Bioinstructive Coatings for Hematopoietic Stem Cell Expansion Based

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Bio-instructive coatings for hematopoietic stem cell expansion based on chemical vapor deposition co-polymerization Anna-Lena Winkler, Meike Koenig, Alexander Welle, Vanessa Trouillet, Domenic Kratzer, Christoph Hussal, Joerg Lahann, and Cornelia Lee-Thedieck Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00743 • Publication Date (Web): 02 Aug 2017 Downloaded from http://pubs.acs.org on August 3, 2017

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Bio-instructive coatings for hematopoietic stem cell expansion based on chemical vapor deposition copolymerization Anna-Lena Winkler†‡, Meike Koenig†‡, Alexander Welle†∥, Vanessa Trouillet∥⊥, Domenic Kratzer†, Christoph Hussal†, Joerg Lahann†§, Cornelia Lee-Thedieck†* †

Institute of Functional Interfaces, ∥Karlsruhe Nano Micro Facility (KNMF) and ⊥Institute for

Applied Materials (IAM-ESS), Karlsruhe Institute of Technology, Hermann-von-HelmholtzPlatz 1, 76344 Eggenstein-Leopoldshafen, Germany §

Department of Chemical Engineering, Biointerfaces Institute, University of Michigan, Ann

Arbor, Michigan, United States ‡

These authors contributed equally to this work

*

E-mail: [email protected]

ACS Paragon Plus Environment

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ABSTRACT

We report the chemical vapor deposition (CVD) of dual-functional polymer films for the specific and orthogonal immobilization of two biomolecules (Notch ligand delta-like 1 (DLL1) and an RGD-peptide) that govern the fate of hematopoietic stem and progenitor cells. The composition of the CVD polymer and thus the biomolecule ratio can be tailored to investigate and optimize the influence of the relative surface concentrations of biomolecules on stem cell behavior. Prior to cell experiments, all surfaces were characterized by infrared reflection adsorption spectroscopy, ToF-SIMS, and X-ray photoelectron spectroscopy to confirm the presence of both biomolecules. In a proof-of-principle stem cell culture study we show that all polymer surfaces are cytocompatible and that the proliferation of the hematopoietic stem and progenitor cells is predominantly influenced by the surface concentration of immobilized DLL1.

KEYWORDS chemical vapor deposition, polymer films, orthogonal biofunctionalization, cell culture, hematopoietic stem cells


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INTRODUCTION Hematopoietic stem and progenitor cells (HSPCs) are at the top of the hierarchy of the bloodforming hematopoietic system. They are responsible for the life-long, daily supply of the body with fresh blood cells. Due to their capacity to reconstitute the blood system by differentiation into the different blood cell types and self-renewal, they are utilized in the treatment of diseases concerning the hematopoietic system including malignancies such as leukemia or lymphoma1,2. During the last decades, the applications of HSPCs obtained from healthy donors have broadened, which resulted in an increased need for healthy HSPCs. Ex vivo expansion of HSPCs – especially from HSPC sources that have limited cell densities such as umbilical cord blood – accommodates this increasing need3,4,5. HSPC behavior, including maintenance of stem cell properties, is naturally controlled by a specific microenvironment, termed the stem cell niche6. Depriving HSPCs of this niche yields in the loss of their stem and progenitor cell potential. All attempts to expand HSPCs ex vivo – and thus outside of the niche – lead not only to proliferation of HSPCs but also a fast onset of differentiation. Therefore, culturing HSPCs without a certain degree of differentiation and thus loss of the stem cell phenotype over time is not possible with nowadays techniques7, 8. Hence, imitation of niche components is a promising strategy, when aiming at ex vivo HSPC expansion. Most research in this area deals with the development of optimal media and appropriate soluble supplements such as cytokines. Other approaches aim at mimicking attributes of the in vivo niche including the extracellular matrix of the niche and the inclusion of supporting cells9,10. The natural niche is a highly complex environment. Simplifying this complexity to identify minimal requirements needed for HSPC expansion is a promising approach. In this study, cell-cell as well as cell-matrix interactions in the niche are mimicked in a highly simplified manner by developing a HSPC growth supporting surface that presents on the


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one hand the cellular Notch ligand delta-like 1 (DLL1) and on the other hand an RGD-peptide, which is a known binding motif of the extracellular matrix protein fibronectin11. DLL1 is naturally presented by osteoblasts in the HSPC niche12. It was shown that DLL1 in combination with the extracellular matrix protein fibronectin supports proliferation of HSPCs (Supplemental Figure S1) expressing the cell surface marker CD3413,3. CD34 is one of the most widely used markers to purify human HSPCs14. Furthermore, the surface density and clustering of DLL1 were demonstrated to play a non-negligible role in CD34+ cell proliferation13,15. Delaney et al. were the first to describe the concentration dependent effects of DLL1 on HSPC proliferation13. In their study they changed the concentration of DLL1 while keeping the amount of a fibronectin fragment – that was identified before to limit the myeloid differentiation-triggering effects of DLL116 – constant. Thus, in this study not only the amount of DLL1 but also the ratio of DLL1 to the fibronectin fragment was varied. In a recent study, we quantified the amount of DLL1 and the bioactive sequence of fibronectin – RGD – with the help of dual functionalized nanopatterned hydrogels15. The ratio of DLL1 and RGD appears to be one parameter that allows controlling HSPC proliferation. Therefore, there is a current need for surface coatings that allow orthogonal biofunctionalization with control over the molar ratio of the immobilized ligands for the development of novel bioreactors for HSPC multiplication. A promising versatile strategy in order to create such bioactive coatings is the vapor deposition polymerization of reactive [2.2]paracyclophanes. Due to the inherent properties of the vapor deposition technique, the resulting polymer films can be deposited on a wide variety of substrate materials and geometries; without the need for solvents or auxiliary reactants, which often result in bridging of polymer coatings, plus the risk of introducing potentially toxic substances from the polymer films to the stem cell cultures is minimized17,18. Coating thicknesses and ratios of co-


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polymer reactants can easily be controlled. Due to the lower probability for side reactions vaporbased polymer films follow cleaner reaction pathways, resulting in polymers with linear polymer chains. Unsubstituted, mono-, and dichloro-substituted poly-p-xylylene (PPX) are commercially available under the brand name parylene N, C, and D, and applied in the industrial production of coatings for implants and other medical devices19. By modification of the precursor paracyclophane, a wide variety of reactive PPX derivatives is available that can be used for the covalent immobilization of biomolecules20. Additionally, coatings with multiple reactive groups for the orthogonal reaction of diverse biomolecules on the same surface are possible by the deposition of copolymers in a randomly mixed, graded or patterned fashion21. Just recently, Chen and co-workers demonstrated the potential advantages of multifunctional vapor-based coatings for controlled stem cell culture and differentiation22, 23. In the present study, copolymer films with aminomethyl and alkyne groups in varying concentrations are utilized for the orthogonal immobilization of RGD and DLL1 in varying molar ratios. The potential of these biofunctionalized surfaces as bio-instructive platforms for guiding HSPC behavior is demonstrated in ex vivo proliferation studies (Figure 1). In contrast to other approaches, the platform developed here, provides the opportunity to coat surfaces independent of their shape.


