Defined three dimensional microenvironment for cell culture
11220669 · 2022-01-11
Assignee
Inventors
- Chan Kim (Gwangju, KR)
- Kyuwon Baek (Seoul, KR)
- Hui-Gwan Goo (Seoul, KR)
- Sangjae Lee (Seoul, KR)
- Bongjin Hong (Pohang-si, KR)
- Song Hee KOO (Seoul, KR)
- In Yong SEO (Seoul, KR)
- Seung Hoon LEE (Paju-si, KR)
- Ji Hyun Lee (Incheon, KR)
- Seonho Jang (Seoul, KR)
- Dong-Sik Seo (Incheon, KR)
Cpc classification
C12N5/0062
CHEMISTRY; METALLURGY
C12N5/0606
CHEMISTRY; METALLURGY
C12N2535/00
CHEMISTRY; METALLURGY
International classification
Abstract
Described is a three-dimensional (3D) microenvironment presenting defined biochemical and physical cues that regulate cellular behavior and use of the microenvironment. A composition to form the 3D microenvironment is provided by combining one or more natural or synthetic polymeric materials and substrate proteins recombinantly or chemically functionalized with a variety of bioactive peptides such as extracellular matrix-derived or growth factor-derived peptides. Also described are devices and methods for screening for optimal combinations of the bioactive motifs in order to create an extracellular microenvironment that can regulate specific cellular behavior such as cell growth, proliferation, migration or differentiation.
Claims
1. A microenvironment for culturing cells, wherein the microenvironment comprises: an adhesion receptor binding motif and a co-receptor binding motif, wherein the adhesion receptor binding motif and the co-receptor binding motif regulate cellular behavior of cells cultured on the microenvironment via combinatorial signaling generating from crosstalk between an adhesion receptor and a co-receptor so as to enhance self-renewal and/or proliferation of cells cultured on the microenvironment, wherein the adhesion receptor binding motif comprises SEQ ID NO: 17, and wherein the co-receptor binding motif is selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
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DETAILED DESCRIPTION
(14) This disclosure is directed to engineered extracellular microenvironments that mimic biochemically and/or physically natural extracellular microenvironments.
(15) A microenvironment comprising at least one of binding motifs selected from the group consisting of adhesion receptor binding motifs and/or co-receptor binding motifs, wherein the binding motifs regulate cellular behavior via combinatorial signaling generating from crosstalk between adhesion receptors, adhesion receptor-co-receptor, co-receptors, or combination thereof, wherein the adhesion receptor binding motif is derived from at least one or more selected from the group consisting of extracellular matrix proteins and cadherin, or its mimetic and wherein co-receptor binding motif is derived from at least one or more selected from the group consisting of cadherin, growth factors, and cytokines. Specifically, the adhesion receptor binding motifs may include an integrin binding motifs, and the adhesion receptor binding motifs is derived from extracellular matrix proteins, cadherin or those mimetics. Also, co-receptor binding motif is derived from cadherin, growth factors or cytokines.
(16) The adhesion receptor binding motifs may comprise at least one or more selected from the group integrin binding motifs and cadherin binding motifs.
(17) Wherein the adhesion receptor binding motif comprises at least one or more selected from the group consisting of integrin α5β1, α6β1 or αvβ3 binding motifs or E-cadherin, N-cadherin, P-cadherin binding motif.
(18) Wherein the adhesion receptor binding motif comprises an integrin α5β1 binding motif. And the integrin α5β1 binding motif comprises a RGD (SEQ ID NO:15) containing peptide, wherein the RGD containing peptide comprises PHSRN-RGDSP (SEQ ID NO:17).
