Selection, Enrichment, and Maintenance of Self-Renewal Liver Stem

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Biomacromolecules 2010, 11, 994–1001

Selection, Enrichment, and Maintenance of Self-Renewal Liver Stem/Progenitor Cells Utilizing Polypeptide Polyelectrolyte Multilayer Films Hsuan-Ang Tsai,† Ruei-Ren Wu,‡ I-Chi Lee,† Hsiao-Yuan Chang,§ Chia-Ning Shen,*,†,‡,§ and Ying-Chih Chang*,† Genomics Research Center, Academia Sinica Taipei 115, Taiwan, R.O.C., Department of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei 112, Taiwan, R.O.C., and Institute of Microbiology and Immunology, National Yang-Ming University, Taipei 112, Taiwan, R.O.C. Received December 23, 2009; Revised Manuscript Received March 11, 2010

Recent progress has led to the identification of liver stem/progenitor cells as suitable sources for generating transplantable liver cells. However, the great variability in methods utilized to isolate liver stem/progenitor cells is a considerable challenge for clinical applications. The polyelectrolyte-multilayer technique can constitute a useful method for selective cell adhesion. Whether enrichment of liver stem/progenitor cells can be achieved utilizing polypeptide polyelectrolyte-multilayer films was investigated in current work. Fetal liver cells isolated from E13.5 mouse embryos were seeded on the poly-L-glutamic acid/poly-L-lysine alternating films, and we revealed that fetal liver stem/progenitor cells were selected and formed colonies. These undifferentiated colonies were maintained on the films composed of four alternating layers, with the topmost poly-L-glutamic acid layer judged by the constitutive expression of stem-cell markers such as Dlk-1, CD49f, and CD133 and self-renew markerβ-catenin. Our work has demonstrated that highly tunable polyelectrolyte-multilayer films were suitable for selective enrichment of liver stem/progenitor cells in vitro.

Introduction Liver transplantation is the most efficient treatment for endstage liver disease.1 However, the major limitation is the shortage of donor livers. Recent success in the isolation of liver stem/progenitor cells has raised expectations that it might be possible for curing end-stage liver disease by transplanting liver stem/progenitor cells or their mature progenies.2,3 Several reports have described the expression of certain surface markers on liver stem/progenitor cells including c-Met, CD49f, Dlk-1, and CD133, which can possibly be utilized for cell purification.2,4-7 However, it is currently infeasible to translate these documented studies for clinical applications because of the great variability in methods used to isolate and culture liver stem/progenitor cells.8,9 Polyelectrolyte multilayer (PEM) films are made by utilizing a layer-by-layer (LbL) technique to form alternative polycationic and polyanionic layers based on nonstochiometric electrostatic interactions.10-13 Through the LbL technique, a wide variety of surface physical properties such as surface charges, film rigidity, hydrophilicity, and functionality can be modulated with high precision through layer sequences, number of depositing layers, and chemical compositions. Strategies to fabricating functional PEM films for the biological applications such as antimicrobial coating, artificial skin, and cell culture coating have been successfully demonstrated.14-16 * To whom correspondence should be addressed. Phone: +886 2 2789930 (Y.-C.C.); +886 2 27899589Ext. 301 (C.-N.S.). Fax: +886 2 27899931 (Y.-C.C.); +886 2 27899587 (C.-N.S.). E-mail:[email protected] (Y.-C.C.); [email protected] (C.-N.S.). † Genomics Research Center. ‡ Department of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University. § Institute of Microbiology and Immunology, National Yang-Ming University.

Among them, PEM films based on alternating cationic and anionic polypeptides, poly-L-lysine (PLL) and poly-L-glutamic acid (PLGA), have been fabricated through the LbL technique as the cell culture coatings materials for chondrosarcoma cells and human gingival fibroblasts.17-20 As cell membranes carried negative charges in the serum-free medium,21 cells consistently exhibited better adhesion on the positively charged PLL-ending than on the PLGA-ending PEM films.18-20 Conversely, in a serum-containing medium, serum proteins adsorbed on the PLLending films, thus, reversing the surface net charges from positive to negative; as a result, cell adhesion force showed a significant reduction on the PLL-ending films.18 Chondrosarcoma cells, human gingival fibroblasts, and liver cells adhered better on the thinner or cross-linked PLL/PLGA PEM films,18-20 suggesting that these cells favored more rigid surfaces mechanically.22 Previously, we have established the characterization scheme to investigate PLL/PLGA surface complexes. We have combined ellipsometry, Fourier transform infrared spectrometery (FTIR), circular dichroism (CD), and atomic force microscopy to characterize the film thickness, refractive index, and secondary conformation of the complexes. We have found that under the physiological condition (pH ) 7.4), individual PLL or PLGA films adopted random coil structures. When the PLGA adsorbed on PLL coating through the electrostatic interaction, the secondary conformation of the surface film underwent transition from random coils to predominantly β-sheet conformation, as measured by both FTIR and CD.23 In addition to the commonly discussed surface properties that can be regulated by PEM process, such as surface charges, serum adsorption, and film rigidity, we would also like to include the conformational effects of the surface coating to the fetal liver cell cultures. In this work, ellipsometry and zeta potentials were the primary tools to establish the correlations of the dielectric and the surface charges

