FOR INDUCING STEM CELL DIFFERENTIATION AND PRODUCING

HOMOGENOUS POPULATION OF A DESIRED CELL TYPE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Nos. 61/230,605 and 61/230,620, both filed July 31, 2009, and both herein incorporated by reference in their entirety.

INTRODUCTION

The study and use of biological cells is of considerable interest in a number of clinical and research applications, particularly stem cell-based therapies. Stem cell therapies rely on the ability of stem cells, which are found in most all multicellular organisms, to renew themselves and differentiate into a diverse range of specialized cell types. The success of stem cell therapies hinges on the ability to influence cell behavior in a selective manner so as to control cell adhesion, growth, cell structure, cell differentiation, and cell survival.

To this end, the art is aware of several methodologies for influencing cell behavior. One such method supplements cell cultures with additives including ligands, small molecular weight compounds, growth factors, cytokines, and/or other exogenous soluble agents. While such additives may influence cell behavior, current methods have significant drawbacks. Many cell culture supplements, and in particular growth factors and cytokines, are expensive, requiring complex purification and/or expression procedures to yield a biologically active agent. Furthermore, many supplements are pleiotropic, and may thus give rise to a range of ill-defined effects depending on the concentrations used and the cells to which these factors are provided.

Consequently, cell populations grown in contact with exogenous agents can develop considerable levels of heterogeneity. Such heterogeneity can prove problematic in applications requiring a substantially pure cell population.

SUMMARY

Embodiments herein include but are not limited to methods, compositions, assays, biochips, kits, materials, tools, instruments, reagents, products, compounds, pharmaceuticals, devices, arrays, programs, computer-implemented algorithms, and computer-implemented methods.

In one aspect, there is provide a method for identifying at least one compound in a chemical library that regulates stem cell differentiation, comprising: (a) immobilizing a plurality of different compounds from said chemical library on a surface; (b) culturing stem cells on said surface for a period of time sufficient for cell differentiation; and (c) analyzing said stem cells to identify at least one compound that regulates stem cell differentiation.

In another aspect, here is provided a high throughput method for regulating stem cells, comprising: (a) screening at least one chemical library for at least one putative compound that regulates stem cell differentiation; (b) identifying and selecting at least one compound from step (a) that expresses at least one molecular or cellular marker associated with a stem cell phenotype; (c) patterning a small surface with each compound selected in step (b) and culturing stem cells on said small surface for a period of time sufficient for stem cell differentiation; (d) identifying and selecting at least one compound from step (c) that expresses at least one molecular or cellular marker associated with a stem cell phenotype, and (e) patterning an edge to edge surface with at least compound from step (d) and culturing stem cells on said edge to edge surface. Patterning can be carried out with use of nanoscopic tips and patterning compounds in steps c and e.

In another aspect, provided herein is a high-throughput assay for identifying at least one chemical functional group and/or a surface pattern for regulating a cell phenotype.

In another aspect, there is a biochip comprising a patterned surface. In one embodiment, the biochip comprises a cell.

In another aspect, there is a cell culture dish comprising a patterned surface. In one embodiment, the cell culture dish comprises a cell.

In one aspect, herein provides a method for regulating stem cell differentiation, comprising: (a) patterning a surface with at least one growth factor and/or polymer to create a patterned surface; and (b) culturing stem cells on said patterned surface for a period of time suitable for growth and/or differentiation of said stem cells.

In one embodiment, patterning is performed by a nanolithography method. In a further embodiment, a nanolithography method is dip pen nanolithography, nanoimprint lithography; direct atomic force microscopy; etching glancing angle deposition; laser ablation; laser deposition; replica molding of x-ray lithography masters; micro contact printing, or etching electron-beam direct-write lithography.

In another embodiment, the growth factor is Bone Morphogenetic Proteins (BMPs), Brain-Derived Neutrophic Factor (BDNF), Ciliary Neutrophic Factor (CNTF), Epidermal Growth Factor (EGF), Erythropoietin (EPO), Fibroblast Growth Factor (FGF), Granulocyte- Colony Stimulating Factor (G-CSF), Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF), Growth Differentiation Factor-9 (GDF9), Hepatocyte Growth Factor (HGF), Insulin-like Growth Factor (IGF), Interleukin (IL), Leukemia Inhibitory Factor (LIF), Myostatin (GDF-8), Nerve Growth Factor (NGF), Neutrophic Factors (NT), Platelet- derived Growth Factor (PDGF), Thrombopoietin (TPO), Transforming Growth Factor alpha(TGF-α), Transforming Growth Factor beta (TGF-β), or Vascular Endothelial Growth Factor (VEGF).

In another embodiment, the polymer is laminin, collagen, poly-lysine L, poly-lysine D, metrigel, fϊbronectin, gelatin, and poly-ornithine.

In another embodiment, the surface is gold.

In another embodiment, the stem cell is an adult stem cell. In other embodiments, the stem cell is an embryonic stem cell.

In another embodiment, the period of time is at least 14 days.

In another aspect, there is provided a method for producing a differentiated stem cell, comprising: (a) patterning a surface with at least one growth factor and/or polymer to create a patterned surface; and (b) culturing stem cells on said patterned surface for a period of time suitable for growth and/or differentiation of said stem cells.

In another aspect, there is provided a method for maintaining an undifferentiated stem cell, comprising: (a) patterning a surface with at least one growth factor and/or polymer to create a nanopatterned surface; and (b) culturing said stem cell on said patterned surface for a period of time suitable for growth and/or differentiation of said stem cell.

