenylindole (scale bar = 100 μm). The phase images of the colonies indicate that the small hHpSCs were tightly bound to one another and had an elevated region at the periphery of the colony, at which the highest numbers of angioblasts were found. In contrast, the colonies of hHBs contained cords of parenchymal cells interspersed by clear channels (bile canaliculi). (C) An ultrastructural image of the hHpSC colony shows that the mesenchymal cells were tightly bound along their length to the hHpSCs, with few desmosomes and only small IF bundles in both the mesenchymal cells and hHpSCs. (F) In comparison, an ultrastructural image of the hHB colony shows that the boundary between the hHBs and mesenchymal cells was partially separated but was held together with desmosomes connecting in the mesenchymal cells to large IF bundles. The arrows show desmosomes; the arrow head shows microvilli. Abbreviation: IF, intermediate filament.

Figure 3.

Colonies of hHpSCs with mesenchymal partners. There are two types of mesenchymal cells associated with hHpSCs: angioblasts within the colonies and stellate cell precursors at the periphery. A colony of hHpSCs is shown (A) in phase and (B) after staining for NCAM (magnification ×10). NCAM (red) was found on angioblasts (NCAM+, CD117+, and vWF+) and also on hHpSCs.2 The angioblasts were scattered throughout the colony and were found especially at the periphery of the colony. (C) The stellate cell precursors21 were found outside and surrounding the colonies and were readily identified via staining for ASMA (green). (D) A merged image of parts A, B, and C shows overlap in the expression of NCAM and ASMA at the periphery of the stem cell colony. (E) A phase contrast image of two adjacent hHpSC colonies. The right colony was associated with angioblasts and was in a quiescent state; under these conditions, hHpSCs self-renewed. The other colony was associated with hepatic stellate cell precursors (and also with endothelial cell precursors, which were present but could not be seen with the staining used), which led to an eruption of cords of hHBs extending from the hHpSC colony. (F) An image of the same colonies shown in part E but stained for CD146 (yellow/green) and ALB (red).

The hHB/stellate cell/endothelial cell precursor partnership resulted in cells that were more loosely bound to one another, as evidenced by both light microscopy and ultrastructural analyses. Transmission electron microscopy (TEM) observations confirmed that hHBs were distinct from hHpSCs: there were striking increases in the number and size of the desmosomes and the intermediate filaments that terminated at the desmosomes in the mesenchymal cells and in the appearance of bile canaliculi. In parallel with morphological changes, hHBs had an antigenic profile that overlapped with that of hHpSCs but showed distinctions in expressing ICAM-1 (not NCAM) and AFP and P450-A7 (data not shown). The activation of angioblasts, which gave rise to hHpSTCs and endothelial cell precursors, was associated with dramatically elevated levels of CD146 (Fig. 3) and with elevated levels of ASMA and desmin (data not shown); this all correlated with the formation of cords of hHBs and committed progenitors from the colonies of hHpSCs.

Later lineage stages of parenchymal cells were partnered with either endothelia (hepatocytes) or hepatic stellate cells, pericytes, and myofibroblasts (cholangiocytes). The data from cultures of these epithelial-mesenchymal partnerships are not shown except in summary form in Supporting Information Fig. 7, although we provide data on the identified paracrine signals from those stages of mesenchymal cells.

Defined Mixes of Signals Can Replace the Feeders

The information obtained from analyses of the feeders was used to define sets of signals eliciting the regulation of hHpSCs under feeder-free conditions. We demonstrated this with four examples: (1) self-renewal, (2) lineage restriction to hepatoblasts, (3) differentiation into hepatocytes, and (4) differentiation into cholangiocytes (a summary is provided in Supporting Information Figs. 5-7)

Self-Renewal (Fig. 4).

Self-renewal occurred with angioblast feeders, which were replaceable with KM and type III collagen and/or uncrosslinked or weakly crosslinked HAs. These conditions resulted in hHpSC colonies with maintenance of the stem cell phenotype for more than 2 months in culture [they were positive for EpCAM, NCAM, and CK19, had weak levels of ALB, and were negative for AFP, P450A7, urea synthesis, and indocyanine green (ICG) uptake)] and in the ability of the cells from those colonies to give rise to both hepatocytes and cholangiocytes if they were transferred to differentiation conditions.