10.1021/bm901461e  2010 American Chemical Society Published on Web 03/25/2010

Self-Renewal Liver Stem/Progenitor Cells

of the films to the depositing number of layers and sequences. The liver stem cell cultures on the series of PLL/PLGA films were also compared with those on the collagen I, collagen IV, laminin, poly-L-ornithine (PLO), and fibronectin. Our primary goal is to select, enrich, and maintain the undifferentiated status of fetal liver stem/progenitor cells (FLSPCs), which is critical for translational applications.

Materials and Methods Isolation and Culture of Mouse Fetal Liver Cells and FLSPCs. All experiments using mice were approved by the Academia Sinica Institutional Animal Care and Utilization Committee. Single cell suspensions of liver cells were prepared from ED13.5 embryos of CD1 mice (BioLASCO, Taipei, Taiwan). Fetal livers were cut into small pieces and digested at 37 °C in Hanks’ balanced salt solution (Gibco, Invitrogen, Gaithersburg, MD) containing 2.5 mg/mL collagenase B (Roche, Basel, Switzerland), 2.4 U/mL dispase (Invitrogen, Grand island, NY), 2.5 mM CaCl2 (Sigma, St Louis, MO), and 2% heatinactive fetal bovine serum (Hyclone, Logan, UT). For isolation of FLSPCs, dissociated fetal liver cells were incubated with PE-conjugated anti-CD49f (BD Biosciences, San Jose, CA) and anti-Dlk-1 (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies and then subsequently incubated with FITC-conjugated antimouse IgG (Vector, Burlingame, CA). After washing with PBS, CD49f+Dlk-1+ FLSPCs were sorted using FACS Vantage (BD Biosciences, San Jose, CA). In some experiments, either FLSPCs or fetal liver cell suspensions were resuspended in DMEM/F12 medium (Invitrogen, Grand island, NY) containing 10% heat-inactive fetal bovine serum, 1× insulin/ transferring/selenium (ITS; Invitrogen, Grand island, NY), 10 mM nicotinamide (Sigma, St. Louis, MO), 20 µg/mL gentamicin, 10 nM dexamethasone (Dex; Merck, Whitehouse Station, NJ), 5 mM Lglutamine (Sigma, St. Louis, MO), and 50 µM β-mercaptoethanol (βME), and then were cultured on 35 mm tissue culture dishes or glass coverslips coated with collagen IV, PLO, laminin, or polypeptide PEM films. To demonstrate the differentiation properties of FLSPCs to hepatocytes, culture medium was replaced with DMEM/F12, 10% FBS, 10 mM nicotinamide, 20 µg/mL gentamicin, 100 nM dexamethasone, 50 µM β-ΜE, 20 ng/mL hepatocyte growth factor (HGF; R&D, Minneapolis, MN), 10 ng/mL oncostatin M (OSM; R&D, Minneapolis, MN), 20 ng/mL epidermal growth factor (EGF; R&D, Minneapolis, MN). Synthesis of Physically Coated Polypeptide PEM Films. The preparation of polypeptide PEM films was followed the procedure as previously described with some modifications.18,20,24 Initially, silicon oxide based substrates (silicon wafers or glass coverslips) were first cleaned with piranha solution and subsequently washed with distilled water and rinsed with acetone. Substrates were then dried under a stream of nitrogen and treated with plasma cleaner. After cleaning, physical deposition of PEM films was performed by batch and static conditions as follows: initially, all polypeptides were dissolved in 10 mM TrisHCl buffer with 0.15 M NaCl, pH ) 7.4. Substrates were then immersed in PLL (MW ) 15000-30000; Sigma, St Louis, MO) solution (1 mg/ mL) for 10 min at room temperature, followed by rinsing with 1 mL of Tris-HCl buffer for 1 min. To couple PLGA, the PLL-coated slide was subsequently immersed in the PLGA solution (MW ) 3000-15000, Sigma, St Louis, MO, 1 mg/mL) for 10 min, followed by rinsing with 1 mL of Tris-HCl buffer for 1 min. Lastly, substrates were cleaned with fresh PBS to remove uncoupled polypeptides. The resulting c-(PLL/PLGA)i, where i was denoted as the number of polyelectrolyte pairs generated by repeating the above steps: i ) 0.5 was referred to c-PLL only, i ) 1 was referred to c-(PLL/PLGA)1, and so on. Analysis of Thickness, Refractive Index, and Surface Electrostatic Potential of PEM Films. Ellipsometric analysis of physical depositing polypeptide PEM films were measured with a Gaertner LSE stokes ellipsometer with a He-Ne laser (λ ) 632.8 nm) at a fixed incident angle of 70°. The thickness and refractive index of