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Figure 1. Schematic representation of the presented biomaterial concept comprising three steps: A) CVD polymerization of copolymers with varying composition, B) bio-functionalization, C) HSPC cultivation.

EXPERIMENTAL SECTION Synthesis of precursors for chemical vapor deposition polymerization. The precursor 4aminomethyl[2.2]paracyclophane (PCP-AM) was synthesized from [2.2]paracyclophane (PCP) (Curtiss Wright Surface Technologies, Galway, Ireland) in a three-step synthesis as described previously24. 4-ethynyl[2.2]paracyclophane (PCP-Alkyne) was synthesized from


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[2.2]paracyclophane (PCP) in a two-step synthesis via 4-formyl-[2.2]paracyclophane (PCPAldehyde) as depicted in Scheme S1. The synthesis of 4-formyl-[2.2]paracyclophane (PCPAldehyde) is described elsewhere25. Bromomethyl triphenylphosphonium bromide ([Ph3PCH2Br]+Br-) had to be prepared freshly prior to the synthesis of 4Ethynyl[2.2]paracyclophane (PCP-Alkyne): In a 250 mL flask, triphenylphoshine (30.0 g, 114 mol, 1.00 equiv.) and dibromomethane (16.0 mL, 39.8 g, 229 mmol, 2.00 equiv.) were dissolved in toluene (160 mL). The solution was heated to 95 °C for 72 h. After cooling to room temperature, the precipitated product was filtered off, washed with diethyl ether (4×150 mL) and dried in vacuum. Bromomethyl triphenylphosphonium bromide was obtained as a white solid (27.3 g, 62.6 mmol, 55%) which was used in the subsequent reaction step without further purification. In a 250 mL three-neck flask equipped with a dropping funnel, bromomethyl triphenylphosphonium bromide (5.00 g, 11.5 mmol, 1.10 equiv.) was dissolved in absolute tetrahydrofuran (60 mL) under an argon atmosphere. After cooling to –78 °C, potassium tertbutoxide (3.10 g, 27.6 mmol, 2.65 equiv.) was added and the suspension was stirred for 1 h. Then, a solution of PCP-Aldehyde (2.46 g, 10.4 mmol, 1.00 equiv.) in absolute tetrahydrofuran (50 mL) was added over a period of 1 h. The reaction mixture was slowly warmed to room temperature and stirred for another 3 h. Next, additional potassium tert-butoxide (2.45 g, 20.3 mmol, 1.95 equiv.) was added and the reaction mixture was heated to 60 °C for 3 h. After cooling to room temperature, water (150 mL) was added and the aqueous phase was acidified with 1 M hydrochloric acid. Then, diethyl ether (100 mL) was added and the phases were separated. The organic phase was washed with saturated sodium chloride solution (3×100 mL) and dried over magnesium sulfate. After removal of the solvent under reduced pressure, the crude product was purified by column chromatography (silica; dichloromethane/hexane, 1:4) to


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yield PCP-Alkyne as a white solid (1.71 g, 7.36 mmol, 71%). Rf = 0.47 (dichloromethane/hexane, 1:4). – 1H-NMR (500 MHz, CDCl3): δ = 7.00 (dd, J = 7.8 Hz, 1.6 Hz, 1H, HAr), 6.56–6.45 (m, 6H, HAr), 3.59 (ddd, J = 13.2 Hz, 10.5 Hz, 2.8 Hz, 1H, HPc), 3.28 (s, 1H, CCH), 3.23 (ddd, J = 13.0 Hz, 10.5 Hz, 5.2 Hz, 1H, HPc), 3.13–2.94 (m, 5H, HPc), 2.86 (ddd, J = 13.0 Hz, 10.5 Hz, 5.2 Hz, 1H, HPc) ppm. The analytical data are in agreement with data known from the literature26. 1H-NMR spectra were recorded on a Bruker Avance III (500 MHz) spectrometer using CDCl3 as solvent. Chemical shifts are expressed in parts per million (ppm, δ) downfield from tetramethylsilane (TMS) and are referenced to CDCl3 (7.26 ppm) as internal standard. All coupling constants are absolute values and J values are expressed in Hertz (Hz). The description of the signals includes: s = singlet, bs = broad singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublet, ddd = double doublet of doublet, m = multiplet. The spectra were analyzed according to first order. The signal abbreviations include: HAr = aromatic proton, and HPc = proton belonging to the [2.2]paracyclophane core. The separation and the purification of products from mixtures were performed by means of column chromatography. Silica gel 60 (Merck KGaA, Darmstadt, Germany) was used as stationary phase. Solvent mixtures are understood as volume/volume. Routine monitoring of reactions were performed by thin layer chromatography (TLC) using silica gel coated aluminum plates (Merck KGaA, Darmstadt, Germany; silica gel 60, fluorescence indicator F254) which were analyzed using an ultraviolet lamp (Merck KGaA, Darmstadt, Germany; 254 nm) as well as potassium permanganate reagent (1.00 g KMnO4 and 5.00 g Na2CO3 in 250 mL water). All solvents, reagents and chemicals were used as purchased, unless stated otherwise. All reactions involving moisture sensitive reactants were executed under argon atmosphere using oven dried glassware, following the common Schlenk technique. For low temperature reactions, the following cooling mixtures were prepared:


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0 °C: ice/water; –78 °C: acetone/dry ice. Solvents, reagents and chemicals were purchased from Acros, ABCR, Alfa Aesar or Sigma-Aldrich. Chemical vapor deposition polymerization. A custom-built setup equipped with two inlet sources was used for the deposition of the copolymer poly(4-aminomethyl-p-xylylene-co-4ethynyl-p-xylylene-co-p-xylylene) (PPX-AM-co-alkyne) as described in an earlier publication27. In this setup, each precursor is sublimated separately in one of the quartz tube sources under a reduced pressure (0.12 mbar) at a temperature close to 100 °C. With the help of argon carrier gas streams that can be adjusted individually, the precursor gases are transported through a pyrolysis furnace, heated to 660 °C. The in situ formed quinodimethane intermediates enter the deposition chamber where they spontaneously copolymerize on the substrates, cooled to 15 °C on the rotating sample holder. A quartz crystal microbalance, located in the deposition chamber, was used to monitor the deposition rate. The deposition rate of the copolymer components is governed by the flux of the argon carrier gas streams. From the average deposition rate measured during separate depositions of each copolymer component at the respective flux, the molar ratio in the copolymer can be estimated, assuming comparable deposition rates in the copolymer and a similar density of the components of the copolymer, as follows:

‫= ܣ×ܦ‬

݊×‫ܯ‬ ߩ

with the deposition thickness D, the covered surface area A, the molar amount of substance n, the molecular weight M and the polymer density ρ. Putting this equation for the 2 components of the copolymer into relation, yields ‫ܦ‬௉௉௑ି஺ெ ݊௉௉௑ି஺ெ × ‫ܯ‬௉௉௑ି஺ெ = ‫ܦ‬௉௉௑ି஺௟௞௬௡௘ ݊௉௉௑ି஺௟௞௬௡௘ × ‫ܯ‬௉௉௑ି஺௟௞௬௡௘


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which leads to the following formula for calculating the molar ratio of the components of the copolymer films:

݊௉௉௑ି஺ெ : ݊௉௉௑ି஺௟௞௬௡௘ =

‫ܦ‬௉௉௑ି஺ெ ‫ܦ‬௉௉௑ି஺௟௞௬௡௘ : ‫ܯ‬௉௉௑ି஺ெ ‫ܯ‬௉௉௑ି஺௟௞௬௡௘

Table 1 summarizes the used copolymer compositions. As substrates, glass slides (15 mm; Carl Roth, Karlsruhe, Germany, for cell culture and ToF-SIMS measurements) or silicon wafers (Siegert Wafer GmbH, Aachen, Germany, for IRRAS and XPS measurements) coated by evaporation deposition with a gold layer were used. Surfaces were cleaned with acetone before use. The thickness of the polymer layer on gold substrates was measured using spectroscopic ellipsometry in dry state (M2000, Woollam Co., Inc., Lincoln NE, USA). Polymer layers with a thickness of 15 to 30 nm were used throughout the experiments. Table 1. Used copolymer compositions with molar composition estimated from the individual deposition rate. argon flow rate [sccm]

deposition rate [Å/s]

calculated molar ratio in copolymer PPX-AM-co-alkyne

PPX-AM

PPX-Alkyne

PPX-AM

PPX-Alkyne

PPX-AM : PPX-Alkyne

4

36

0.1

1

1:10

5

15

0.15

0.6

1:4

20

20

0.5

0.6

1:1

36

4

0.8

0.15

5:1

Infrared Reflection Adsorption Spectroscopy (IRRAS). IR spectra were recorded using a Bruker Vertex 80 spectrometer (Bruker Optics, Ettlingen, Germany), equipped with a liquid nitrogen cooled mid band MCT detector and a grazing incidence reflection unit, in between 600 – 4000 cm-1 with a resolution of 4 cm-1 at an incidence angle of 80°. As a reference, selfassembled monolayers of perdeuterated 1-hexadecanethiol on gold were used. A baseline and smoothing correction as well as carbon dioxide compensation were applied to the resulting


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spectra. For comparison of coatings with slightly varying thickness, all spectra were scaled using the aromatic C-H stretch band at 3010 cm-1 as a reference. Biofunctionalization of Surfaces. After coating with PPX-AM-co-alkyne, samples were sterilized for 30 min in Ethanol (70% (v/v)) and subsequently washed with ultrapure water. The RGD-peptide, carrying a PEG spacer at the amino terminus of the lysine and an azide moiety at the PEG terminus (Supplemental Scheme S2A; sequence RGDSK-PEG-azide, MW = 931.37 g/mol, synthesized by H. Kalbacher, University of Tübingen, Germany), was immobilized on the surfaces via copper-catalyzed alkyne-azide cycloaddition (CuAAC; Supplemental Scheme S2B). For the CuAAC, sodium ascorbate (20 mM; Sigma-Aldrich, Saint Louis, Missouri, USA), CuSO4 (0.1 mM; Sigma-Aldrich, Saint Louis, Missouri, USA) and RGD-peptide (35 µM for cell culture experiments with proliferation and CD34 expression analysis; 100 µM for XPS analysis) were dissolved in ultrapure water and placed (100 µl droplets) on the bottom of 12 well plates (Greiner Bio-One, Frickenhausen, Germany). The samples were placed immediately upside-down onto the droplets and incubated overnight at 4 °C (5 d for XPS analysis). Surfaces were washed 6 times for 15 min in ultrapure water and equilibrated in PBS. DLL1 was covalently bound to the surface via N-(3-dimethylaminopropyl)N’-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) coupling (Supplemental Scheme S2C). For that purpose the surfaces were placed for 1 h at 4 °C onto 100 µl droplets of a solution containing EDC and NHS in equimolar concentrations (391 mM each) as well as the protein DLL1 (2.5 µg/mL; PeproTech, Princeton, New Jersey, USA) in PBS (pH 7.4; Sigma-Aldrich, Saint Louis, Missouri, USA). To test for unspecific protein binding by ToF-SIMS, a DLL1 solution (10 µg/mL in PBS) without EDC/NHS was used. For the surfaces solely functionalized with RGD, a PEG-COOH (20 mM, JenKem Technology USA, Plano,


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Texas, USA) was used instead of the DLL1 to passivate free amino groups. In the following, the last mentioned surfaces are designated as “RGD” for reasons of simplicity. Finally, surfaces were washed with PBS before further cell experiments. For XPS and ToF-SIMS measurements the samples were washed with ultrapure water and dried with nitrogen. A table summarizing the details of the different samples prepared for characterization and cell experiments is included into the supplementary material (Supplemental Table S1). X-ray photoelectron spectroscopy (XPS). XPS measurements were performed using a K-Alpha XPS spectrometer (Thermo Fisher Scientific, East Grinstead, UK). The kinetic energy of the electrons was measured by a 180° hemispherical energy analyzer operated in the constant analyzer energy mode (CAE) at 50 eV pass energy for elemental spectra. Data acquisition and processing using the Thermo Avantage software is described elsewhere28. All samples were analyzed using a microfocused, monochromated Al Kα X-ray source (400 µm spot size). The KAlpha charge compensation system was employed during analysis, using electrons of 8 eV energy, and low-energy argon ions to prevent any localized charge build-up. The spectra were fitted with one or more Voigt profiles (BE uncertainty: +0.2 eV) and Scofield sensitivity factors were applied for quantification29. All spectra were referenced to the C 1s peak (C-C, C-H) at 285.0 eV binding energy controlled by means of the well-known photoelectron peaks of metallic Cu, Ag, and Au, respectively. ToF-SIMS (Time-of-Flight Secondary Ion Mass Spectrometry) was performed on a TOF.SIMS5 instrument (ION-TOF GmbH, Münster, Germany). This spectrometer is equipped with a field emission bismuth cluster primary ion source and a reflectron type time-of-flight analyzer. Main chamber pressure was < 2×10-8 mbar. For high mass resolution the Bi source was operated in the “high current bunched” mode providing short Bi3+ primary ion pulses at 25 keV energy and a