(19) Further, the adhesion receptor binding motif may comprise an integrin α6β1 binding motif, and wherein the integrin α6β1 binding motif may comprise at least one or more selected from the group consisting of laminin α1 LG domain-derived motif, laminin α5 LG domain-derived motif, and γ1 chain-derived motif. Wherein the integrin α6β1 binding motif comprises at least one or more selected from the group consisting of NRWHSIYITRFG (SEQ ID NO:34), GKNTGDHFVLYM (SEQ ID NO:22), VVSLYNFEQTFML (SEQ ID NO:23), VLVRVERATVFS (SEQ ID NO:27), and RNIAEIIKDI (SEQ ID NO:51).
(20) This may include the co-receptor binding motifs and the co-receptor motifs may comprise at least one or more selected from the group consisting of cadherin binding motif, growth factor receptor binding motif and cytokine binding motif.
(21) The co-receptor binding motif may comprise cadherin binding motifs, wherein the cadherin binding motif comprises at least one or more selected from the group consisting of LFSHAVSSNG (SEQ ID NO:52), ADTPPV (SEQ ID NO:53), DQNDN (SEQ ID NO:54) and LRAHAVDING (SEQ ID NO:55).
(22) Further, wherein the co-receptor binding motif may comprise growth factor receptor binding motif. Wherein the growth factor receptor binding motif is selected from fibroblast growth factor (FGF)- or transforming growth factor (TGF)-receptor binding motif.
(23) Provided is a microenvironment, wherein the FGF receptor binding motif is at least one or more selected from the group consisting of TGQYLAMDTDGLLYGS (SEQ ID NO:35), WFVGLKKNGSCKRG (SEQ ID NO:36), ANRYLAMKEDGRLLAS (SEQ ID NO:37), ERGVVSIKGV (SEQ ID NO:38), WYVALKRTGQYKLG (SEQ ID NO:39), HFKDPKRLYCK (SEQ ID NO:40), FLPMSAKS (SEQ ID NO:41), KTGPGQKA (SEQ ID NO:42), SRFFVAMSSKGKLYGS (SEQ ID NO:43) and MFIALSKNGKTKKG (SEQ ID NO:44).
(24) The TGF receptor binding motif may comprise at least one or more selected from the group consisting of LTGKNFPMFHRN (SEQ ID NO:45) and MHRMPSFLPTTL (SEQ ID NO:46).
(25) Further, wherein the co-receptor biding motif of the microenvironment may comprises cytokine binding motif and the cytokine binding motif may comprise at least one or more selected from the group comprising WNT-derived peptide motif and LIF-derived peptide motif. Further to the WNT-derived peptide motif may comprise at least one or more selected from the group consisting of LCCGRGHRTRTQRVTERCNC (SEQ ID NO:47) and LGTQGRLCNKTSEGMDGCEL (SEQ ID NO:48). The LIF-derived motif comprises at least one or more selected from the group consisting of IVPLLLLVLH (SEQ ID NO:49) and YTAQGEPFPNNVEKLCAP (SEQ ID NO:50).
(26) Provided is a biomaterial composition for electrospun matrix, comprising a) a synthetic hydrophobic polymer for electrospinning, b) hydrophilic protein comprising at least one or more selected from the group consisting of integrin binding motif, cadherin binding motif, growth factor receptor binding motif, and cytokine receptor binding motif at C-, N-, or C- and N-terminus of the hydrophilic protein. The hydrophobic polymer may be a polyvinylidene fluoride (PVDF).
(27) The hydrophilic protein comprises proteins comprising at least one or more selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:14NO:14.
(28) Provided is a three-dimensional microenvironment for growing, self-renewing, or proliferating stem cells comprising: a) an electrospun nanofiber matrix that comprises a hydrophilic protein comprising at least one or more binding motifs selected from the group consisting of integrin-, cadherin-, growth factor receptor, and cytokine receptor binding motifs; and b) chemically defined media, wherein the binding motif surfaced on a hydrophobic polymer supports growth and self-renewal of a stem cell.
(29) The hydrophilic protein further comprises at least one or more antimicrobial peptide motif is immobilized on the substrate.