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PEM films were then calculated by the software of Gaertner Ellipsometer. To measure the thickness and refractive index of the films in aqueous conditions, a liquid cell was used (Gaertner Inc.). The Ψ and ∆ angles were recorded until equilibrium was reached. The measurement of Ψ and ∆ was then used to calculate the film thickness and refractive index. The refractive index of air and buffer solution was assigned as 1 and 1.334, respectively. The zeta potential analysis (Anton Pear-Electro Kinetic Analyzer, Graz, Austria) was applied after polypeptide PEM films immersed in a stream potential of 10-3 M KCl solution (pH ) 7.4). Statistical Analysis. The data for thickness and refractive index measurements was displayed as a mean ( standard deviation from an average of at least five measuring spots on each sample. For zeta potential analysis, each experiment was independently repeated six times. RNA Extraction and RT-PCR. Total RNA was extracted from FLSPC colonies using Trireagent (Sigma, St. Louis, MO) and reverse transcribed using Superscript III (Invitrogen, Grand Island, NY) according to the manufacturer’s manual. A total of 5 µg of the purified total RNA was used for reverse transcription to generate cDNAs for PCR reactions. Details of the sequence of primers are listed in the Supporting Information. Immunofluoresent Staining. Immunofluoresent staining was performed as follows: briefly, FLSPC colonies were fixed in 4% paraformaldehyde for 30 min at room temp followed by permeabilization utilizing 0.1% Triton X-100. After blocking with 2% Roche blocking reagent, cells were incubated with primary antibody overnight at 4 °C and with secondary antibody for 2 h at room temp. The primary antibodies were used at the following dilutions: Dlk-1 (1/50; Santa Cruz Biotechnology, Santa Cruz, CA), β-catenin (1/500; BD Biosciences, San Jose, CA), albumin (ALB; Sigma, St. Louis, MO), R-fetoprotein (AFP; Dako Cytomation, Glostrup, Denmark), cytochrome P450 3A1 (CYP3A1; Chemicon International, Temecula, CA), and glutamine synthetase (Gln Syn; Bd Biosciences, San Jose, CA). The secondary antibodies were used at the following dilutions: fluorescein antirabbit IgG (H+L; 1/300; Vector Laboratories, Servion, Switzerland), fluorescein antimouse IgG (H+L; 1/300; Vector Laboratories, Servion, Switzerland), and fluorescein antigoat IgG (H+L; 1/300; Vector Laboratories, Servion, Switzerland). Images of the immunostained specimen were obtained either using a SPOT camera mounted on a Zeiss Axioplan 2 microscope or using confocal microscopy Leica TCS SP2 AOBS.

Results Isolation, Verification, and In Vitro Study of Mouse FLSPCs. Based on the FLSPCs isolated from rodent, several recent studies have suggested their potential to differentiate both hepatocytes and ductal epithelial cells.25 For example, Sharferitz and colleagues recently demonstrated isolated FLSPCs were fully capable of repopulating the adult liver.2 Flow cytometry analysis of FLSPCs has revealed the surface expression of Dlk1, CD49f, and CD133 in these cell populations.5-8,26,27 As shown in Figure 1A, 1.7% of the unfractionated fetal liver cell population was positive for both FLSPC markers, Dlk-1 and CD49f. The characteristics of CD49f+Dlk-1+ fetal liver cells were further determined by RT-PCR (Figure 1B), which further revealed these cells expressing other FLSPC markers: CD34, CD133, c-Met, AFP, and ALB.4,5,7,28 We also confirmed that CD49f+Dlk-1+ fetal liver cells did not express the late hepatic marker-tyrosine aminotransferase (TAT), indicating this cell population was the FLSPC. We then tried to explore whether the growth capability of CD49f+Dlk-1+ FLSPCs can be maintained in vitro. When CD49f+Dlk-1+ FLSPCs were seeded on laminin-coated dishes, only approximately 0.3% of the sorted cells were able to form

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Figure 1. Characterization of Dlk-1+CD49+ fetal liver cells. (A) E13.5 fetal liver cells were sorted by FACS system. Sorting gates were first set for the propidium iodide (PI) stained negative region and then Dlk-1+CD49f+ and Dlk-1-CD49f+/hi subpopulations were sorted and isolated. (B) RT-PCR analysis was used to determine the expression of CD34, CD133, Dlk-1, CD49f, c-Met, AFP, ALB, and TAT in sorted and unsorted fetal liver cells. GAPDH was performed as a control. (C) Phase-contrast images of colonies formed from Dlk-1+CD49f+ cells cultured on a laminincoated dish. (D) Dlk-1+CD49f+ cells were treated with OSM-containing differentiation medium for 5 days, and then cells were fixed and stained for ALB (green) and cell nuclei were stained with Hoechst 33342 dye (Blue). (E) The phase-contrast images of (D).