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lateral resolution of approx. 4 µm. The short pulse length of 1.1 ns allowed for high mass resolution. Primary ion doses were kept below 1011 ions/cm2 (static SIMS limit) for all measurements. Spectra were calibrated on the omnipresent C-, CH-, CH2-, OH-; or on the C+, CH+, CH2+, and CH3+ peaks. In vitro study of HSPC proliferation. Umbilical cord blood was obtained from the DRKBlutspendedienst (Mannheim, Germany) or the DKMS Nabelschnurblutbank gemeinnützige GmbH (Dresden, Germany) after informed consent of the parents and with approval by the local ethics committee (Landesärztekammer Stuttgart, Germany; reference number: B-F-2013-111). CD34+ cells were isolated from umbilical cord blood according to the manufacturer’s instructions (CD34 MicroBead Kit, human; Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of the isolated CD34+ cells was tested via flow cytometry using anti CD34-FITC antibodies (Invitrogen, Carlsbad, USA) and was around 98% in all experiments. Biofunctionalized surfaces were placed in 24 well-plates (Greiner Bio-One, Frickenhausen, Germany), equilibrated for 10 min at 37 °C in 1 mL stem cell medium (Stem Span SFEM II supplemented with cytokine cocktail CC100; both Stemcell Technologies, Vancouver, British Columbia, Canada and 1% (v/v) penicillin/streptomycin; Sigma-Aldrich, Saint Louis, Missouri, USA). The plain well surfaces served as a tissue culture plastic (TCP) control. 2×104 cells were seeded on the surfaces and medium exchange and cell dilutions (reseeding of 2×104 cells per mL) were done on day 4, 7 and 11. Thus, cells were replated on the surfaces every 3 to 4 days. The total numbers of living cells were assessed on days 4, 7, 11 and 14 via trypan blue staining (0.4% (v/v); Sigma-Aldrich, Saint Louis, Missouri, USA) and manual counting with a hemocytometer (VWR International, Radnor, USA). To analyze the proliferation, the dilution factor was calculated for each time point and multiplied with the counted cell number.


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HSPC cultures were carried out on PPX-AM-co-alkyne(5:1) and (1:10) coatings that were dual functionalized with RGD and DLL1. As controls, the pristine copolymer coatings and PPX-AMco-alkyne single functionalized with RGD (without DLL1) were applied. Surfaces single functionalized with DLL1 (without RGD) were not included into the study, as DLL1 alone does not only support HSPC proliferation but also myeloid differentiation16, which was contradicting the aim of the present study – ex vivo HSPC expansion with as little differentiation as possible. Flow cytometric analysis. The proportion of CD34+ cells within the cell populations was assessed directly after isolation and after 14 days of cultivation on the different functionalized surfaces. For that purpose, minimal 2×104 cells were incubated for 1 h at 4 °C with 2.5 µL of anti CD34-FITC antibodies and the respective isotype controls (both Invitrogen, Carlsbad, USA) in PBS containing FBS (0.01% (v/v)). For studying the cytotoxic and apoptotic potential of the chemical vapor deposition-coated surfaces, a minimum of 5×104 cells was stained with Sytox AADvanced Dead Cell Stain and counterstained with Annexin V FITC (both from Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer’s instructions. Sytox AADvanced Dead Cell Stain dye binds to the DNA of dead cells with defective membranes, while the Annexin V staining binds phosphatidylserines, which are presented by apoptotic cells on the outer leaflet of their cell membranes30. After analysis populations were discriminated into early apoptotic, late apoptotic, necrotic, and live cell subpopulations. As positive and negative controls for the Sytox AADvanced Dead Cell staining living cells were mixed with cells, which were killed by treatment at 70 °C for 10 min. For Annexin V staining, beads (AbC Anti-Mouse Bead Kit; Thermo Fisher Scientific, Waltham, Massachusetts, USA) incubated with a FITC labeled antimouse isotype control (Invitrogen, Carlsbad, USA) and the in the AbC Anti-Mouse Bead Kit


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supplied negative beads were used as controls. The analysis was performed with an Attune Acoustic Focusing Cytometer (Applied Biosystems) and the acquired data were subsequently analyzed with FlowJo V10.0.7 (Tree Star Inc., Ashland, Oregon, USA). Colony forming unit assay. The early myeloid differentiation potential of cultured HSPCs was assessed by colony forming unit assays as described previously15. Briefly, 500 cells were plated in triplicate in semisolid methyl-cellulose containing, growth factor supplemented media (MethoCult H4434 (Stemcell Technologies, Vancouver, Canada)). After 14 days of cultivation different types of colonies (CFU-GEMM: colonies risen from progenitors which can differentiate to granulocytes, erythrocytes, macrophages and megakaryocytes; CFU-GM: colonies of progenitors with differentiation potential towards granulocytes and macrophages; BFU-E: colonies grown from progenitors that can differentiate to erythrocytes) were microscopically determined and counted. MTT test. 105 CD34+ cells isolated from umbilical cord blood were cultured for 48 h on the tested substrates in Stem Span SFEM II supplemented with cytokine cocktail CC100 (both Stem Cell Technologies) and penicillin/streptomycin (Sigma-Aldrich). For the MTT assay 3-(4,5Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (Sigma-Aldrich) was freshly added to the culture medium in a final concentration of 1.2 mM. After 2 h of incubation, cells were pelleted, lyzed in 100 µL dimethyl sulfoxide with 0.6 % acetic acid and 10 % sodium dodecyl sulfate and the absorption was recorded at 570 nm with an EnSpire 2300 Multimode Plate Reader (Perkin Elmer, Waltham, USA). For analysis background absorption at 630 nm was subtracted from the measured absorption values. The data were normalized to the values obtained from the cell cultured on TCP.


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Data analysis and statistics. Reported results are the mean values ± standard error of the mean (SEM) or mean values ± standard deviation (SD) and are adjusted for outliers. The MannWhitney test was used to test the significance between the results of two groups, in not indicated otherwise in the figure legend. If the resulting P values were ≤ 0.05 the differences between the results were regarded as statistically significant. Results of the Mann-Whitney tests are indicated in the figures with one to two asterisks as follows: P ≤ 0.05 with * and P ≤ 0.01 with **, ‘ns’ indicates ‘not significant’. Data processing was performed with Microsoft Office Excel (Microsoft, USA) and GraphPad Prism 6.0 (GraphPad Software, Inc., USA).

RESULTS AND DISCUSSION Copolymer films with varying ratios of functional groups were produced using a custom-built two-source CVD setup27. In this setup, the variation of the composition is achieved by adjusting the argon flow of each source, resulting in variation of the transport rate of the sublimated dimers to the deposition chamber. By rotation of the sample stage a homogeneous coating of the sample is ensured. The molar ratio of the copolymer components can be estimated from the deposition rate of each monomer at the respective argon flow rate (Table 1). Figure 2A displays IRRAS spectra of coatings produced with three different argon flow rate conditions (ࢂሶ (PCP-AM)-ࢂሶ (PCP-Alkyne): 36 sccm-4 sccm; 20 sccm-20 sccm; 4 sccm-36 sccm) leading to different ratios of the copolymer components (named as PPX-AM-co-alkyne(5:1), PPX-AM-co-alkyne(1:1), PPX-AM-co-alkyne(1:10)). With increasing flow rate of the PCP-AM source, an increase in signal intensity of the N-H bend vibration between 1650-1580 cm-1 is detected. In parallel, the decrease of the flow rate of the PCP-Alkyne source, results in a decrease


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in the signal intensity of the alkyne C-H stretch vibration at 3300 cm-1 and the alkyne C-C stretch vibration at 2100 cm-1. Additionally, XPS was used to measure the nitrogen content of different copolymer coatings). The N 1s peak can be deconvoluted in 2 components (see Supplemental Figure S2): a main one at 399.5 eV attributed to amine31 and one of weak intensity at 401.5 eV attributed to protonated amine. With increasing flow rate of the PCP-AM source, an increased nitrogen content of the coating was detected (Figure 2B).