Figure 4.

Conditions for self-renewal. HHpSCs self-replicated when they were maintained as monolayers (A) on feeders of angioblasts or (B) on a substratum of purified type III collagen or (C) when they were embedded into weakly crosslinked HA hydrogels in which the hHpSCs formed aggregates or balls of cells. Under all three sets of conditions, hHpSCs could self-renew for many weeks at division rates as high as one division every 36 hours on culture plastic and as high as one division every 20 to 24 hours on type III collagen.13 (D,E) TEM images of hHpSCs in the hydrogels indicate aggregates of cells with an outer layer interfacing with the hydrogels through numerous microvilli, which are outlined in part E with dashes. The cell diameters were approximately 7 to 9 μm, and the nucleocytoplasmic ratio was high. At a high magnification, the interface cells appeared to have small amounts of undeveloped ER, mitochondria (M), and rough and free ribosomes (R) beneath the microvilli; these are characteristics of immature, undifferentiated hepatic parenchymal cells.

Ultrastructural studies of the cells in weakly crosslinked HA hydrogels showed tightly aggregated hHpSCs enveloped by the mesenchymal cells and having quite distinctive desmosomes and tight junctions. At a low magnification, the surface layer of cells was seen to form an interface with the hydrogel that was characterized by numerous short microvilli. At a higher magnification, the microvilli were shown to be irregular in size and spacing. Beneath the microvilli were clusters of mitochondria and free and bound ribosomes. This outer cell layer of mesenchymal cells enveloped a large aggregate of hHpSCs.

Lineage Restriction to hHBs (Fig. 5).

Feeders of stellate cell precursors or activated stellate cells caused hHpSCs to be lineage-restricted to hHBs within 24 hours and to express AFP and glycogen. The feeders were proved to be replaceable by KM and matrix components produced by these cells, including type IV collagen and laminin, crosslinked HA hydrogels, or combinations of these. hHBs did not take up ICG, although the cultures contained some committed progenitors that did demonstrate some uptake. The lineage restriction to hHBs was associated with a separation between the cells, the formation of bile canaliculi, and increases in the presence of desmosomes and in the size of the bundles of intermediate filaments in the mesenchymal cells.

Figure 5.

Lineage restriction to hHBs. The hHpSCs were restricted to hHBs within 24 hours when grown on stellate cell precursor feeders. The feeders (4) could be replaced with (B) substrata of type IV collagen or laminin (inset) or (C) HAs that were crosslinked. Combinations of these matrix components produced the same result and yielded cells with (C) AFP expression, (D) glycogen synthesis as evidenced by periodic acid-Schiff staining, and (E) a lack of ICG uptake and secretion in those cells expressing a hepatoblast phenotype. (C) Crosslinked HA hydrogels also yielded this result, and this indicated that mechanical forces conferred by the rigidity of the gel were factors in lineage restriction. (F) Ultrastructural images of hHBs showed that they were much more loosely connected to one another than hHpSCs, and the BCs were separated from the remainder of the intercellular space by tight junctions and sparse microvilli on the surfaces of the cells. The cells displayed oval-shaped nuclei, a paucity of cytoplasm, immature ER, mitochondria, and ribosomes.

Differentiation into Mature Parenchyma.

This required MKM (described in the Materials and Methods section) and all variables and factors that were previously defined as critical for mature parenchymal cell metabolism2 and used in the lineage restriction of embryonic stem cells to liver fates.6 However, the ability to drive the cells to the hepatocytic pathways versus the biliary pathways necessitated distinctions in both the hormonal constituents of the media and the matrix chemistry.

Selective differentiation into hepatocytes occurred with feeders of mature endothelia (Fig. 6), which were replaceable with 3D cultures in MKM-H and in HA hydrogels composed of type IV collagen (60%). Partial effects were observed in monolayer cultures on matrix-coated plates. The cells grew in size to >18 μm, demonstrated a cordlike morphology in the colonies with classic bile canaliculi, lost expression of EpCAM, NCAM, and AFP, and acquired expression of ALB, glycogen storage, ICG uptake, and urea secretion. In ultrastructural studies, the cells acquired the classic hepatocyte features of large numbers of mitochondria, rough endoplasmic reticulum (ER), and Golgi complexes.