hepatic colony-forming units in cultures (Figure 1C). Further induction of the sorted cells with the OSM-containing differentiation medium would result in spreading out of colony cells. These cells expressed ALB by the immunostaining analysis (Figure 1D) and exhibited an epithelial-like morphology (Figure 1E). Because colony-forming efficiency was too low for further studies, we next attempted to determine whether we could enrich stem cell population from unfractionated liver cell suspensions by coating culture wares with different extracellular matrix proteins or polymers. We seeded unfractionated liver cells on culture dishes coated with collagen I, collagen IV, laminin, PLO, and fibronectin, respectively. We found that hepatic colony-forming units expressing Dlk-1, AFP, and selfrenewal marker-β-catenin could only be observed in culture dishes that were coated with collagen IV or PLO (Figure 2A-I). RT-PCR confirmed that some colony-forming cells expressed CD34, CD133, ALB, and AFP. However, we also found a large amount of stromal cells also adhering to those dishes coated with collagen IV (Figure 2J), which possibly resulted in an inability to maintain the undifferentiated status as colony cells quickly spread out and appeared to have a hepatocyte-like morphology (arrow cells, Figure 2K). Results from RT-PCR showed that some colony-forming cells could be maintained and express CD34, CD133, AFP, and ALB in the undifferentiated state for about 4 days, and then FLSPC colonies spontaneously differentiate into late hepatic lineages, as judged by the expression of TAT (Figure 2L) after 8 days in cultures. The results were very similar to differentiated colony cells induced by the OSM treatment (Figure 2L).

For a further comparison, the FLSPC colonies were maintained on the PLO-coated surface for longer durations, up to 6 days before differentiation. PLO has been applied in other stem cell cultures successfully.29-32 The capability of forming undifferentiated FLSPC colonies on the PLO-coated surface may imply that artificial polymers can be utilized to generate a suitable surface for adhesion and growth of FLSPCs. Because the PEM technique provides a higher tunable surface through nanoscale control of electrostatic interactions, it is feasible to further investigate whether tuning the surface properties of PLL/ PLGA PEM films can offer better selectivity and maintenance of FLSPCs. Preparation and Characterization of PEM Films. The PEM films started with the PLL adsorption on a negatively charged silicon wafer or glass coverslip, forming a physically coated PLL film (c-PLL, sample 1), followed by PLGA adsorption generating c-(PLL/PLGA)1 (sample 2), and so forth. In this study, the films were prepared up to 5 cycles, with 10 samples in total. Ellipsometry has been utilized to measure the film thickness and refractive index simultaneously for all samples. It has shown that the film thickness was increased with the number of layers, where the 14 nm of c-PLL was the thinnest and 145 nm of c-(PLL/PLGA)5 was the thickest. On the other hand, the refractive indices of the films were varied from 1.419 to 1.397. The film hydration (Table 1, column 4) can be calculated by an approximate formula from pervious studies.33,34 We found the hydration of the c-form PEM films vary from 60% up to 80%. These data confirmed the successful LbL deposition and



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Figure 2. Effect of extracellular matrix proteins on culturing unfractionated fetal liver cells. Unfractionated fetal liver cells isolated from E13.5 embryos were either cultured on collagen IV- (A, D, G, J, K), laminin- (B, E, H), and PLO-coated (C, F, I) dishes, respectively. Expression of Dlk-1 (A-C), AFP (D-F), and β-catenin (G-I) was determined by immunostaining. The phase-contrast images of the FLSPC colony formed on collagen IV-coated dishes on days 2 (J) and 4 (K) in cultures. (L) RT-PCR was utilized to determine the expression of FLSPC markers (CD34, CD133, AFP, and ALB) and hepatic differentiation markers (TAT) in fetal liver cells cultured on collagen IV-, laminin-, and PLO-coated dishes and in OSM-containing differentiation medium. Table 1. Physical Properties (Thickness, Refractive Index, Hydration, and Zeta Potential) of PEM Films in pH ) 7-7.4 Buffer Solution vs FLSPC Colony Maintenance Ratio on Different Types of PEM Films on Day 8 sample No.

sample type

d (Nf)a (nm)

film hydrationb (%)

zeta potentialc (mV)

1 2 3 4 5 6 7 8 9 10

c-PLL c-(PLL/PLGA)1 c-(PLL/PLGA)1-PLL c-(PLL/PLGA)2 c-(PLL/PLGA)2-PLL c-(PLL/PLGA)3 c-(PLL/PLGA)3-PLL c-(PLL/PLGA)4 c-(PLL/PLGA)4-PLL c-(PLL/PLGA)5

14 (1.419) 24 (1.402) 43 (1.391) 51 (1.388) 64 (1.377) 74 (1.377) 93 (1.380) 115 (1.378) 129 (1.388) 145 (1.397)