In another aspect, there is a method for patterning at least one growth factor and/or polymer on a surface, comprising depositing said growth factor and/or polymer on said surface using a nanolithography technique. In another aspect, here is provided a patterned surface comprising at least one growth factor and/or polymer. In an embodiment, the patterned surface comprises a cell.

In another aspect, there is a patterned surface comprising a biological cell. In an embodiment, the cell is a stem cell or a non stem cell.

In another aspect, there is provided a kit comprising a patterned surface and at least one growth factor and/or polymer.

In another aspect, there is provided a cell culture dish comprising a patterned surface and at least one growth factor and/or polymer. In an embodiment, the cell culture dish comprises at least one cell.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 provides a schematic outline of a high throughput screening (HTS) system for identifying and optimizing pattern surfaces for stem cell regulation.

Figure 2(a) shows the chemical structure of thiol inks for 1-hexadecane thiol (HDT), 16- mercaptohexadecanoic acid (MHA) , 11-mercaptoundecylamine (AUT), and 11- mercaptoundecanol (MUO).

Figure 2(b) shows representative images of the nano features patterned using dip pen nano lithography (DPN) at various pitches. Image (i) shows a pattern at pitch of da = 1000 nm; Image (ii) shows a pattern at pitch da = 280 nm; and Image (iii) shows a pattern at pitch of dα = 140 nm.

Figure 3 shows STRO-I expression in MSC stem cells grown on ODT modified and control substrates at 24 hours (left), 14 days (middle) and 28 days (right), visualization using confocal microscopy.

DETAILED DESCRIPTION

It has been shown that stem cell differentiation is heavily influenced by the cellular environment, as specific micro- and nano-scale environments are capable of producing changes in cell alignment, polarization, elongation, migration, proliferation and gene expression, and can control the dynamics of stem cell growth and differentiation. Recent evidence suggests that nanometer level changes in the extracellular matrix (ECM) has significant effects on the timing and direction of adult stem cell differentiation [Martinez et al., 2009, Annals of Anatomy - Anatomischer Anzeiger, 191, 126-135].

In 2006, Curran et al. demonstrated that various substrates (clean glass, PLLA and PCL) modified with -CH ₃ , -OH, -COOH and -NH ₂ groups, via silane modifications, have the ability to trigger and maintain MSC differentiation in vitro. The ability of the surfaces to induce differentiation was extensively researched on silane modified surfaces, but cellular responses were heterogeneous across the surface, largely due to the heterogeneous nature of the modifications (especially at the nanometer scale).

In recognizing that current protocols fail to produce a differentiated and homogeneous stem cell population, the present inventors have developed approaches for regulating stem cell growth and differentiation.

For example, and as explained in greater detail below under subheading "A, " the present inventors developed a high throughput screening assay for identifying chemical groups and/or surface patterns controlling stem cell differentiation. Stem cells and non stem cells alike can be grown on these surfaces and the effect of these surfaces are examined in terms of stem cell self-renewal, differentiation, survival, adhesion, and migration.

Similarly, and as discussed below under subheading "B," the present inventors contemplate using at least one growth factor and/or polymer for coating a surface at a desired scale, pattern, size, and shape, and then culturing stem cells on said surface.

All technical terms used herein are terms commonly used in cell biology, biochemistry, molecular biology, and nanolithography and can be understood by one of ordinary skill in the art to which this invention belongs. These technical terms can be found in the current editions of Molecular Cloning: A Laboratory Manual, (Sambrook et al., Cold Spring Harbor); Gene Transfer Vectors for Mammalian Cells (Miller & Calos eds.); and Current Protocols in Molecular Biology (F. M. Ausubel et al. eds., Wiley & Sons). Cell biology, protein chemistry, and antibody techniques can be found in Current Protocols in Protein Science (J. E. Colligan et al. eds., Wiley & Sons); Current Protocols in Cell Biology (J. S. Bonifacino et al., Wiley & Sons) and Current Protocols in Immunology (J. E. Colligan et al. eds., Wiley & Sons.). Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, ClonTech, and Sigma-Aldrich Co. Cell culture methods are described generally in the current edition of Culture of Animal Cells: A Manual of Basic Technique (R. I. Freshney ed., Wiley & Sons); General Techniques of Cell Culture (M. A. Harrison & I. F. Rae, Cambridge Univ. Press), and Embryonic Stem Cells: Methods and Protocols (K. Turksen ed., Humana Press). Other texts include Creating a High Performance Culture (Aroselli, Hu. Res. Dev. Pr. 1996) and Limits to Growth (D. H. Meadows et al., Universe Publ. 1974). Tissue culture supplies and reagents are available from commercial vendors such as Gibco/BRL, Nalgene-Nunc

International, Sigma Chemical Co., and ICN Biomedicals.

Also, cells may be cultured and grown as disclosed in U.S. Application No:

12/458,425, filed July 10, 2009; U.S. Application No 12/656,310, filed January 25, 2010; U.S. Application No. 12/656,311, filed January 25, 2010; U.S. Application No. 12/656,312, filed January 25, 2010; U.S. Application No. 12/656,313, filed January 25, 2010; US provisional application 61/295,133 filed January 14, 2010; and WO 2010/047939, filed October 5, 2009, each of which is herein incorporated by reference in its entirety.