Figure 2. (A) IRRAS spectra of copolymers with three different compositions achieved by variation of the argon carrier gas flow rates. PPX-AM-co-alkyne ratios overlay with 5:1 as black line, 1:1 as red line, and 1:10 as blue line;


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(B) Nitrogen content of copolymers measured by XPS. The stars represent the theoretical values derived from the calculated values of the molar component ratios.

The measured nitrogen concentrations in the different copolymers (0.3 at% in PPX-AM-coalkyne(1:10), 1.1 at% in PPX-AM-co-alkyne(1:4), 2.9 at% in PPX-AM-co-alkyne(1:1) and 3.9 at% in PPX-AM-co-alkyne(5:1)) agree well with the theoretical values calculated on the basis of the molar ratio of the copolymer compounds (0.5 at% in PPX-AM- co-alkyne(1:10), 1.1 at% in PPX-AM- co-alkyne(1:4), 2.8 at% in PPX-AM- co-alkyne(1:1) and 4.6 at% in PPX-AM-coalkyne(5:1)). The theoretical values were calculated based on a molar ratio of the components PPX-AM : PPX-Alkyne = a : b and the amount of nitrogen and carbon atoms N of the monomers PCP-AM and PCP-Alkyne as follows: ܿ௡௜௧௥௢௚௘௡ =

ܽ × ܰ௡௜௧௥௢௚௘௡ (ܲ‫ ܲܥ‬− ‫)ܯܣ‬ ∗ 100 ܽ × ܰ௡௜௧௥௢௚௘௡ (ܲ‫ ܲܥ‬− ‫ )ܯܣ‬+ ܽ × ܰ௖௔௥௕௢௡ (ܲ‫ ܲܥ‬− ‫ )ܯܣ‬+ ܾ × ܰ௖௔௥௕௢௡ (ܲ‫ ܲܥ‬− ‫)݁݊ݕ݈݇ܣ‬

with the concentration c in at% and the number of atoms N of the elements in the two monomers. Since the polymer shows a negligible content of oxygen due to oxidation or contamination layer, it’s possible here to consider directly the atom concentration and not only the ratio N/C. The calculated and the estimated nitrogen concentration are summarized in Supplemental Table S3. Results of both, the IRRAS and the XPS characterization, indicate the successful synthesis of copolymer-coatings with varying ratios of functional groups. In the following, the PPX-AM-co-alkyne surfaces were functionalized to enhance their bioactivity towards HSPCs. For this purpose, the surfaces were chemically equipped with two biologically relevant biomolecules which are present in the in vivo stem cell niche. Firstly an RGD-peptide (minimal adhesive amino acid sequence in many molecules of the extracellular matrix such as fibronectin) that was modified with an azide group was ‘clicked’ to the alkyne groups of the surface via CuAAC. Secondly, the naturally cell membrane bound Notch ligand


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DLL1 was coupled via its carboxylic groups to the amino groups of the copolymer by EDC-NHS chemistry. In order to confirm the orthogonal functionalization with both biomolecules, XPS and ToF-SIMS measurements were performed. Figure 3A compares the high resolution spectra of the N 1s peak of (from bottom to top) a copolymer coating (PPX-AM-co-alkyne(1:4)), before and after the reaction of the alkyne groups with RGD-azide without and with copper catalyst in the reaction mixture. Topmost, the measurement of the pure peptide on gold is shown for comparison. Before the reaction, one peak is detected that can be fitted with a single component at 399.5 eV attributed to the amino groups in the copolymer32. The spectrum remains unchanged if no copper is added to the reaction, indicating that no unspecific adsorption of RGD-peptide is taking place. After the reaction with additional copper, the N 1s peak can only be fitted using two additional components. Similar to the spectrum of the pure peptide, the main component is detected at 400.5 eV, which can be attributed to the amide groups in the peptide chain33. From the comparison of the reaction with and without the copper catalyst, the specific reaction of the RGD-azide to the alkyne groups can be concluded.


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Figure 3. XPS N 1s (A) and ToF-SIMS (B, C) measurements of biofunctionalized PPX-AM-co-alkyne surfaces. (A) High resolution XPS N 1s spectra of (from bottom to top) PPX-AM-co-alkyne(1:4) polymer coating on gold slides, RGD without Cu2+, RGD with Cu2+, and RGD-azide reference (100 µM; RGD-azide in water without Cu2+ and sodium ascorbate), showing the specific immobilization of RGD to the CVD copolymer. All spectra were normalized to the maximum of intensity. ToF-SIMS results of CNO- signal (B) of the RGD-peptide (35 µM) and DLL1 protein (2.5 µg/mL) backbone and (C) cysteine (S-, sulfur) amino acid signal of human DLL1, proving the immobilization of both biomolecules on the surfaces. PPX-AM-co-alkyne polymer (black line), RGD and PEG (blue line), RGD and DLL1 (purple line).

The successful covalent immobilization of both biomolecules — RGD in combination with DLL1 — on glass surfaces with PPX-AM-co-alkyne(5:1) coating, was examined with ToF-SIMS analysis (Figure 3B, C). Different samples were compared: (i) the unmodified copolymer, (ii) a copolymer exposed to RGD only (35 µM, blue line), and (iii) a copolymer exposed to the combination of RGD and DLL1 (2.5 µg/mL, purple line). The CNO- signal, indicative of the amide backbone of peptides and proteins is shown in Figure 3B. The highest CNO- signal was