59 ( 1 67 ( 1 71 ( 1 72 ( 2 78 ( 1 77 ( 2 76 ( 3 77 ( 3 72 ( 2 68 ( 2

30 ( 3 -21 ( 1 25 ( 1 -14 ( 1 17 ( 1 -12 ( 2 16 ( 1 -6 ( 1 14 ( 1 -6 ( 2

zeta potential with serumd (mV) -17 ( 1 -18 ( 2

-4 ( 1 -10 ( 1

day 8 colony maintenance ratioe (%) 3(2 2(1 10 ( 8 10 ( 2 9(2 44 ( 5 15 ( 5 52 ( 7 15 ( 8 40 ( 7

a PEM films measured by ellipsometry in Tris-HCl buffer, pH ) 7.4 at 25 °C (d is thickness and Nf is refractive index. The maximum standard deviation to the range of thickness and refractive index are (3 nm and (0.005, respectively.) b An approximation of the film hydration is calculated by the equation: θ Np + (1 - θ)NS ) Nf33,34 (θ is the volumetric fraction of the polymer; Np and NS are the refractive indices of the dehydrated polymer and solvent, respectively). c PEM films were immersed by zeta potential measurement in 10-3 M KCl(aq). d PEM films were immersed in 10% serum-containing medium for 1 h before zeta potential measurement in 10-3 M KCl(aq). e The day 8 maintenance ratio is defined as the ration of the day 8 colony number to the day 2 colony number.

showed that the film thickness (or the rigidity) was readily controlled by the number of layers, with the span from the order of 10 to 100 nm. Zeta potential measurements were also performed to monitor the charge accumulation near surfaces. As summarized in Table 1 (column 5), all PLL-ending PEM films displayed positive zeta potential, and all PLGA-ending PEM films displayed negative zeta potential. These results confirmed that the net surface charge signs were identical to the charge signs of the topmost layers, indicating charge overcompensation. Nevertheless, for all films, the absolute values of zeta potentials were initially quickly decreased with each additional layer (i ) 1-3), then reaching

a plateau for the more layer build-ups (i ) 4 and 5). These observations were in good agreement with those in previous studies.18,35 To further understand the surface charge distribution in the FLSPCs culturing condition, the PEM films were immersed in the serum-containing medium for 1 h, followed by zeta-potential measurements. As shown in Table 1 (column 6), the PLGA-ending PEM films remained negative with a slight increase of zeta potentials, suggesting possible adsorption of a small amount of positively charged serum protein. However, all PLL-ending PEM films switched from positive to negative values, suggesting a considerable amount of negatively charged serum protein adsorption. Our results are consistent with the

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Figure 3. Average numbers of FLSPC colonies formed on laminin, collagen IV, PLO, glass, and PEM films were calculated on days 2, 4, 6, and 8, respectively. Four samples were examined for each coating. Data are mean ( s.d.

previous studies.18 In summary, both ellipsometry and zeta potential measurements of PEM films in serum-free conditions have confirmed the successful PEM film construction. The zetapotential measurements in serum-containing medium reflected the actual surface charges that cells encountered. In this case, both PLGA-ending and PLL-ending PEM films carried negative charges in the serum-containing medium. We have also immersed all PEM films in PBS buffer for 8 days to examine their stability in solutions. The ellipsometric study showed negligible film depletion. For example, c-(PLL/ PLGA)4 film changed its thickness from the original 115 nm (Nf ) 1.378) to 110 nm (Nf ) 1.379) after 8 days in PBS buffer. The film reduction was less than 5%. Culture of FLSPCs on PEM Films. We then used these PEM films, together with the controlled coatings, laminin, collagen IV, PLO, and glass, for fetal liver cells cultures. With initial 1 × 106 fetal liver cells seeding, semiattached cell colonies with an average size around 40-50 µm, through a rough estimation, appeared on all coatings on day 2. Figure 3 summarizes the colony counts per cm2 on all samples. On day 2, colony numbers ranged from 150 to 350 colonies/cm2. Among the samples, more colonies were present on the PLGA-ending PEM films, laminin, collagen IV, and PLO coatings consistently than on the c-PLL, PLL-ending PEM films, or glasses. Specifically, all PLGA-ending PEM samples obtained ∼300 colonies/ cm2 compared to ∼200 colonies/cm2 on the PLL-ending PEM films. Colony numbers were decreased on all samples on days 4, 6 and 8, conceivably due to the spontaneous differentiation and cell loss during medium washes and exchanges. Interestingly, while laminin, collagen IV, and PLO were initially good materials to enrich colonies on day 2, the maintenance ability was poor. Virtually no colonies were left on these coatings by the end of day 8. Quite on the contrary, with the exception of c-PLL, all PLL/PLGA complexes demonstrated excellent maintenance ability. Table 1 (column 7) tabulated their counting ratios () number of colonies on day 8/number of colonies on day 2, called “day 8 maintenance ratios”) for further elucidation. We found that thicker PLGA-ending PEM films (e.g., c-(PLL/ PLGA)3-5) were the most effective surfaces in maintaining FLSPC colonies. Among them, 146 colonies (on day 8) out of