Nanolithography methods, such as direct-write technologies can be carried out by methods described in, for example, Direct-Write Technologies for Rapid Prototyping Applications: Sensors, Electronics, and Integrated Power Sources, Ed. by A. Pique and D. B. Chrisey, Academic Press, 2002. Chapter 10 by Mirkin, Demers, and Hong, for example, describes nano lithographic printing at the sub- 100 nanometer length scale, and is hereby incorporated by reference (pages 303-312). Pages 311-312 provide additional references on scanning probe lithography and direct-write methods using patterning compounds delivered to substrates from nanoscopic tips which can guide one skilled in the art in the practice of the present invention. Nanolithography and nanofabrication is also described in Marc J. Madou's Fundamentals of Micro fabrication, The Science of Miniaturization, 2.sup.nd Ed., including metal deposition at pages 344-357. See, e.g., U.S. Pat Nos. 6,827,979; 6,635,311; and 6,867,443.

Although this specification provides guidance to one skilled in the art to practice the present technology, including reference to technical literature, mere reference does not constitute an admission that the technical literature is prior art.

A. Large Scale Screening of Chemicals/Compounds Involved in Stem Cell Regulation

1. Chemical Libraries and Immobilization One can screen a large number of compounds from chemical libraries to identify optimum functional groups for surface patterning. See, for example, Figure 1, step 1.

A chemical library or compound library is a collection of stored chemicals usually used ultimately in high-throughput screening or industrial manufacture. The chemical library can consist in simple terms of a series of stored chemicals. Each chemical has associated information stored in some kind of database with information such as the chemical structure, purity, quantity, and physiochemical characteristics of the compound. Methods for generating chemical libraries are known in the art and chemicals are

commercially available. See , e.g., U.S. Pat. No. 6,677,160.

For example, and in no way limiting the invention, a chemical library may comprise compounds/chemicals shown to impact stem cell growth and differentiation, such as peptides, cytokines, erythropoietin etc. and alkylthiols that have chemical functional groups Of-CO ₂ H, -NH ₂ , -CH ₃ , -OH, -SH, which have been shown to influence MSC adhesion and differentiation [Curran, J., Chen, R. & Hunt, J. The guidance of human mesenchymal stem cell differentiation in vitro by controlled modifications to the cell substrate. Biomaterials 27, 4783-4793 (2006)]. Chemicals and compounds tested will vary as new libraries are identified, and those shown to have some effect on stem cell growth/differentiation are modified and tested further.

A chemical functional group refers to a specific group of atoms within a molecule that are responsible for the characteristic chemical reactions of those molecules. The same functional group will undergo the same or similar chemical reaction(s) regardless of the size of the molecule it is a part of. Non- limiting exemplar chemical functional groups include CO ₂ H, NH ₂ , CH ₃ , OH, or SH, (-0-CH ₂ -CH ₂ -OH). Such chemical functional groups can be added to any molecule of interest, including but not limited to mercaptoundecanol, mercaptohexadecanoic acid, 11-mercaptoundecyl amine, hexadecane thiol, octadecane thiol, mercaptohexadecanoic acid, 2-{2-[2-(l-mercaptoundec-l l-yloxy)-ethoxy]-ethoxy}-ethanol, and thiotic acid-2-{2-[2-(l-mercaptoundec-l l-yloxy)-ethoxy]-ethoxy}-ethanol.

For example, compounds from chemical libraries can be assayed using glass 96 well plates, or polystyrene, polypropylene, cyclo-olefm, and polycarbonate well plates that are modified directly with organosilane or silane promoter followed by organosilane. The organosilane-modified surfaces are linked to a large numbers of chemicals from chemical libraries, thereby immobilizing the chemical compounds on the surface of the plates. Adult or embryonic stem cell suspensions are added to the 96 well plates and cells are cultured in direct contact with the chemically modified surfaces for a period of time sufficient for growth and/or differentiation (14 and 28 days). Cells, growth of cells, cell differentiation, and stem cells are generally known in the art. See, for example, Essentials of Stem Cell Biology, (Ed. R. Lanza), 2006; Cell Biology, 2 ^nd Ed., Pollard and Earnshaw, 2008; Gilbert, Developmental Biology, 5 ^th ed., 1997; Cell Lineage Specification and Patterning of the Embryo, (ed. Etkin, Jeon), 2001. See Curran et al., Biomaterials, 27 (2006), 4783-4793; Curran et al., Biomaterials 26 (2005), 7057-7067. Exemplary cells include those disclosed in Curran et al. Biomaterials 27 (2006), 4783-4793.

At time periods of, for example, 14 and 28 days post-culture, the cells can be analyzed using various molecular and cellular biology techniques known in the art. For example, an in no way limiting the invention, reverse transcription PCR (RT-PCR) may be used to detect a specific genetic marker indicative of cell differentiation. Likewise, a western blot and/or Bradford protein assay may be performed to analyze protein expression commensurate with a specific cell type. Similarly, various cell sorting techniques such as fluorescence-activated cell sorting (FACS) may used to determine cell differentiation.

Compounds identified as regulating stem cell growth and/or differentiation are selected for further study.

2. Testing Small Pattern Areas for Stem Cell Regulation

Compounds identified from the chemical library screen that regulate stem cell growth and/or differentiation are used to pattern an array of dots to cover a small area.