(30) The antimicrobial peptide is at least one or more selected from the group consisting of KLWKKWAKKWLKLWKA (SEQ ID NO:59), FALALKALKKL (SEQ ID NO:60), ILRWPWWPWRRK (SEQ ID NO:61), AKRHHGYKRKFH (SEQ ID NO:62), KWKLFKKIGAVLKVL (SEQ ID NO:63), LVKLVAGIKKFLKWK (SEQ ID NO:64), WSILAPLGTTLVKLVAGIGQQKRK (SEQ ID NO:65), GIGAVLKVLTTGLPALISWI (SEQ ID NO:66), SWLSKTAKKGAVLKVL (SEQ ID NO:67), KKLFKKILKYL (SEQ ID NO:68), GLKKLISWIKRAAQQG (SEQ ID NO:69) and GWLKKIGKKIERVGQHTRDATIQGLGIAQQAANVAATAR (SEQ ID NO:70).
(31) Also provided is biochemically and physically defined surface that regulates cell surface receptors specifically, selectively, simultaneously, or sequentially to regulate cellular behavior such as cell attachment, migration, growth, proliferation, or differentiation of cells.
(32) Also provided is microenvironments that comprise at least one or more selected from the group consisting of integrin binding motif, cadherin binding motif, growth factor receptor and cytokine binding motif.
(33) As used herein “microenvironment” refers to physical and/or biochemical cues, surrounding a cell in an organism or in the laboratory. Molecules, including small molecules such as compounds and soluble factors, macromolecules such as insoluble polymers, nutrients, growth factors, fluids, cytokines and parameters such as pH, ionic strength and gas composition, and the like surrounding the cell are the biochemical cues. The molecules for biochemical cues may be, reversibly or irreversibly in response to biological or physiological conditions, immobilized to the substrate.
(34) A microenvironmentally, namely biochemically and physically, defined cell culturing substrate is provided for regulating cellular behavior in serum-free and feeder-free conditions for extended periods of time in culture. The microenvironmentally defined culture surface hereof promotes more efficient attachment and expansion of cells such as pluripotent stem cells as well as adult stem cells such as mesenchymal or neural stem cells in an undifferentiated state, as compared to standard culture substrates such as tissue culture-treated or serum coated surfaces. In some embodiments, human embryonic or induced pluripotent cells may be expanded on the microenvironmentally defined cell culture surface.
(35) Biochemically defined surface is a surface that presents ECM-, growth factor-, or cytokine-derived peptide motif or its mimetic, alone or in combination, to mimic in vivo microenvironment in order to regulate cellular behaviors.
(36) ECM, growth factor, or cytokine signaling environments are the important mechanisms for regulating cell fate; and, these microenvironmental stimuli are processed through combinatorial signaling pathways. The interactions between signaling pathways are critical in determining cell fate (C. J. Flaim et al., Stem Cells Dev. 2008, 17(1):29-39).
(37) The biochemically defined, peptide motif-presenting surfaces described herein are useful in a variety of contexts and applications. For example, for stem cell culture, the defined surfaces can be used for maintaining pluripotent cells in an undifferentiated state. In addition, the surfaces can be used for expanding a population of pluripotent cells without loss of differentiation potential in serum free or feeder-free conditions. The biochemically defined, peptide-presenting surfaces are also useful for culturing pluripotent cells that are subsequently induced to differentiate by, for example, adding one or more differentiation agent to the media. Differentiated cells derived from pluripotent cells can be maintained on the biochemically defined surfaces.
(38) Suitable pluripotent cells for use herein include ESCs and iPS cells, which preferably are from a primate, especially a human primate. As used herein, “embryonic stem cells” or “ESCs” mean a pluripotent cell or population of pluripotent cells derived from an inner cell mass of a blastocyst.
(39) Suitable adult stem cells for use herein include mesenchymal stem cell, neural stem cell, hematopoietic stem cell, which preferably are from a primate, especially a human primate.