Figure 6.

Conditions yielding mature hepatocytes. (A,B) Full differentiation into hepatocytes occurred within 7 to 10 days when the cells were plated on to MKM supplemented further with factors for hepatocytes (MKM-H) and with feeders of endothelia. (C) The feeders could be replaced via the embedding of the cells into a mix of hydrogels (HA or Matrigel; 40%) and type IV collagen and laminin (60%) in combination with MKM-H. C1, C2, C4 = cells suspended in the matrix; C3 = cells in sandwich culture in the matrix. The cells demonstrated (C2,E) strong ALB expression, (C4) glycogen, (D) ICG uptake and excretion, and (E) urea secretion. (B) A TEM image of the hepatocytes showed typical microstructures, such as large numbers of spherical, elongated mitochondria (M), Golgi complexes (G), lysosomes (L), and ER in the cytoplasm. (E) Quantitative measures of ALB secretion and urea synthesis are shown (Student t test, n = 3, mean ± standard error of the mean, *P < 0.05 and **P < 0.01 for comparisons).

Selective differentiation into cholangiocytes occurred with feeders of mature stellate cells and myofibroblasts from adult livers. Feeder-free conditions that yielded equivalent results consisted of the embedding of hHpSCs into hydrogels containing type I collagen (60%) and HAs (or Matrigel; 40%) and the use of MKM-C. The cells formed branches and ducts, especially in 3D cultures, and the cells within the ducts expressed secretin receptors (SRs) and CK19 (Fig. 7).

Figure 7.

Conditions yielding mature cholangiocytes. (A) The formation of bile duct structures within 7 to 10 days required hHpSCs to be cultured in MKM-C and plated onto mature stellate cells (pericytes) or myofibroblasts. (B-E) The feeders could be replaced via the embedding of the cells into a mix of type I collagen gel (60%) and hydrogel (HAs or Matrigel; 40%) and into MKM. (E) The cells formed ramifying ducts and tubes that expressed CK19 (green) and SR (red) as well as other markers (e.g., aquaporins and 4-aminobutyrate aminotransferase; data not shown) for cholangiocytes. (B) The image is a composite of multiple images showing a large colony within the hydrogel/matrix mixture. (C,D) The images are enlargements of some of the ducts from the colony in part B.

Discussion

Liver development is induced in a stepwise process with signals from the cardiac mesoderm and then from subpopulations of mesenchymal cells.14 During liver organogenesis, endodermal cells are induced by the cardiac mesoderm to differentiate into hHpSCs within the ventral endoderm. Subsequently, newly specified hepatic cells delaminate, migrate into the surrounding septum transversum mesenchyme, and intermingle with endothelia, which remain in contact with hepatic cells throughout development.14 Thus, mutant mouse embryos with fetal liver kinase 1 (a receptor for VEGF essential for the formation of endothelia), lacking endothelia, show initial hepatic induction but not the proliferation of hepatic cells into the surrounding septum transversum mesenchyme; this indicates the importance of endothelia for liver organogenesis.15

At the time of hepatic induction, septum transversum mesenchymal cells surround the developing cardiac region near the ventral foregut endoderm and are the source of inductive signals including fibroblast growth factors and bone morphogenetic proteins, angiogenesis, and intense hedgehog signaling, which is also a key regulator of murine and human hepatic progenitors throughout life.14 The liver is organized into physiological units that contain all developmental stages of hepatic cells, and the stem cell niche in vivo has been shown to be the ductal plates in fetal and neonatal livers and the canals of Hering in pediatric and adult livers.8, 16 These niches contain type III collagen, HAs, a form of laminin binding to α6β4 integrin (assumed to be laminin 5), and a novel form of CS-PG found to have minimal sulfation.8, 17, 18 In contrast, the in vivo microenvironment associated with hHBs is composed of type III, IV, and V collagens, laminin isoforms binding to α3β1, CS-PGs with normal levels of sulfation, and various forms of HS-PGs.8, 17, 18 The matrix chemistry found in the space of Disse (the space between differentiated hepatocytes and endothelium) forms a gradient from the periportal region (zone 1) to the pericentral region (zone 3).19 The portal triads are dominated by fibrillar collagens (types I and III), forms of laminin (weak levels), vimentin, HAs, and less sulfated forms of CS-PGs and HS-PGs transitioning in a gradient fashion through the space of Disse to a matrix chemistry around the central vein composed of type IV and VI collagens (with weak expression of type III), syndecans 1 and 4, highly sulfated proteoglycans [especially heparin proteoglycans (HP-PGs)], and no HAs or laminin. In addition, elastin is found generally throughout the acinus, as is type I8 collagen, a form of HS-PG; both are closely associated with the blood vessels.