Any lithography technique by which molecules are delivered to a substrate of interest in a positive printing mode may be used. Lithography can be done at the micro level or nano scale, as is the case for nanolithography. Non-limiting exemplary nanolithography methods include but are not limited to dip pen nanolithography (DPN), nanoimprint lithography; direct atomic force microscopy; etching glancing angle deposition; laser ablation; laser deposition; replica molding of x-ray lithography masters; micro contact printing, and etching electron-beam direct-write lithography.

For example, and in no way limiting the invention, dip pen nanolithography (DPN) may be used for depositing at least one growth factor and/or polymer on a surface for cell growth. In its broadest sense, DPN utilizes a solid substrate as the "paper" and a scanning probe microscope tip (e.g., an atomic force microscope (AFM) tip) as the "pen". The tip is coated with a patterning compound (the "ink"), and the coated tip is used to apply the patterning compound to the substrate to produce a desired pattern. As presently understood, the molecules of the patterning compound are delivered from the tip to the substrate by capillary transport.

DPN™ , DIP PEN NANOLITHOGRAPHY™, and are trademarks of Nanolnk, Inc. and are used accordingly herein (e.g, DPN printing or DIP PEN NANOLITHOGRAPHY printing). DPN methods and equipment are generally available from Nanolnk, Inc. (Chicago, 111.), including the NLP™ and NScriptor™, which can be used to carry out nanolithography.

As used herein, the term patterning means any micro- or nano-scale lithography technique for depositing at least one growth factor and/or polymer on a surface in a pattern. The resultant delivery of said growth factor and/or polymer produces a pattern, which may be defined by spot size, shape, and pitch. As circumstances may require, a pattern may be altered for a given purpose or need. Nanopattern refers to patterns generated at the nano- scale level, whereas micropattern refers to patterns generated at the micro-scale level.

Exemplary nanopatterned surfaces for stem cells are disclosed in Curran et al. J. Mater Sci: Mater Med 21 :1021-1029 (2010); and Curran et al. Lab Chip 10, 1662-1670 (2010).

For example, and in no way limiting, the small area may be 500x500 μm ² of 6 different center-to-center dot spacing (pitch size, dα, of 100, 200, 400, 600, 800 and lOOOnm), on a 5mm ² gold surface (dot diameter and spacing will be confirmed by topographical atomic force microscopic (AFM) imaging). In other embodiments, the patterned areas may consist of 70 nm dot diameter (dβ) or smaller, or lesser than 100 or 200 nm, or the 500x500 μm ² area will be filled with arrays of nanodots of various dimensions such 5x5, 10x10, 20x20, 30x30 μm ² and separated by the same or large distances.

Human adult or embryonic stem cells are grown on these surfaces for 14 and 28 days in basal culture media (media without supplements that may stimulate differentiation) and then analyzed using high content fluorescent analysis to evaluate the cellular response to the nanopattern on the biochips. The expression(s) of key stem cell and differentiated stem markers are analyzed to ascertain cellular phenotype and any induced differentiation solely attributable to the cell-to-surface interaction. Surfaces that successfully demonstrate the ability to control stem cell dynamics, i.e. maintaining the cells in an undifferentiated state or inducing controlled differentiation into a desired cell type, are further developed.

3. Edge-to-Edge Patterned Surfaces for Producing Differentiated Stem Cells

Following identification of patterns (chemical groups and center to center dot spacing) that produce a desired cell type(s), the patterns are further developed through the production and testing (e.g. adherence, growth, differentiation determined by cell morphology and stem cell marker analysis) of edge-to-edge 5 mm nanopattern substrates.

Stem cells are grown on these surfaces for 1, 14, and 28 days in basal culture media and then analyzed using various molecular and cellular methodologies, including qPCR, high content fluorescence analysis, FACS analysis, and ELISA (enzyme-linked

immunosorbent assay) to evaluate the cellular response on the nanopattern biochips. For example, the expressions of key adipocyte markers (e.g. adiponectin, adipocyte lipid binding protein, fatty acid transporter) can be quantified to ascertain production of homogenous populations of fat cells from MSCs.

The assay is also useful for identifying chemical functional groups and topographic patterns capable of producing desired cell types from adult and embryonic stem cells.

Depending on circumstance and need, patterned surfaces as described herein, may be used to regulate or control stem cell growth and differentiation. For example, a patterned surface comprising at least one functional group may be used to promote cell

differentiation. Likewise, a patterned surface comprising a different functional group may be used to maintain cells in an undifferentiated state.

4. Illustrative Patterned Products

The patterned surfaces provided herein may be used in a variety of products, including but not limited to biochips, kits, reagents, and cell culture dishes and related tools.

Specific examples are presented below of methods for identifying chemical functional groups and pattern dimensions for regulating a desired cell type. They are exemplary and not limiting. EXAMPLE 1

Preparation of small area nanopatterned substrates and their effect on stem cells

To evaluate the influence of controlled nanopatterns with specific chemical functionalities, 4 thiol based inks are deposited onto gold (Au) surfaces, in defined nanopatterns (700μM x 35μM), using a DPN based NScriptor system and a one- dimensional pen array. The nanopatterns include arrays of 70nm (dβ) dots separated by defined spacings (dα) of 140, 280, and 1000 nm with terminal chemical functionalities of carboxyl, amino, methyl and hydroxyl (MHA, AUT, HDT, and MUO, respectively; see Figures 2 A and 2B). ODT can be also used.