(40) Suitable adult cells for use herein include, primary or cell lines such as immortalized HUVEC cells, epithelial cell, endothelial cell, neural cell, mesenchymal cell, etc.
(41) Regardless of the cell used, the biochemically defined surfaces described herein can be constructed according to known methods. For example, one can use contact spotting of peptides onto glyoxylyl-functionalized glass slides (see, e.g., J. Falsey et al., “Peptide and small molecule microarray for high throughput cell adhesion and functional assays,” Bioconjug. Chem. 12, 346-353 (2001)); contact printing of peptides onto acrylamide-coated glass slides; and spotting combinations of peptides onto a glass slide followed by in situ polymerization (see, e.g., Anderson et al., Nanoliter-scale synthesis of arrayed biomaterials and application to human embryonic stem cells, Nat. Biotechnol. 22:863 (2004). In addition, one can use streptavidin-coated plates treated with a biotinylated peptide of interest or even polyacrylamide gels cross-linked to a peptide of interest. See, e.g., Klein et al., Cell adhesion, cellular tension, and cell cycle control, Meth. Enzymol. 426:155 (2007).
(42) The biochemically defined surface presents a plurality of adhesion receptor binding peptide motifs and a plurality of co-receptor binding peptide motifs to activate at least two different receptors of cells. The adhesion receptor binding peptide motifs can bind to integrin or cadherin, and the co-receptor binding peptide motif activates to co-receptor such as growth factor receptor or cytokine receptor that coordinates with the adhesion receptor signaling.
(43) Integrin, cadherin, and growth factor receptor mediated signaling are essential for fundamental cellular functions including cell adhesion, migration, proliferation, differentiation, and survival. These cell surface receptors cross-talk with each other in the regulation of such cellular functions.
(44) Typical examples are integrin-integrin and integrin-cadherin crosstalk. Crosstalk between integrins has been well known in immune system and angiogenesis. For example, in human lymphocytes, αLβ2 (LFA-1) integrin binding to ICAM-1 decreases adhesion of α4β1 integrin to VCAM-1 and fibronectin, facilitating detachment of α4β1 integrin from the apical surface of endothelial cells. The decreased α4β1 integrin activity leads to an enhancement of α5β1 integrin mediated migration on fibronectin, a process that promotes transmigration through an endothelium (J. C. Porter, N. Hogg, Integrin cross talk: activation of lymphocyte function-associated antigen-1 on human T cells alters alpha4beta1- and alpha5beta1-mediated function, J. Cell Biol. 1997, 138(6):1437-47). The coordinate modulation of the cellular functions of cadherins and integrins plays an essential role in fundamental physiological and pathological processes, including morphogenesis, tissue differentiation and renewal, wound healing, immune surveillance, inflammatory response, tumor progression, and metastasis (see, Luca Goitre et al., Journal of Signal Transduction Volume 2012 (2012), 12 pages).
(45) For another example, it has been known that crosstalk between integrins and growth factor receptors by two mechanism, i) two separate signals merge with one another in multiple levels inside the cells (see, Legate, et al., Genetic and cell biological analysis of integrin outside-in signaling, Genes Dev. 2009, 23, 397-418), or ii) FGF1 directly binds to integrin αvβ3 in order to induces the FGFR1-FGF1-integrin αvβ3 ternary complex (S. Mori et al., Direct binding of integrin αvβ3 to FGF1 plays a role in FGF1 signaling, J. Biol. Chem. 2008, 283, 18066-18075). In one embodiment, the extracellular microenvironment surface simultaneously activates two different receptors integrin α5β1 and FGFR to support self-renewal of pluripotent or multi-potent stem cell.
(46) An extracellular component such as a cell adhesion molecule such as ECM protein or cadherin, growth factors, or cytokines can be a natural or recombinant extracellular matrix protein, ECM- or cadherin-derived domain including core motif that binds to specific adhesion receptor such as integrin or cadherin or its mimetic, growth factor (GF)-derived domain containing core motif that bind to specific binding sites of such growth factor receptor, or its mimetic, and cytokine-derived domain containing core motif that binds to cytokine receptor, or its mimetic. The mimetic comprises of a recombinant protein or polypeptide functionalized with at least one or more peptide motifs derived from a variety of extracellular components described above.