The behavior of hHpSCs and feeders parallels that observed during liver development and that occurring between the parenchyma and mesenchymal cells in the space of Disse.14 Our data on matrix components in immunoselected angioblasts from fetal livers show that they produce low levels of collagens (only type III collagen was found by immunohistochemistry) as well as one isoform of laminin (A4), elastin, HAs, syndecan, and CS-PG (only CS-PG was detected by immunohistochemistry). Those from adult livers have higher levels of syndecan, type IV collagen, elevated levels of laminin A4, and fibronectin. The endothelial cells (CD31+) from fetal livers make all the tested forms of HS-PGs, low levels of type I, III, and V collagens, and laminin B2. Those from adult livers express the highest observed levels of HS-PG2 and syndecan, type I and IV collagens, high levels of the laminins A4 and some fibronectin, and very high levels of elastin.

There are multiple stellate cell subpopulations. The stellate cell precursors appear to be derived from angioblasts, as evidenced by the proximity of the precursors at the edges of the angioblast colonies, by the sharing of markers such as vascular cell adhesion molecule 1, β3-integrin, and CD146,20, 21 and, if serum is present transiently or permanently, by the transition of primary cultures of immunoselected angioblasts to cultures dominated by activated stellate cells within a few days in culture. Although we cannot exclude culture selection for a preexisting, initially minor subpopulation of activated stellate cells, we propose that the net sum of the evidence implicates a lineage connection between angioblasts and stellate cells. Efforts are ongoing to assess this hypothesis.

The stellate cell/myofibroblast subpopulations are in a maturational lineage with overlapping but also distinct characteristics, which include the cell length or size, position of the nucleus, level of expression of CD146 and other markers (e.g., ASMA and desmin), extent of intermediate filaments and desmosomes, and compositions and levels of their matrix components. Those from fetal livers have the previously reported characteristics,21 and those from adult livers have a phenotype of pericytes or myofibroblasts.11 Activation of either fetal or adult subpopulations occurred at high cell densities (confluence of the cells), after sustained serum exposure, or with rigidity of the substratum (e.g., highly crosslinked HA hydrogels).22

Mature stellate cells produced both network and fibrillar collagens (large amounts of type I collagen and lower levels of type III, IV, and V collagens), large amounts of elastin, and both HS-PGs and CS-PGs. The levels of all of these were the highest observed in the activated stellate cells and myofibroblasts obtained from adult livers. A primary biological activity of activated hHpSTCs is matrix synthesis, and this includes the production of diverse collagen types (types I, III, IV, and V) and multiple types of basal adhesion molecules (fibronectin and laminin α1 and laminin γ1 chains).23 Disease states such as fibrosis and cirrhosis are associated with highly activated stellate cells, which contribute to scar tissue formation throughout the liver. Indeed, mice defective in the LIM homeobox 2 gene experience early and inappropriate activation of stellate cells and spontaneous cirrhosis.24

CS-PGs, detected by immunohistochemical assays, were present in feeders derived from human fetal livers or hHpSC colonies. They can form complexes with growth factors and chemokines, albeit more weakly than those found for HS-PGs.18, 25, 26 A recent report identified unique forms of CS-PGs with little or no sulfation present in stem cell niches, including the liver.18 The liver's stem cell niche is dominated by HAs and by forms of CS-PGs that make a nonsulfated (or minimally sulfated) glycosaminoglycan (GAG) barrier minimizing the presentation of signals (i.e., those bound to GAGs) to the stem cells. When the stem cells migrate from the niche, they come into contact with GAGs and proteoglycans with more extensive sulfation and stably bound growth factors that are known to influence the stem cells either with respect to growth or with respect to differentiation into various mature cell fates.27