Human MSCs are grown in complete MSC growth media (Lonza MSC growth medium blue kit PT-3001), at 37°C under 5% CO ₂ . The media is replaced every 3-4 days, and cells are subcultured once when approximately 90% confluent. Plain gold and fabricated gold substrates are sterilized by washing with 200 proof ethanol, rinsed twice with PBS, and stored in fresh PBS until shortly before cell plating. Fourth passage cells are trypsinized and resuspended at a density of 50,000 cells/ml. One ml of cell suspension is placed in each well of a 24 well plate containing one 5mm ² gold substrate/well. MHCs are grown on these surfaces for 24 hours. The cells were then fixed and stained for actin cytoskeleton and for vinculin.

MHA and MUA patterns yield similar results: on surfaces with a pitch of 280 nm the cells attached and formed dense spread clusters with focal contacts spread throughout the body of the cell cluster, while on surfaces with a pitch of 140 and 1000 nm there was no viable cell adhesion. In contrast, cells attached well to AUT patterned areas at all three pitches. On surfaces with a pitch of 280 nm, the cells were relatively elongated and polarized, with stress fibers running throughout the body of the cells in parallel to the direction of orientation; while on surfaces with a pitches of 140 and 1000 nm the cells were well spread with focal contacts associated with stress fibers running in all directions throughout the body of well spread cells.

This data provides evidence that the combination of specific chemistry with defined positioning and density has a direct effect on cell adhesion and function. This control was provided by controlling initial adhesion, focal contact formation, clustering, distribution, and spreading of cells on surfaces. There were many important subtle variations in adhesion between well adhered and fully spread to non-adherent within which cells can be directed to become differentiate into those with the desired phenotype and function. Even significant inhibition of cell adhesion, using chemistry and spacing to inhibit focal contact formation, can be utilized to control cell populations; for example MHA and MUO chemistries provided the optimal chemistry for chondrogenic differentiation, AUT for osteoblast differentiation and ODT for maintaining MSC phenotype. This provided the principle of controlling cell growth by defined nanopatterns through the use of specific chemical functionalities that can be chosen for different cell phenotypes and functions.

EXAMPLE 2

5mm ² patterned biochips with uniform edge-to-edge and their effect on stem cells

In order to characterize the effects of the nanopatterns on MSC cell growth and differentiation, it is important to generate uniform nanofeatures that cover the entire substrate surface. Alkythiol based inks are deposited onto gold (Au) surfaces, in defined nanopatterns, using a DPN based NScriptor system and a two-dimensional pen array (2DnanoPrintArray™) consisting of 55,000 tips in a lcm chip. The 2D nanoPrintArray is level with respect to the substrate surface, thereby providing uniform contacts between the cantilevers and the surface which leads to reproducible, accurate, and homogeneous edge- to-edge patterning of alkylthiol nanostructures across large areas.

Nanopatterns include arrays of 70 nm (dβ) dots separated by defined spacing's (dα) of 140, 280 and 1000 nm with terminal functionalities of carboxyl, amino, methyl and hydroxy (MHA, AUT, HDT, ODT, and MUO, respectively). In order to achieve uniform patterning across the surface, DPN parameters such as homogeneous tip coating (by vapor coating), temperature and humidity are controlled. Calibration and determination of the diffusion co-efficient for each ink is carried out prior to deposition of the patterns and the tips are tested for writing ability at the end of each deposition cycle to ensure sufficient ink.

Nanopattern homogeneity, dot diameter,and spacing is confirmed by topographical AFM imaging. Typically, tip dwell times (time the tip rests on the surface) of 0.01 s (HDT, ODT, MHA, MUO and AUT) are used to produce dots of 65-70 nm (dβ), using different temperature for each alkylthiol compound.

Human MSCs are grown and plated on these substrates as described above in Example 1. The cells are fed complete growth medium every 2-3 days for the duration of a 28-day experiment. MSCs are grown on these surfaces for 1, 14, and 28 days and then analyzed using qPCR, immunofluorescence, and FACs analysis to evaluate cellular response to the nanopatterns. The expression of key markers is quantified to ascertain cellular phenotypes and any induced differentiation solely attributable to the cell-substrate interaction. Plain gold control substrates without chemical patterns are run in parallel.

EXAMPLE 3

28 day experimental results with ODT 280nm (dα) 5mm ² nanopattern samples A. High-content Immunofluorescence Imaging

Nanopatterned substrates are removed from cell culture plates and the cells are fixed in 4% formaldehyde/2% sucrose for 10 minutes at room temperature, rinsed with PBS, and permeabilized in PBS / 0.2% Triton for 10 minutes at 4°C. The cells are rinsed again with PBS and blocked in PBS/1% BSA for 1 hour at room temperature, incubated in the indicated primary antibodies in PBS/1% BSA overnight at room temperature. Cells are then washed three times in PBST (PBS plus 0.1% Tween-20), and then incubated with 1 :400 dilutions of AlexaFluor-488, -546 or -647 labeled secondary antibodies (Invitrogen) in PBS/1% BSA for 1 hour at room temperature. The cells are again washed three times in PBST and mounted on slides using ProLongGold plus DAPI (Invitrogen). Cells grown on gold-coated silicon substrates are mounted using 0.5 mm thick, 20 mm diameter Press-to- Seal™ silicone isolators with adhesive (Invitrogen). Finally, cells are examined using a Zeiss Axiolmager Zl microscope.

Primary antibodies include: STRO-I, vinculin, nucleostemin, osteocalcin, CBFAl, collagen 2, SOX9, GaIC, beta-tubulin III, and ADRP.