(47) Any suitable natural extracellular matrix proteins including but not limited to fibronectin, laminin, vitronectin may be used as an extracellular component to activate integrins. Preferably, the extracellular matrix protein is fibronectin. More preferably, the fibronectin can be used alone or the combination with laminin, vitronectin or cadherin.
(48) Any suitable natural or recombinant cadherin such as E-cadherin or N-cadherin may be used as an extracellular component to activate adhesion receptors. Preferably, the cadherin is E-cadherin.
(49) Any suitable natural growth factors are fibroblast growth factor (FGF) or transforming growth factor (TGF) may be used as an extracellular component to activate such growth factor receptors. Preferably, the growth factor can be used along or the combination of FGF and TGF.
(50) Generally any extracellular mimetic component including extracellular matrix mimetic, cadherin mimetic, growth factor mimetic, or cytokine mimetic comprises a substrate protein recombinantly or chemically functionalized with peptide motif derived from the extracellular components.
(51) Any suitable substrate protein including but not limited to fibrin, elastin, mussel adhesive protein may be used as the substrate protein to present extracellular component. Preferably, the protein is a recombinant mussel adhesive protein.
(52) Any suitable recombinant mussel adhesive protein may be used as the extracellular component herein. Examples of commercially available substrate proteins include MAPTrix™ ECM marketed by Kollodis BioSciences, Inc. (North Augusta, S.C.). An optional third component is a biocompatible polymer (e.g., polyethylene glycol or polyvinylalcohol), which may be added to the compositions to enhance their physicomechanical characteristics such as physical or mechanical properties of a customizable microenvironment.
(53) The MAPTrix™, developed by Kollodis BioSciences Inc. (North Augusta, S.C.), are predesigned mussel adhesive protein or barnacle-based extracellular component mimetics. The mussel adhesive proteins were recombinantly functionalized with a variety of ECMs-, GFs-, or other ligand-derived peptides in order to mimic the bioactivity of naturally occurring ligands such as ECMs, GFs, or cytokines including, but not limited to, IL-3, LIF, or WNT which were demonstrated to have a similar bioactivity to natural or recombinant ECMs, GFs, or cytokines in primary cell cultures as compared to various natural or recombinant ECM, GF or cytokine proteins. The pre-designed MAPTrix™ mimetics are highly advantageous for creating extracellular microenvironments. For example, it provides for the design of cell-specific or user-defined regulation of extracellular microenvironments to emulate the native microenvironment in terms of biochemical cues.
(54) The MAPTrix™ is a fusion protein comprising a first peptide of mussel foot protein FP-5 that is selected from the group consisting SEQ ID NOS:1-4, or barnacle-derived adhesive protein consisting SEQ ID NO:5 and a second peptide of at least one selected from the group consisting of mussel FP-1 selected from the group consisting of SEQ ID NOS:6-8, mussel FP-2 (SEQ ID NO:9), mussel FP-3 selected from the group consisting of SEQ ID NOS:10-11, mussel FP-4 (SEQ ID NO:12), mussel FP-6 (SEQ ID NO:13) and fragment thereof, and the second peptide is linked to C-terminus, N-terminus or C- and N-terminus of the FP-5. Preferably, the second peptide is the FP-1 comprising an amino acid sequence of SEQ ID NO:6.
(55) Extracellular components including integrin binding motif or co-receptor binding motif such as fibroblast growth factor receptor (FGFR), transforming growth factor receptor (TGFR), insulin-like growth factor receptor (IGFR)-derived peptide motif, cytokine receptor binding motif such as WNT and/or LIF (leukemia inhibitor factor)-derived core motif may also be incorporated into the mussel adhesive protein to further enhance the beneficial effect of the extracellular environment mimic on self-renewal and pluripotency of a stem cell.