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obtained for the surface with DLL1 and RGD followed by the surface with RGD and the unmodified PPX-AM-co-alkyne polymer film. Since DLL1 contains more amino acids as compared to the RGD derivative, a stronger CNO- signal, being independent of the amino acid composition, is recorded for sample (iii). To verify the obtained results, a measurement of the CH4N+ signal was conducted (Supplemental Figure S3A). The surfaces with DLL1 plus RGD showed the same additive effect in CH4N+ signal intensity, compared to RGD and the polymer coating, as observed in the CNO- measurement. The obtained signal from the copolymer background (black lines in Figure 3B and C) arises from the presence of amino groups. In the sample with only RGD, the amide and triazole groups after grafting lead to the additional signal intensity. The increased signal observed for the surfaces presenting both RGD and DLL1 through EDC/NHS (Supplemental Scheme S2C) as compared to surfaces decorated with RGD only via CuAAC (Supplemental Scheme S2B) suggests that both biomolecules were successfully immobilized on the surface. To discriminate immobilized DLL1 from RGD, specific amino acids (Cys, Ala, Leu/Ile) that are characteristic for the DLL1 protein, were monitored using ToFSIMS. When analyzing the S- fragment, which is characteristic of Cys, only the DLL1 treated sample showed increased S- levels (Figure 3C). SIMS does not differentiate between leucine and isoleucine, therefore only one diagram for both is depicted. Moreover, the signals for alanine (C2H6N+), and leucine/isoleucine (C5H12N+) (Supplemental Figures S3B and S3C) showed increased signals for surfaces co-presenting DLL1 and RGD as compared to the unmodified PPX-AM-co-alkyne surface. Both results, from CNO-, CH4N+ and the amino acids cysteine, alanine, and leucine/isoleucine confirm successful co-immobilization of both biomolecules after CuAAC reaction and EDC/NHS coupling. To exclude possible influences of scratches or drying artifacts on the sample with DLL1, spatially resolved measurements were done (Supplemental


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Figure S3D). The analysis proved that the surfaces were homogeneously coated with protein and/or peptide hence measurements of peptides and proteins were valid. To show that covalent immobilization, rather than unspecific protein binding, was responsible for the observed results for DLL1, PPX-AM-co-alkyne(1:4) surface coatings were functionalized with RGD followed by DLL1 incubation with and without EDC/NHS and compared to the plain copolymer surface as a control (Supplemental Figure S4A-C). As indicated in Supplemental Figure S4A and Supplemental Figure S4C, the CNO- and CH4N+ signal of peptide backbones were higher on samples incubated with EDC/NHS yielding covalent immobilization of DLL1 compared to surfaces treated with protein solution without EDC/NHS. The same effect was observed with regard to the SH- signal of cysteine (Supplemental Figure S4B). The results indicate that the majority of protein was covalently immobilized and only small amounts were bound by unspecific adsorption to the surface. Following the characterization of PPX-AM-co-alkyne coatings and their biofunctionalization with both biomolecules, the obtained surfaces were tested in HSPC culture experiments. While the concentrations of applied RGD and DLL1 solutions for biofunctionalization were kept constant at 35 µM and 2.5 µg/mL, respectively, the ratio of amino to alkyne groups in the functionalized polymer coatings were changed (PPX-AM-co-alkyne(1:10 and 5:1)) to examine whether substrates with a higher density of functional amino groups, and therefore DLL1, were more suitable to expand HSPC (CD34+ cells). Besides studying the CD34+ cells’ response and behavior regarding proliferation and stem cell marker CD34 expression, the cytotoxic potential of the applied biofunctionalized surfaces was investigated (Figure 4A). The utilization of CuAAC with copper as catalyst prompts the suspicion that residues of copper ions on the surface after reaction can impair the cell vitality since these metal ions are known to form reactive


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oxygen species which can force apoptosis and cell death34,35. Therefore, the potential cytotoxicity of the samples was assessed by staining for dead and apoptotic cells (Figure 4B and Supplemental Figure S5). Tissue culture plastic (TCP) and non-treated PPX-AM-co-alkyne were utilized as controls. We could show that all surfaces were not detrimental to HSPCs. The amounts of live, dead or apoptotic cells on RGD functionalized surfaces, which were in contact with ascorbic acid and copper during CuAAC were comparable to the results obtained on the control samples (Figure 4A) that were not in contact with copper ions. The proportion of live cells was at 76.8 ± 4.3% on all surfaces. Only 10.3 ± 2.5% of the cells were in early apoptosis 11.5 ± 2.5% in late apoptosis, and the smallest proportion of cells (1.4 ± 0.5%) was necrotic (mean ± SD). This result – that no negative effect of eventual residuals of copper ions after functionalization could be observed – was backed by an additional XPS analysis of RGDfunctionalized surfaces to identify metal traces of copper after CuAAC and several washing steps with ultrapure water (Supplemental Figure S6). The results showed that copper was not detectable in verifiable amounts, which indicates that copper surface concentrations are well below the known cytotoxic concentrations (LD50 = 220.5 ± 23.8 µg/mL upon 48 h exposure on HepG2 cells34). Therefore, the obtained results from XPS measurements are in accordance with the results received from the Annexin V/Sytox AADvanced cytotoxic assay, and indicate that our rinsing process to remove copper ions from the samples is adequate and effective. Furthermore, it appears that the developed, dual functionalized copolymer coatings are cytocompatible for HSPCs.


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Figure 4. HSPC proliferation, apoptosis and CD34 expression on various surfaces. (A) Cytotoxicity of the different applied surfaces with and without RGD biomolecule coupling via CuAAC. Shown are the mean values of N=3, in case of plain copolymers N=1, independent experiments. Error bars represent the standard deviation (SD). ‘PPXAM-co-alkyne’ is abbreviated by ‘PPX’. (B) Exemplary contour plot (Annexin V-FITC plotted against Sytox AADvanced) with gating of the different subpopulations. (C-F) Results of the total cell count relative to the number of cells on TCP found over time (C, E) and bar diagrams of the number of CD34+ cells normalized to the number of cells found on TCP on day 14 (D, F). (C-F) Mean values of N=3 independent experiments. Error bars represent standard error of the mean (SEM).


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Having shown the cytocompatibility, the coatings were tested for their ability to influence HSPC proliferation as well as the expression of the HSPC surface marker CD34. For that purpose, freshly isolated HSPCs were cultivated for 14 days on the different surfaces, the total cell count and percentage of CD34+ cells were assessed and compared to standard TCP. Normalization to the TCP control sample was applied to correct for donor-dependent individual variations. The results show that the total number of cells was higher on the plain PPX-AM-co-alkyne(5:1) surface than on standard TCP (Figure 4C) suggesting a slight proliferation triggering effect of the polymer coating itself. The same effect was observed for the number of CD34+ cells (Figure 4D). It is known from earlier studies that amino groups themselves e.g. on polyethersulfone nanofiber meshes and films are able to support CD34+ cell expansion after 10 day cultivation36. Hence the proliferative effect of the amino groups of the PPX-AM-co-alkyne(5:1) surfaces utilized here on HSPCs is conceivable. On plain PPX-AM-co-alkyne(1:10), with more alkyne and less amino groups than the PPX-AM-co-alkyne(5:1) on the surface, cell proliferation and CD34+ cell count were not significantly increased compared to TCP (Figure 4C,D). Alkyne groups are highly chemoselective, as they undergo mainly reactions with azides. Additionally they are hydrophobic, and non-adhesive for peptides, as it was shown in XPS measurements of surfaces incubated with RGD without copper, which did not show any binding (Figure 3A). Therefore, we propose that amino rather than alkyne groups of the copolymer coatings are responsible for the observed positive effect of the pure polymer coating on HSPC proliferation. As a next step, we investigated the effect of the single functionalization with RGD on HSPC expansion. For that purpose, the alkyne groups of the copolymer were functionalized with RGDazide and the remaining amine groups were passivated by EDC/NHS coupling with PEG-COOH. In comparison to TCP, these surfaces significantly enhanced the total cell proliferation