The feeders with the most extensive effects on differentiation are those with the highest levels of HS-PGs, which are renowned for operating as high-affinity chemical scaffolds for growth factors. HS-PGs have been purified from rodent livers by Gallagher and associates28 and from human livers by Linhardt and associates27 and characterized extensively. The maturation of liver parenchymal cells is induced by HS-PGs with a higher degree of sulfation, especially O-sulfation (as found in heparin chains), which in both humans and rodents is associated with the most mature parenchymal cells in the liver.29

The extent of differentiation also correlated with the three-dimensionality, the ratio of type I collagen to other collagen types, the ratio of fibronectin to laminin isoforms, the presence of proteoglycans with moderate to high levels of sulfation (e.g., HS-PG isoforms), and the rigidity of the hydrogels. The least differentiated were in a monolayer; intermediate levels were in type I collagen sandwich cultures that resulted in a mix of hepatocytic and biliary lineages; and the most differentiated were cells suspended three-dimensionally into hydrogels with matrix components required for driving cells differentially toward hepatocytic or biliary fates. Preferential differentiation toward cholangiocytic fates occurred under conditions of higher rigidity (and higher levels of CS-PGs), whereas less rigidity and higher levels of HS-PGs or HP-PGs correlated with differentiation toward hepatocytic fates. The effects of CS-PGs versus HS-PGs are assumed to be due to their distinctions in growth factor binding. The relevance of mechanical forces on differentiation is now the focus of ongoing experiments.

Although the data presented here emphasize the role of the changes in the matrix chemistry along with certain known soluble signals, we have identified more than a dozen other soluble signals that change qualitatively and quantitatively with differentiation (J. Uronis and L. Reid, unpublished data, 2010). Matrix molecules such as proteoglycans and especially HS-PGs and HP-PGs have many growth factor–binding sites that determine growth factor storage, release, conformation, stability, and affinities for specific receptors as well as other aspects of the signal transduction processes. Therefore, completion of the ongoing studies seeking to define the lineage-dependent, soluble paracrine signals should allow future studies on mechanisms by which paracrine signaling, involving synergies between the soluble signals and the matrix components, dictates the cell responses.

In summary, the interdependency of parenchymal cells and their mesenchymal companions is a stringent constraint on stem cell and maturational lineage biology, and it has been mimicked by the use of feeders. The uniformity of the cell population within a feeder cell line facilitates the analyses of cell-cell and cell-matrix interactions but ignores that mesenchymal cells mature coordinately with epithelia. This maturation is associated with changes in the paracrine signaling. In addition, feeder cell lines stably maintained in an animal serum have muted effects with respect to those kept serum-free and are barriers for clinical programs and commercial and research applications because of concerns about unidentified factors and pathogens in the serum. Thus, the identification of the matrix and soluble signals that control the fate of stem cells is critical for translating the use of normal cells into the realms of reproducibility and effectiveness. Our success in generating cultures of stem cells with specific biological fates is possible because of the use of specific paracrine signals (both matrix and soluble) and the recognition that serum has to be eliminated to the extent possible. In addition, the ability to generate reproducibly uniform cultures of liver parenchymal cells maintained at a precise maturational lineage stage represents an important step for the development of safe stem cell–based therapy and drug development as well as model systems for analyzing development.

Acknowledgements

The authors thank Lucendia English for her technical assistance, Dr. Michael Chua and the staff of the Michael Hooker Microscopy Core Facility (University of North Carolina), the staff of Cell Services (University of North Carolina), Dr. Victoria Madden of the Microscopy Services Laboratory in Pathology and Laboratory Medicine (University of North Carolina), and the staff of the Histology Core Facility (University of North Carolina). The findings of some of these studies have been included in patent applications that belong to the University of North Carolina.

Ancillary

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