(56) There are 24 known integrin heterodimers comprised of one of 18α subunits and one of 8β subunits and these have a diverse range of functions mediating cell-cell adhesion, growth factor receptor responses and intracellular signaling cascades for cell migration, differentiation, survival and proliferation. A number of ECM molecules or domains are capable of assisting in the maintenance of undifferentiated hESC alone or in combination, including laminin 511 (see, T. Miyazaki et al., Recombinant human laminin isoforms can support the undifferentiated growth of human embryonic stem cells, Biochem. Biophys. Res. Commun., 375 (2008), pp. 27-32), fibronectin and vitronectin (see, Melkoumian et al., Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells, Nat. Biotechnol. 28 (2010), pp. 606-610; Braam et al., Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via alphavbeta5 integrin, Stem Cells 26 (2008), 2257-2265).
(57) The extracellular domain of integrins can bind ECM proteins used in hESC support such as collagen, fibronectin, laminin and vitronectin as well as members of the SIBLING family (Small Integrin Binding Ligand, N-Linked Glycoproteins, e.g., osteopontin and bone sialoprotein). Integrin clustering occurs after ECM adhesion promoting lateral association with other cell surface receptors and increases in the cytoplasmic concentration of cell signaling molecules such as PI3-kinase and MEK-ERK, which are involved in hESC maintenance (see, J. Li et al., MEK/ERK signaling contributes to the maintenance of human embryonic stem cell self-renewal, Differentiation 75 (2007), 299-307).
(58) Recently, the Hubbell laboratory developed and tested various synthetic substrates for their capacity to maintain mouse ES cell self-renewal and concluded that simultaneous ligation of α5β1-, αvβ5-, α6β1, and α9β1 integrins promotes stemness of ES cells. These integrins have also been implicated in the regulation of mouse and human ES cell self-renewal in a number of other studies performed under various growth conditions (see, Sandhanakrishnan Cattavarayan et al., α6β1- and αv-integrins are required long-term self-renewal of murine embryonic stem cells in the absence of LIF, BMC Cell Biology 2015, 16:3; Y. Meng et al., Characterization of integrin engagement during defined human embryonic stem cell culture, FASEB J. 2010, 24(4):1056-65; S. R. Braam et al., Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via αvβ5 integrin, Stem Cells 2008; 26(9):2257-65).
(59) Also provided is a microenvironmentally defined surface that activates α5β1, α6β1 and/or αvβ5 simultaneously or sequentially in order to regulate signaling pathway for self-renewal and pluripotency maintenance of a stem cell. Any suitable substrate protein containing peptide ligand to activate integrin α5β1-, αvβ5-, α6β1, or α9β1 simultaneously or sequentially to support self-renewal and pluripotency of a stem cell. In one embodiment, the microenvironment surface provides a substrate protein presenting α5β1 integrin activating motif or heparin binding motif derived from fibronectin domain III. Any suitable α5β1 integrin activating- or heparin binding motif can be selected from RGD (SEQ ID NO:15), GRGDSP (SEQ ID NO:16), PHSRN-RGDSP (SEQ ID NO:17), SPPRRARVT (SEQ ID NO:18), WQPPRARI (SEQ ID NO:19), KNNQKSEPLIGRKKT (SEQ ID NO:20), or its combination of α5β1 integrin activating motif and heparin binding motif.