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(Supplemental Figure S7A), however, no significant effects could be observed on the CD34+ cell count compared to the TCP (Supplemental Figure S7B). The relative amount of bound RGD seemed not to influence the effect on the total cell proliferation – the total cell count relative to TCP was comparable on both types of substrate (PPX-AM-co-alkyne(1:10) and (5:1) functionalized with PEG and RGD). Thus it appears that RGD on the PPX surfaces enhanced cell proliferation in general, but the proliferation of the more immature CD34+ cells was unaffected by RGD. RGD can be recognized by cells via different integrin receptors. Integrins are known to be crucial regulators of processes including cell adhesion and proliferation37. The elicited effects, however, depend on the type of integrin that is activated. E.g. it was shown that the proliferation of human adipose-derived stem cells is differently regulated by the RGDbinding αV and α5 integrins. While αV appeared to be involved in the regulation of proliferation, α5 was not38. This might explain the different effects that we observed for RGD on total and CD34+ cell proliferation in the present study. While in the total cell population RGD recognition might occur via both integrins, human CD34+ HSPCs recognize RGD via α5 integrins according to their integrin receptor expression repertoire: CD34+ HSPCs (freshly isolated from cord blood – as applied in the current study) express the RGD binding integrin αVβ3 not or only in very low amounts, but they do express α5 integrins.39 Thus, RGDrecognition by CD34+ cells is limited to α5 integrins, which do not influence cell proliferation, while in the total cell population αV and α5 integrins might be involved and, thus, proliferation is affected. These results are also in line with a previous study showing that monofunctionalized RGD cell culture substrates have no effect on of HSPC proliferation and differentiation40. Finally, the dual functionalized copolymer films – decorated with RGD and DLL1 in varying ratios – were evaluated in HSPC culture. In these experiments, a significantly enforced HSPC


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proliferation was observed on PPX-AM-co-alkyne(5:1) coated surfaces with DLL1 and RGD (Figure 4E and 4F). On these surfaces a ~ 7.8-fold increase of total cells and ~ 24.1-fold increase of CD34+ cells was detected in comparison to the control TCP. For comparison, this significant increase was not observed on the PPX-AM-co-alkyne(1:10) copolymer films which behaved more similar to the unmodified copolymer films (Supplemental Table S2). At the same time, early myeloid differentiation was not significantly affected by the biofunctioned surfaces, as shown by colony forming unit assays (Figure S8). The metabolic activity of HSPCs on all different applied copolymer surfaces was not significantly different from the one measured on TCP, underscoring the cytocompatibility of the developed copolymer coatings (Figure S9). As the cells were cultured for 14 days on the biofunctionalized substrates, it appears likely that the surfaces were modified during the course of culture by factors that were released by the cells and the adsorption of media components, which might affect the activity of the biofunctionalization. However, the observed effects of the biofunctionalized surfaces in comparison to the pristine CVD coatings show that the duration of activity is sufficient to stimulate HSPCs. All in all, the proliferative effect of DLL1 on HSPCs in presence of RGD was the highest on surfaces carrying the higher concentration of DLL1. We conclude that the amount of DLL1 molecules on the PPX-AM-co-alkyne(5:1) was sufficient for triggering cell expansion while the amount of DLL1 on substrates with less amines (PPX-AM-co-alkyne(1:10)) was not. These results are in agreement with previous studies showing that the surface concentration of DLL is an important determinant for the proliferative effects of this molecule on HSPCs15,3. Thus, the developed surfaces are suitable to mimic crucial aspects of how the niche controls HSPC behavior via cell-cell and cell-matrix interactions. Neighboring cells in the niche present ligands such as DLL1 that bind to Notch receptors in the HSPC membrane. The activation of Notch


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leads to the cleavage of the intracellular domain of Notch that directly travels to the nucleus and acts there as a transcription regulator41. The resulting changes in gene expression are known to enhance proliferation and myeloid differentiation of HSPCs16. As the aim of the current study was to develop surfaces that allow HSPC proliferation but limit differentiation, the second stimulus – RGD – was included into the system. The RGD peptide – recognized by human CD34+ HSPCs via integrin α5β139 – is a bioactive sequence of the fibronectin fragment that was shown before to limit myeloid differentiation of HSPCs in the presence of DLL116. The interplay of Notch and integrins is well known42. In the present study it appears that the crosstalk of the targeted receptors – Notch and integrin α5β1 – yields enhanced proliferation of HSPCs without triggering myeloid differentiation. Therefore, mimicking cell-cell stimulation via DLL1 as well as cell-matrix interactions via RGD is a promising strategy for the development of artificial stem cell niches that allow in vitro proliferation of HSPCs.

CONCLUSION The interactions of HSPCs and their environment in the stem cell niche are highly complex and very dynamic. Components of the extracellular matrix and adjacent supporting cells as well as soluble factors such as cytokines influence stem cell fate. In the present study, we developed a convenient, polymer based, and biocompatible surface with orthogonally functionalizable chemical groups to covalently bind DLL1 and RGD in various surface ratios. PPX-AM-coalkyne copolymers with different ratios of amino and alkyne groups were coated onto surfaces by CVD with two inlet sources and postfunctionalized with biomolecules via CuAAC and via EDC/NHS conjugation.


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In a proof-of-principle stem cell culture study, all surface films proved to be cytocompatible and the RGD-DLL1-dual functionalized PPX-AM-co-alkyne(5:1) surface was found to significantly enhance HSPC proliferation. Thus, we developed a versatile platform that allows orthogonal dual functionalization of cytocompatible surfaces with two biomolecules of choice and has future potential for both, fundamental stem cell studies as well as ex vivo expansion of human HSPCs.

ASSOCIATED CONTENT Supporting Information: -

Supporting tables S1 to S3

-

Supporting schemes S1 and S2

-

Supporting Figures S1 to S9

This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]


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Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally.

Funding Sources The research was funded by the BMBF NanoMatFutur Program (FKZ 13N12968), the Program “BioInterfaces in Technology and Medicine” of the Helmholtz Association and the German Research Foundation (DFG) (SFB 1176, Project B3).

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The project was funded by the BMBF NanoMatFutur Program (FKZ 13N12968). A.W., M.K., C.H., J.L. and C.L.T. acknowledge support by the program “Biointerfaces in Technology and Medicine” of the Helmholtz Association. C.H. and J.L. thank the German Research Foundation (DFG) for financial support within the frame of the collaborative research center SFB 1176 (Project B3). The authors thank Saskia Kraus for excellent technical assistance.