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283 colonies (on day 2), or 52% of colonies were maintained on the c-(PLL/PLGA)4 films for 8 days. The stem cell characteristics of colonies cultivated on c-(PLL/ PLGA)4 films after a 2-day or 8-day culture were confirmed by immuno-staining and RT-PCR analysis. As shown in Figure 4, these colonies were positive for markers of FLSPCs, including Dlk-1, CD133, CD49f, AFP, ALB, and the self-renewal marker β-catenin. It is noted that the average size of the remaining colonies on day 8 is markedly larger than those colonies on day 2. Based on colony image analysis, we found that the average area of c-(PLL/PLGA)4 colonies on day 8 was 1.6fold larger than those colonies on day 2. The results clearly demonstrated that these cells were expanding. Differentiation of FLSPCs into Hepatocytes on PEM Films. Differentiation potentials of FLSPC colonies on c-(PLL/ PLGA)4 were further investigated by replacing with OSMcontaining serum medium after 4-day cultures. After induction with OSM-containing medium for 4 (Figure 5A), 7 (Figure 3A, Supporting Information), and 14 day periods (Figure 3B, Supporting Information), the cytochrome 450 type 3A1 (CYP3A1), a detoxification enzyme expressed by matured hepathocytes, were observed clearly. The staining of glutamine synthetase (Gln Syn), a marker of perivenous hepatocytes, was observed in those cells spreading out from the colonies (Figure 5B). As a comparison, both CYP3A1 and Gln Syn staining on the FLSPC colonies on collagen IV with OSM for 4-day induction periods were shown on Figure 5C,D. The results show that FLSPC colonies can be successfully inducted to mature hepatocytes by OSM induction medium on both PLL/PLGA complex and collagen IV surfaces.

Discussion Isolation of specific cell populations having stem cell characteristics is one of the most important tasks in stem cell research. Fluorescence-activated cell sorting (FACS) and its derivatives36-38 have been widely used for stem cell isolation; however, FACS is sometimes not practical for many applications due to the inherent complexities such as the difficulty to remove antigen-antibody on cell membranes afterward.38,39 A labelfree system that can selectively enrich certain cell populations could be a better strategy for stem cell isolation. For example, hepatocytes were found to adhere efficiently on sulfonic PEM films.40 Positively charged surfaces were also commonly used to adhere primary neuronal cells.30 Our approach has shown that by properly tuning the surface properties, PLL/PLGA-based PEM films are excellent to purify FLSPCs from the primary culture. As a rough estimation, there were about 45000 stem cells (based on the 350 colony counting with approximately 125 cells in each colony) obtained from the initial 106 seeding on c-(PLL/PLGA)1 on day 2. The amount harvested based on our strategy was comparable with that obtained from FACS and by far better than any tested conventional coatings such as laminin, collagen IV, PLO, or glass. It is of our interest to further identify surface properties of PEM films that play a role in determining selection, enrichment, and maintenance of FLSPCs. By examining the cell types and cell population appearance both on day 2 and day 8, we are able to roughly categorize these samples into four groups. (1) c-PLL films: On day 2, very few FLSPCs were present. Fetal hepatocytes and liver stromal cells were the majority (Figure 6A). On day 8, no FLSPCs were present, and fetal hepatocytes and stromal cells were expanded all over the plates (Figure 6A1). The c-PLL films appear to be poor in both selecting and



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Figure 4. Characterization of FLSPC colonies formed on PEM films. Immunotaining of FLSPC colonies including Dlk-1, AFP, and β-catenin and phase-contrast images on c-(PLL/PLGA)4 PEM films were shown after 2 days (A-D) and 8 days (E-H) culture, respectively. Cell nuclei were stained with Hoechst 33342 dye (Blue). The bar is 50 µm. (I) RT-PCR analysis was utilized to determine expression of FLSPC markers (CD133, Dlk-1, CD49f, AFP, and ALB) and hepatic differentiation markers (TAT) from E13.5 liver and day-8 colony cells formed on c-(PLL/PLGA)4 PEM films, respectively.

Figure 5. Hepatic differentiation potential of FLSPC colonies formed on PEM films and collagen IV-coated surface for 4 days, followed by induction medium treatment. (A, B) CYP3A1 and Gln Syn (green) expression from differentiated FLSPC colonies cultured on c-(PLL/ PLGA)4 with OSM-containing medium induction for 4 days. (C, D) CYP3A1 and Gln Syn (green) expression from differentiated FLSPC colonies cultured on collagen IV-coated surfaces with OSM-containing medium induction for 4 days. Cell nuclei were stained with Hoechst 33342 dye (blue). The bar is 50 µm.