At 24 Hours:

1. As shown in Figure 3, STRO-I (an MSC marker) expression increased on ODT modified surfaces compared with plain gold controls. Qualitative evaluation shows that every cell on the ODT surface is STRO-I positive, indicating a more homogenous population than observed on control substrates.

2. Collagen I (MSC marker) expression was up-regulated and homogenous on cells cultured in contact with ODT modified surfaces, i.e. every cell expressed collagen I, whereas cells cultured on plain gold control surfaces were positive for collagen I in only small areas. In terms of the overall cell response, based on morphology and expression of markers of interest, the response was homogenous across the test substrates.

At 14 Days:

1. As shown in Figure 3, STRO-I expression is enhanced on ODT modified surfaces with 100% homogenous expression across the surface (not observed on control substrates, see Figure 3).

2. Collagen I is homogenously expressed in cells on ODT modified surfaces, at levels higher than on control substrates.

3. Nuleostemin expression is observed in small clusters on ODT modified surfaces. As MSCs display contact inhibition, nucleostemin expression may be lost as the cells become confluent, with only small clusters of expression maintained.

At 14 days cells on ODT surfaces show enhanced MSC phenotype as demonstrated by an increased expression of STRO-I and the absence of differentiation markers.

At 28 Days:

1. As shown in Figure 3, increased, and homogenous expression of STRO-I is observed on ODT modified surfaces (even though the cells are over confluent). On the control surface STRO-I expression is heterogeneous across the surface (see Figure 3).

2. Expression of collagen I is homogenous on ODT surfaces, with evidence of extracellular collagen I. This pattern is also evident on the control surface.

3. Samples were negative for nucleostemin, this is attributable to the cells filling the available space and are therefore not proliferating in the current environment. Regardless of this the cells on the ODT surface are still positive for STRO-I indicative of MSC phenotype.

The expression profile observed at 14 days is maintained at 28 days, with MSCs cultured on ODT surfaces showing enhanced and homogenous expression of STRO-I and collagen I compared to controls (cells on control surface have lost the ability to express STRO-I). This was also true for collagen I. Cells were also negative for all differentiation markers tested, including: collagen II and X, aggrecan, adiponectin, osteocalcin, and CBFAl. 3.2. Real-time polymerase chain reaction (RT-PCR).

Cells are trypsinized, collected, and washed with phosphate buffered saline (PBS). Total RNA is extracted from cells with TaqMan Gene Expression Cells-to-CT Kit

(Ambion). Total RNA is treated with DNase I to remove genomic DNA and reverse transcribed with appropriate mixture of oligo (dT)/random primers according to

manufacturer's instructions. Real-time PCR master mix is prepared by combining TaqMan gene expression master mix, cDNA, and nuclease-free water according to manufacturer's instructions (Ambion). The samples are loaded into custom made TaqMan arrays (32- format) (Applied Biosystems). Signals are detected with a 7900HT Fast Real-Time PCR System (Applied Biosystems). The relative expression levels of genes is determine by the 2-ΔCT method and normalized against RPLPO. Undifferentiated MSCs (passage 4) is used for calibration.

The following genes are used in custom made TaqMan arrays as markers for different cell types: STROl, GNL3, CD29, CD44, CD34, CD45 and COLl (MSC); COL2, COL4, COLlO, SOX9 and Aggrecan (Chondrocyte), Osteocalcin, Osteopontin, Osteonectin and CBFAl (Osteoblast); Nestin, NEFM, MAP2 and TUBB3 (Neuron); Adiponectin, FUS, FABP4, GLUT and PPARG (Adipocyte); Desmin, MYLK2, MYH 14, and GATA4

(Myocyte).

RT-PCR results indicate that ODT maintains MSCs as a healthy, growing (i.e. active cell division), undifferentiated culture. Edge-to-edge patterned substrates produced a homogenous response on cell function across the substrates, thus indicating the importance of homogenous edge-to-edge nanopatterned surfaces on control of stem cell differentiation into homogenous populations of defined cell type(s). There was no change in expression in 24 h, 14 day, and 28 day samples.

EXAMPLE 4

AUT 280nm (dα) 5mm ² nanopattern samples

24 hour and 14 day samples were analyzed by immunofluorescence for expression of the genes indicated below

24 Hours:

1. Every cell on the AUT substrate was CBFAl positive and no CBFAl staining was observed on the control (non-patterned) gold substrate. 2. Cells on both AUT substrates and control gold substrates were negative for Osteocalcin, as expected at 24 hours

14 Days:

1. Increased expression of the Osteoblast markers CBFAl and osteocalcin and decreased expression of STRO-I (MSC marker) were observed on amine substrates. Expression of CBFAl (intracellular marker) on AUT substrates was homogenous.

2. AUT sample was negative for β-tubulin and adiponectin and control gold sample was positive for the MSC marker STRO-I.

The expression profile observed at 14 days indicates continued expression of CBFAl, as observed at 24 hours, with the addition of an increase in expression of CBFAl and osteocalcin and a decrease in expression of STROl . Therefore, AUT induces the differentiation of MSCs into osteoblasts.

Thus, the results of both the ODT and AUT MSC trials indicate that defined, homogenous, edge-to-edge nanopatterned surfaces provide a profound level of control over stem cell dynamics.