(60) In another embodiment, the microenvironment surface provides a substrate protein presenting α6β1 integrin activating motif-derived laminin α1 or laminin α5 LG domain to support self-renewal and pluripotency of a stem cell. Any suitable α6β1 integrin activating motif can be selected from GKNTGDHFVLYM (SEQ ID NO:22), VVSLYNFEQTFML (SEQ ID NO:23), RFDQELRLVSYN (SEQ ID NO:24), RLVSYSGVLFFLK (SEQ ID NO:25), ASKAIQVFLLGG (SEQ ID NO:26), VLVRVERATVFS (SEQ ID NO:27), TVFSVDQDNMLE (SEQ ID NO:28), RLRGPQRVFDLH (SEQ ID NO:29), FDLHQNMGSVN (SEQ ID NO:30), QQNLGSVNVSTG (SEQ ID NO:31), SRATAQKVSRRS (SEQ ID NO:32), TWYKIAFQRNRK (SEQ ID NO:33), NRWHSIYITRFG (SEQ ID NO:34), RNIAEIIKDI (SEQ ID NO:51).
(61) In another embodiment, the microenvironment surface provides a substrate protein presenting a combinatorial motif of α5β1 integrin activating motif and α6β1 binding motif at the same time to support self-renewal and pluripotency of a stem cell. Suitable combinatorial motif is a combination of PHSRN-RGDSP (SEQ ID NO:17) and NRWHSIYITRFG (SEQ ID NO:34) to support self-renewal and pluripotency of a stem cell.
(62) Generally embryonic stem cells grow as individual colonies, maintained via E-cadherin-mediated cell-cell contact. Transcription profiling studies have revealed that over 60% of genes are expressed in ES cells (compared to only 10-20% in somatic cells) and most of these are involved in signal transduction and regulation, making ES cells very responsive to the microenvironment (see, C. E. Eckfeldt, E. M. Mendenhall, C. M. Verfaillie, The molecular repertoire of the “almighty” stem cell, Nat. Rev. Mol. Cell Biol. 2005, 6:726-737; N. Sato, I. M. Sanjuan, M. Heke, M. Uchida, F. Naef, A. H. Brivanlou, Molecular signature of human embryonic stem cells and its comparison with the mouse, Dev. Biol. 2003; 260:404-413).
(63) Provided is a microenvironmentally defined surface that binds to cadherin to form colony for self-renewal and stemness maintenance in defined conditions. Any suitable cadherin binding motif can be selected from LFSHAVSSNG (SEQ ID NO:52), ADTPPV (SEQ ID NO:53), DQNDN (SEQ ID NO:54), or LRAHAVDING (SEQ ID NO:55).
(64) Fibroblast growth factors (FGFs) are essential for maintaining self-renewal in human embryonic stem cells and induced pluripotent stem cells. Recombinant basic FGF (bFGF or FGF2) is conventionally used to culture pluripotent stem cells. Today FGF family consists of 23 members including acidic and basic fibroblast growth factor, and each FGF has canofin, hexfin, and decafin domain (S. Li et al., Fibroblast growth factor-derived peptides: functional agonists of the fibroblast growth factor receptor, J. Neurochem. 2008 February 104(3):667-82; S. Li et al., Agonists of fibroblast growth factor receptor induce neurite outgrowth and survival of cerebellar granule neurons, Dev. Neurobiol. 2009, 69(13):837-54; Li Shizhong et al., Neuritogenic and Neuroprotective Properties of Peptide Agonists of the Fibroblast Growth Factor Receptor, Int. J. Mol. Sci. 2010; 11(6):2291-2305).
(65) FGFRs are transmembrane glycoproteins with three extracellular domains, Ig1, Ig2 and Ig3. An FGFR fragment Ig2 and Ig3 is the minimal unit sufficient for specific ligand binding (see, V. I. Manfè et al., Peptides derived from specific interaction sites of the FGF 2-FGF receptor complexes induce receptor activation and signaling (see, J. Neurochem. 2010, 114(1):74-86; S. K. Olsen et al. (2004), Insights into the molecular basis for fibroblast growth factor receptor autoinhibition and ligand-binding promiscuity, Proc. Natl Acad. Sci. USA 101 935-940).