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REFERENCES 1. Wayne, A. S.; Baird, K.; Egeler, R. M., Pediatric Clinics of North America: Hematopoietic Stem Cell Transplantation Stem Cell Transplantation for Leukemia. Pediatr Clin North Am 2010, 57, 1-25. 2. Cortez, A. J. P.; Dulley, F. L.; Saboya, R.; Mendrone Júnior, A.; Amigo Filho, U.; Coracin, F. L.; Buccheri, V.; Linardi, C. d. C. G.; Ruiz, M. A.; Chamone, D. d. A. F., Autologous hematopoietic stem cell transplantation in classical Hodgkin's lymphoma. Revista Brasileira de Hematologia e Hemoterapia 2011, 33, 10-14. 3. Delaney, C.; Heimfeld, S.; Brashem-Stein, C.; Voorhies, H.; Manger, R. L.; Bernstein, I. D., Notch-mediated expansion of human cord blood progenitor cells capable of rapid myeloid reconstitution. Nat Med 2010, 16, 232-6. 4. Boitano, A. E.; Wang, J.; Romeo, R.; Bouchez, L. C.; Parker, A. E.; Sutton, S. E.; Walker, J. R.; Flaveny, C. A.; Perdew, G. H.; Denison, M. S.; Schultz, P. G.; Cooke, M. P., Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells. Science 2010, 329, 1345-8. 5. Rohrabaugh, S. L.; Campbell, T. B.; Hangoc, G.; Broxmeyer, H. E., Ex vivo rapamycin treatment of human cord blood CD34+ cells enhances their engraftment of NSG mice. Blood Cells Mol Dis 2011, 46, 318-20. 6. Boulais, P. E.; Frenette, P. S., Making sense of hematopoietic stem cell niches. Blood 2015, 125, 2621-9. 7. Dahlberg, A.; Delaney, C.; Bernstein, I. D., Ex vivo expansion of human hematopoietic stem and progenitor cells. Blood 2011, 117, 6083-90. 8. Walasek, M. A.; van Os, R.; de Haan, G., Hematopoietic stem cell expansion: challenges and opportunities. Annals of the New York Academy of Sciences 2012, 1266, 138-50. 9. Tiwari, A.; Tursky, M. L.; Mushahary, D.; Wasnik, S.; Collier, F. M.; Suma, K.; Kirkland, M. A.; Pande, G., Ex vivo expansion of haematopoietic stem/progenitor cells from human umbilical cord blood on acellular scaffolds prepared from MS-5 stromal cell line. J Tissue Eng Regen Med 2013, 7, 871-83. 10. Jing, D.; Fonseca, A. V.; Alakel, N.; Fierro, F. A.; Muller, K.; Bornhauser, M.; Ehninger, G.; Corbeil, D.; Ordemann, R., Hematopoietic stem cells in co-culture with mesenchymal stromal cells--modeling the niche compartments in vitro. Haematologica 2010, 95, 542-50. 11. Pytela, R.; Pierschbacher, M. D.; Ruoslahti, E., Identification and isolation of a 140 kd cell surface glycoprotein with properties expected of a fibronectin receptor. Cell 1985, 40, 191-8. 12. Nobta, M.; Tsukazaki, T.; Shibata, Y.; Xin, C.; Moriishi, T.; Sakano, S.; Shindo, H.; Yamaguchi, A., Critical regulation of bone morphogenetic protein-induced osteoblastic differentiation by Delta1/Jagged1-activated Notch1 signaling. J Biol Chem 2005, 280, 15842-8. 13. Delaney, C.; Varnum-Finney, B.; Aoyama, K.; Brashem-Stein, C.; Bernstein, I. D., Dosedependent effects of the Notch ligand Delta1 on ex vivo differentiation and in vivo marrow repopulating ability of cord blood cells. Blood 2005, 106, 2693-9. 14. Civin, C. I.; Strauss, L. C.; Brovall, C.; Fackler, M. J.; Schwartz, J. F.; Shaper, J. H., Antigenic analysis of hematopoiesis. III. A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG-1a cells. J Immunol 1984, 133, 157-165. 15. Winkler, A.-L.; von Wulffen, J.; Rödling, L.; Raic, A.; Reinartz, I.; Schug, A.; GrallaKoser, R.; Geckle, U.; Welle, A.; Lee-Thedieck, C., Significance of Nanopatterned and Clustered DLL1 for Hematopoietic Stem Cell Proliferation. Adv Funct Mater 2017, 27, 1606495.


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Table of content


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Figure 1: Schematic representation of the presented biomaterial concept comprising three steps: A) CVD polymerization of copolymers with varying composition, B) bio-functionalization, C) HSPC cultivation. 129x135mm (300 x 300 DPI)


Biomacromolecules

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Figure 2. (A) IRRAS spectra of copolymers with three different compositions achieved by variation of the argon carrier gas flow rates. PPX-AM-co-alkyne ratios overlay with 5:1 as black line, 1:1 as red line, and 1:10 as blue line; (B) Nitrogen content of copolymers measured by XPS. The stars represent the theoretical values derived from the calculated values of the molar component ratios. 96x158mm (300 x 300 DPI)


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Figure 3. XPS N 1s (A) and ToF-SIMS (B, C) measurements of biofunctionalized PPX-AM-co-alkyne surfaces. (A) High resolution XPS N 1s spectra of (from bottom to top) PPX-AM-co-alkyne(1:4) polymer coating on gold slides, RGD without Cu2+, RGD with Cu2+, and RGD-azide reference (100 µM; RGD-azide in water without Cu2+ and sodium ascorbate), showing the specific immobilization of RGD to the CVD copolymer. All spectra were normalized to the maximum of intensity. ToF-SIMS results of CNO- signal (B) of the RGDpeptide (35 µM) and DLL1 protein (2.5 µg/mL) backbone and (C) cysteine (S-, sulfur) amino acid signal of human DLL1, proving the immobilization of both biomolecules on the surfaces. PPX-AM-co-alkyne polymer (black line), RGD and PEG (blue line), RGD and DLL1 (purple line). 168x133mm (300 x 300 DPI)


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Figure 4. HSPC proliferation, apoptosis and CD34 expression on various surfaces. (A) Cytotoxicity of the different applied surfaces with and without RGD biomolecule coupling via CuAAC. Shown are the mean values of N=3, in case of plain copolymers N=1, independent experiments. Error bars represent the standard deviation (SD). ‘PPX-AM-co-alkyne’ is abbreviated by ‘PPX’. (B) Exemplary contour plot (Annexin VFITC plotted against Sytox AADvanced) with gating of the different subpopulations. (C-F) Results of the total cell count relative to the number of cells on TCP found over time (C, E) and bar diagrams of the number of CD34+ cells normalized to the number of cells found on TCP on day 14 (D, F). (C-F) Mean values of N=3 independent experiments. Error bars represent standard error of the mean (SEM). 179x189mm (300 x 300 DPI)


Bioinstructive Coatings for Hematopoietic Stem Cell Expansion Based

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