B. Surface patterning for controlling stem cells.

Methodologies are provided herein for producing a differentiated and pure population of cells. More specifically, the methodology produces homogenous and differentiated population of stem cells by surface patterning with growth factors and polymers. Surface patterning may be performed on any level or scale, including but not limited to micron and nanopatterning

In recognizing that current protocols fail to produce a differentiated and homogeneous cell population, the present inventors turn to surface patterning methods for controlling stem cell self-renewal, differentiation, and survival. Specifically, and as explained in greater detail below, the inventors contemplate using at least one growth factor and/or polymer for coating a surface at a desired scale, pattern, size, and shape, and then culturing stem cells on said surface. Stem cells and non stem cells can be grown on these surfaces and the effect of these surfaces are examined in terms of stem cell self-renewal, differentiation, survival, adhesion, and migration.

1. Stem Cells Stem cells are cells found in most, if not all, multi-cellular organisms. They are characterized by the ability to renew themselves through mitotic cell division and differentiating into a diverse range of specialized cell types. The two broad types of mammalian stem cells are: embryonic stem cells that are isolated from the inner cell mass of blastocysts, and adult stem cells that are found in adult tissues. In a developing embryo, stem cells can differentiate into all of the specialized embryonic tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing specialized cells, but also maintain the normal turnover of regenerative organs, such as blood, skin or intestinal tissues.

Cells, growth of cells, cell differentiation, and stem cells are generally known in the art. See, for example, Essentials of Stem Cell Biology, (Ed. R. Lanza), 2006; Cell Biology, 2 ^nd Ed., Pollard and Earnshaw, 2008; Gilbert, Developmental Biology, 5 ^th ed., 1997; Cell Lineage Specification and Patterning of the Embryo, (ed. Etkin, Jeon), 2001. See Curran et al, Biomaterials, 27 (2006), 4783-4793; Curran et al, Biomaterials 26 (2005), 7057-7067.

Suitable source cells for culturing stem cells include established lines of pluripotent cells derived from tissue formed after gestation. Exemplary cells include mesenchymal stem cells, which can be obtained by methods known in the art. For example, and in no way limiting the invention, mesenchymal stem cells can be obtained from raw, unpurified bone marrow or ficoll-purified bone marrow monocytes plated directly into cell culture plates or flasks. Exemplary cells include those disclosed in Curran et al. Biomaterials 27 (2006), 4783-4793.

2. Growth Factors and Polymers

The present inventors contemplate coating a surface at a desired pattern with at least one growth factor and/or polymer. Such patterned surfaces may be used for many purposes, including culturing stem cells and non stem cells.

As used herein, a growth factor is a naturally occurring substance capable of stimulating cellular growth, proliferation, and cellular differentiation. Generally, a growth factor is a protein or a steroid hormone, and typically act as signaling molecule between cells. Relevant to stem cells, growth factors may promote cell differentiation and maturation, which varies based on the particular growth factors employed. For example, bone morphogenic proteins (BMPs) stimulate bone cell differentiation, while fibroblast growth factors and vascular endothelial growth factors (VEGF) stimulate blood vessel differentiation (angio genesis).

Non-limiting exemplary growth factors include Bone Morpho genetic Proteins (BMPs), Brain-Derived Neutrophic Factor (BDNF), Ciliary Neutrophic Factor (CNTF), Epidermal Growth Factor (EGF), Erythropoietin (EPO), Fibroblast Growth Factor (FGF), Granulocyte-Colony Stimulating Factor (G-CSF), Granulocyte -Macrophage Colony

Stimulating Factor (GM-CSF), Growth Differentiation Factor-9 (GDF9), Hepatocyte Growth Factor (HGF), Insulin-like Growth Factor (IGF), Interleukin (IL), Leukemia Inhibitory Factor (LIF), Myostatin (GDF-8), Nerve Growth Factor (NGF), Neutrophic Factors (NT), Platelet-derived Growth Factor (PDGF), Thrombopoietin (TPO),

Transforming Growth Factor alpha(TGF-α), Transforming Growth Factor beta (TGF-β), and Vascular Endothelial Growth Factor (VEGF).

Independently or in conjunction with a growth factor, a polymer may be used for coating a surface with a desired pattern. In instances where both a surface is coated with at least one growth factor and at least one polymer, the polymer is not a growth factor (i.e. the growth factor and polymer are different). A suitable polymer may be any natural or synthetic polymer, as well as any natural or synthetic polymer with any modification(s). Non-limiting exemplary polymers include any polymer or peptide sequence that can be used for growing cells, particularly undifferentiated and differentiated stem cells. Such polymer and peptide sequences are know to effect stem cell and non stem cell survival,

differentiation, adhesion, and/or migration. Exemplary polymers and peptides include but are not limited to laminin, collagen, poly-lysine (L and D), metrigel, fibronectin, gelatin, and poly-ornithine.

3. Coated Surfaces for Patterning and Culturing Stem Cells

The present inventors contemplate depositing at least one growth factor and/or polymer on a surface to produce a coated surface having a desired pattern.

It is well known in the art that stem cells, particularly embryonic stem cells, may undergo spontaneous cell differentiation. Further compounding the problem of maintaining stem cells in an undifferentiated state, previous studies demonstrate that stem cells grown on plastic and plastic-coated surfaces can also undergo spontaneous differentiation. Consequently, when the cells are passed or simply grown in a plastic or plastic-coated dish, the cell population homogeneity may decrease with time.