maintaining FLSPCs. (2) PLL-ending PEM films: On day 2, FLSPC colonies and stromal cells were present in low density. No fetal hepatocytes were observed (Figure 6B). On day 8, FLSPC colonies were spread out, indicating spontaneous differentiation (Figure 6B-1). The PLL-ending PEM films appear to be excellent in selecting but poor in maintaining FLSPCs. (3) Thin PLGA-ending PEM films: On day 2, FLSPC colonies and stromal cells were present in high density and no fetal hepatocytes were observed (Figure 6C). On day 8, around 2∼10% of FLSPC colonies and stromal cells were maintained (Figure 6C-1). The thin PLGA-ending PEM films appear to be excellent in selecting and enriching but less capable of

Figure 6. Three featured groups obtained from fetal liver cells cultured on different PLL/PLGA PEM films based on morphologic appearance. (A, A-1) The phase-contrast images of fetal liver cells cultured on c-PLL films. Images were either observed on day 2 (A) or day 8 (A1). (B, B-1) The phase-contrast images of fetal liver cells cultured on PLL-ending PEM films. Images were either observed on day 2 (B) or day 8 (B-1). (C, C-1, C-2) The phase-contrast images of fetal liver cells cultured on PLGA-ending PEM films. Images were either observed on day 2 (C) or day 8 (C-1, C-2). The film thickness was below (C-1) and above 50 nm (C-2). Scale bar is 100 µm.

maintaining FLSPCs. (4) Thick PLGA-ending PEM films: On day 2, the cell images are similar to those of thin PLGA-ending PEM films. However, over 40% of FLSPC colonies and stromal cells were maintained on these films over 8 days (Figure 6C2). Thick PLGA-ending films are excellent in selecting, enriching, and maintaining FLSPCs among all films. FLSPCs Selectivity. Among four categories, only c-PLL film has no capability to select FLSPCs. Instead, it provides an environment that promotes adhesion and growth of fetal hepatocytes and liver stromal cells. However, when forming the PLL/PLGA molecular complex, whether they were PLLending or PLGA-ending, the surface conditions seem to discourage the growth of hepatocytes and stromal cells, instead, allowing FLSPCs to adhere and to form semiattached colonies. Further examination of the film thickness, refractive index, and zeta potential of all samples (Table 1) suggested that c-PLL

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is the thinnest, highest charged surface, however, the differences from other samples are marginal. Previous studies have demonstrated that specific surface domains could promote selective cell adhesion. For example, fibroblasts could effectively adhere to the surface of the PEM film that was comprised of (GVGVP)40 segments, a sequence recognizing cell membrane protein receptor elastonectin.41 Tissue cell attachments were improved on a self-assembled peptide nanofiber, RADA16, coupling with different functional peptide motifs.42,43 In our case, it was known that c-PLL adopted random coils by forming multivalent attachments with the SiOx surfaces. However, the surface complexes of PLL and PLGA self-assembled to predominantly an anti-β-sheet structure.23,44-46 Meanwhile, previous studies have shown polypeptide PEM films were successfully applied to the chiral separation of amino acids and drugs through their specific molecular interactions.47-49 This may provide one plausible explanation that PLL/PLGA complex forming distinctive structures that effectively mimicked specific domain features to target surface molecules of FLSPCs. On the other hand, the random coiled c-PLL failed to form such a domain, therefore, less effective in selecting FLSPCs. FLSPCs Enrichment. As summarized in Table 1, we found that while both PLL-ending and PLGA-ending films supported FLSPCs selection and colony formation, as shown in day 2 results, the number of colonies formed on PLGA-ending was distinctively higher than those on PLL-ending PEM films. This indicates that ending layer can further modulate forming efficiency of FLSPC colonies. Based on zeta potential analysis, PLL-ending films have positively charged surfaces in KCl solution but in reality carried large negative charges in serumcontaining medium. On the other hand, there is little zeta potential value changes for the PLGA-ending films in serumcontaining medium (Table 1).18 To interpret the serum effects, we further measured the ellipsometric thickness/refractive indices of the PEM films before/after immersing in 10% serum or 1 mg/mL bovine serum albumin (BSA). The film thickness changes of the PLGA-ending sample before and after serum or BSA adsorption are negligible, while the thickness changes of the PLL-ending sample were increased substantially, from 130 to 150 nm (after serum treatment) and to 140 nm (BSA only; Table 2, Supporting Information). Ellipsometry data support the changes of zeta potentials, suggesting significant amount of negatively charged serum proteins adsorbed to the positively charged PLL-ending PEM films, thus, reversing the surface charges from positive to negative. Both zeta potential and ellipsometry data clearly suggested that both surfaces carried negative charges. On the “PLL-ending” films, however, the surface was in fact covered with serum proteins. This offers an explanation why the FLSPC colony density on the PLL-ending is less than that on the PLGA-ending PEM films on day 2, and the FLSPCs on all PLL-ending films are prone to differentiate regardless of the film thickness, as evidenced by the low maintenance ratio on day 8. The serum protein adlayers may have blocked the specific polypeptide domains of PLL/PLGA complexes; the “PLL-ending” PEM films are less likely to be recognized by the FLSPCs than on the PLGA-ending complexes. FLSPCs Maintenance. The thicker PLGA-ending PEM films were found to perform better for maintaining FLSPCs for a longer period in vitro than the thinner PLGA-ending PEM films (Figure 6C-2 and Table 1). As shown in Table 1, on day 8, over 40% of FLSPC colonies were still maintained on the thicker films, c-(PLL/PLGA)3-5 (74, 115, and 145 nm). On the contrary, merely 2∼10% of colonies were maintained on the thinner films,