Thus acknowledging the problems associated with spontaneous cell differentiation, which may be amplified by the choice of surface material, the present inventors employ micro- and nano lithography techniques for culturing cells on novel surfaces. Any solid material may be used, including metals, polymers, composites, natural materials, and ceramics. In some embodiments, the surface is gold, silica, glass, nitrocellulose, polycaprolactone (PCL), PolyLLactic acid (PLLA), PolyGlycolic acid (PGA), Poly(urethane), hydroxyapatite, tricalcium phosphate, titanium and it's alloys, shape memory alloys, and natural materials like bone and hydroxyapatite, and stainless steel.

A suitable substrate surface may be selected with reference to the intended purpose of the material. For example, materials that are intended for use in cell or tissue culture may typically make use substrates may be relatively inflexible, and may include gold, silica, or glass. Likewise, materials that are intended for medical use (for example as implantable medicaments, or the like) may utilize substrates, that are well tolerated within the body, and do not give rise to a chronic inflammatory response. Suitable substrates for use in materials of this sort may be bioresorbable (that is to say the substrate, and/or the material, may be broken down by the body over a period of time), and ultimately replaced with the body's own tissues. Other considerations for selecting a substrate surface include the use of a three dimensional substrates in materials that are to be used in regenerative medicine. Such substrates may serve as scaffolds for the generation of replacement tissues such as bone, cartilage, fat, muscle and neural tissues.

4. Surface patterning of Growth Factors and/or Polymers

Any lithography technique by which molecules are delivered to a substrate of interest in a positive printing mode may be used. Lithography can be done at the micro level or nano scale, as is the case for nanolithography. Non-limiting exemplary nanolithography methods include but are not limited to dip pen nanolithography, nanoimprint lithography; direct atomic force microscopy; etching glancing angle deposition; laser ablation; laser deposition; replica molding of x-ray lithography masters; micro contact printing, and etching electron-beam direct-write lithography.

For example, and in no way limiting the invention, dip pen nanolithography (DPN) may be used for depositing at least one growth factor and/or polymer on a surface for cell growth. In its broadest sense, DPN utilizes a solid substrate as the "paper" and a scanning probe microscope tip (e.g., an atomic force microscope (AFM) tip) as the "pen". The tip is coated with a patterning compound (the "ink"), and the coated tip is used to apply the patterning compound to the substrate to produce a desired pattern. As presently understood, the molecules of the patterning compound are delivered from the tip to the substrate by capillary transport.

DPN™ , DIP PEN NANOLITHOGRAPHY™, and are trademarks of Nanolnk, Inc. and are used accordingly herein (e.g, DPN printing or DIP PEN NANOLITHOGRAPHY printing). DPN methods and equipment are generally available from Nanolnk, Inc. (Chicago, 111.), including the NLP™ and NScriptor™, which can be used to carry out nanolithography.

As used herein, the term patterning means any micro- or nano-scale lithography technique for depositing at least one growth factor and/or polymer on a surface in a pattern. The resultant delivery of said growth factor and/or polymer produces a pattern, which may be defined by spot size, shape, and pitch. As circumstances may require, a pattern may be altered for a given purpose or need. Nanopattern refers to patterns generated at the nano- scale level, whereas micropattern refers to patterns generated at the micro-scale level.

Exemplary nanopatterned surfaces for stem cells are disclosed in Curran et al. J. Mater Sci: Mater Med 21 :1021-1029 (2010); and Curran et al. Lab Chip 10, 1662-1670 (2010).

5. Cell Culture on Patterned Surface

Following patterning of a desired surface with at least one growth factor and/or polymer, a biological cell may be cultured on a patterned surface. In one embodiment, stem cells may be cultured on the patterned surface; in other embodiments, non-stem cells can be cultured on the patterned surface. Cells are cultured on said surface in a medium that does not interfere with differentiation state, such as a basal medium. To determine the effect of a patterned surface on cell growth and differentiation, cells are cultured for a period of time sufficient for cell growth and/or differentiation. While specific culture conditions may vary with cell type and need, generally, cell are cultured for at least 14 days. For example, stem cells may be cultured for 14 days before analyzing any characteristic, including for example cell growth, differentiation, adhesion, migration, or survival. Likewise, a non stem cell may be cultured for at least 14 days before analyzing any characteristic, including for example cell growth, differentiation, adhesion, migration, or survival.

While it is generally known in the art, cell differentiation refers to a cells ability to differentiate into a more specialized cell type. For example, and in no way limiting the invention, a stem cell could differentiate into an osteoblast or a chondrocyte.

Various methods may be used for analyzing stem cell growth and differentiation, which may be used for screening a population of stem cells for selecting a differentiated cell. In no way limiting the invention, a variety of molecular and/or cell biology techniques may be used to assess stem cell differentiation. For example, RT-PCR may be used to detect a specific genetic marker indicative of cell differentiation. Likewise, a western blot and/or Bradford protein assay may be performed to analyze protein expression

commensurate with a specific cell type. Similarly, various cell sorting techniques such as fluorescence-activated cell sorting (FACS) may used to determine cell differentiation.

Depending on circumstance and need, patterned surfaces as described herein, may be used to regulate or control stem cell growth and differentiation. For example, a patterned surface comprising at least one growth factor and/or polymer may be used to promote cell differentiation. Likewise, a patterned surface comprising at least one growth factor and/or polymer may be used to maintain cells in an undifferentiated state.

6. Illustrative Patterned Products

The patterned surfaces provided herein may be used in a variety of products, including but not limited to kits, reagents, and cell culture dishes and related tools.