Tsai et al.

c-(PLL/PLGA)1,2 (24 and 51 nm). FLSPC colonies on the thinner PLGA-ending PEM films spread out on day 8, as evidenced by the morphological changes (Figure 6C-1), but were maintained on the thicker PLGA-ending PEM films (Figure 6C2) For the thinner films with the thickness below 50 nm, the hardness of the underneath glass substrate played an important role to the overall surface mechanical property.50 Second, the higher content of water adsorption in films led to softer films, which were indicated by the lower refractive index values of the films.33,51,52 Both glass substrate effect and the water adsorption have resulted in softer surfaces on the thicker film samples than on the thinner film samples. Previously, the softness of films was thought to suppress cell adhesion.51,53-55 In our case, the softness of the thicker PEM films (80% of hydration) seemed to effectively reduce the number of other cells such as fibroblast-like cells while promoting the undifferentiated state of FLSPC colonies. These observations were in good agreement with those in previous studies.56,57 This could be explained by three effects: (1) In this work, adhesion of fibroblasts-like cells was effectively suppressed by the softer surfaces, thus, FLSPCs becoming more competitive for the space. (2) Maintenance of FLSPCs requires that FLSPC colonies do not fully adhere. The spreading of FLSPC colonies, that is, adhesion of FLSPC colonies, in fact leads to differentiation. Therefore, soft surfaces that suppressed cell adhesion in turn facilitating FLSPC colony maintenance. (3) Apart from the surface effects, the interaction between the FLSPC colony and the fibroblast cells might have played a role in influencing the maintenance of FLSPC colonies. Culturing isolated FLSPC colonies on the respective surfaces is, upon further investigation, to verify the influences of the cell-cell interactions.

Conclusion PLL/PLGA PEM films displaying certain physical characteristics were successfully fabricated through tuning the number of depositing layers and surface compositions and were utilized for FLSPCs selection, enrichment, and maintenance. In current work, we have shown that mouse FLSPCs could selectively adhere to the specific PLL/PLGA PEM films and form colonies. We revealed that both layer sequences and the thickness of PLL/ PLGA PEM films played important roles in forming FLSPC colonies. We were able to identify the optimal conditions for maintenance and differentiation of FLSPCs on the PLL/PLGA PEM films. In particular, thick (i ) 3-5) PLGA-ending PLL/ PLGA films were found to have the best properties for the isolation, enrichment, and long-term maintenance of FLSPCs, among all currently tested conventional surfaces. We anticipate that this new platform can facilitate transplantable human liver stem cells in the future, and we will continue to apply this strategy as the materials design guideline for stem cell research. Acknowledgment. H.-A.T. was the main contributor to the work on PEM films. R.-R.W. and H.-Y.C. contributed to the isolation and characterization of FLSPCs, and I.-C.L. performed RT-PCR on analyzing stem cell markers of FLSPCs. H.-A.T., C.-N.S., and Y.-C.C. conceived and designed the experiments, analyzed the data, and wrote the paper. The authors thank Ms. Wen-Wen Chen, Wan-Yu Mao, and Dr. Chung-Lieng Li (GRC, Academia Sinica) for the flow cytometric analysis, and Dr. KueiHsien Chen and his group (IAMS, Academia Sinica) for zeta potentials. Financial support was obtained from Genomics Research Center, Academia Sinica, Taiwan, Summit Project Grants of Academia Sinica #5202402020-0, National Science


Council Grants 95-2311-B-001-057-MY3 & 98-3111-B-001005 to C.-N.S., 94-2120-M-003-001, 95-2120-M-003-002, and 96-2120-M-003-003 to Y.-C.C., and Postdoctoral Grants 952811-M-001-082, 96-2811-M-001-009 to H.-A.T. Supporting Information Available. Day 2 and day 8 fetal liver cell morphologies, as observed on all PEM films, respectively; Hepatic differentiation potential of FLSPC colonies formed on PEM films after long-term induction by OSMcontaining differentiation medium; Summary graph of physical properties (thickness, hydration, and zeta potential) of PEM films and numbers of FLSPC colonies formed on different PLL/PLGA PEM films; List of primer sequence used in RT-PCR; Thickness and refractive indices of PLL-and PLGA-ending PEM films before/after immersing in 10% serum or 1 mg/mL BSA measured by ellipsometry measurement. This material is available free of charge via the Internet at http://pubs.acs.org.

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BM901461E

Selection, Enrichment, and Maintenance of Self-Renewal Liver Stem

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