(66) Bell et al. (see, 2000 Rotational coupling of the transmembrane and kinase domains of the Neu receptor tyrosine kinase, Mol. Biol. Cell 11:3589-3599) demonstrated that activation of receptor tyrosine kinases requires specific orientations of the kinase domains in a formed receptor dimer. The ligand binding mediates the optimal rotational positioning of the individual monomers within the dimer and thus the specific orientation of the catalytic domains. Binding of different agonists, such as FGF2 and canofins resulted in different modes of orientation of catalytic domains yielding differences in receptor activation (see, V. Manfe et al., Peptides derived from specific interaction sites of the fibroblast growth factor 2—FGF receptor complexes induce receptor activation and signaling, J. Neurochem. 2010, 114(1):74-86).
(67) When a growth factor binds to the extracellular domain of a receptor tyrosine kinase (RTK), its dimerization is triggered with other adjacent RTKs. Dimerization leads to a rapid activation of the protein's cytoplasmic kinase domains and the activated receptor as a result then becomes autophosphorylated on multiple specific intracellular tyrosine residues, resulting in signal transduction cascade.
(68) Recent studies have demonstrated that the immobilization of soluble factors such as FGF, TGF or cytokines to the ECM plays an important role in mediating their biological effects (see, C. C. Rider (2006), Heparin/heparan sulphate binding in the TGF-beta cytokine superfamily, Biochem. Soc. Trans. 34:458-460). Presentation of soluble factors in an immobilized fashion alters their local effective concentration, bioavailability, and stability, and thereby modulates their effects on target cells. For example, NSC-proliferative regions in the SVZ are situated in proximity to regions, in which growth factors including basic fibroblast growth factor-2 are concentrated by heparan sulfate proteoglycan (HSPG) (see, F. Mercier et al. (2002), Anatomy of the brain neurogenic zones revisited: fractones and the fibroblast/macrophage network, J. Comp. Neurol. 451:170-188).
(69) Provided is the FGF mimetic comprises recombinant mussel adhesive protein functionalized with FGF-derived peptide motif derived from hexafin domain or canofin domain. Preferably, FGF1 mimetic peptide motif can be selected from TGQYLAMDTDGLLYGS (SEQ ID NO:35), WFVGLKKNGSCKRG (SEQ ID NO:36), and FGF2 mimetic peptide motif can be selected from hexafin domain-derived ANRYLAMKEDGRLLAS (SEQ ID NO:37), ERGVVSIKGV (SEQ ID NO:38) or decafin domain-derived WYVALKRTGQYKLG (SEQ ID NO:39), canofin domain-derived HFKDPKRLYCK (SEQ ID NO:40), FLPMSAKS (SEQ ID NO:41), KTGPGQKAIL (SEQ ID NO:42), and FGF4 mimetic peptide motif can be selected from SRFFVAMSSKGKLYGS (SEQ ID NO:43), MFIALSKNGKTKKG (SEQ ID NO:44).
(70) In one embodiment hereof, a microenvironment surface that combinatorially regulates the activity of both integrin and growth factor receptor to support self-renewal and pluripotency of murine embryonic stem cell is provided. The microenvironment surface comprises a substrate protein functionalized with a peptide such as fibronectin-derived peptide PHSRN-RGDSP (SEQ ID NO:17) to target α5β1 and FGF2-derived peptide selected from ANRYLAMKEDGRLLAS (SEQ ID NO:37), ERGVVSIKGV (SEQ ID NO:38), WYVALKRTGQYKLG (SEQ ID NO:39), HFKDPKRLYCK (SEQ ID NO:40), FLPMSAKS (SEQ ID NO:41), KTGPGQKAIL (SEQ ID NO:42) to target FGF receptor; FGFR2IIIc.
(71) Also provided is a microenvironment surface to activate TGF receptor to induce signaling pathway to activate transcriptional factors for self-renewal and pluripotency of pluripotent stem cell. A recombinant mussel adhesive protein as a substrate protein containing TGF mimetic peptide to bind to TGFβ receptor domain TβRI or TβRII can be used herein. Preferably, TGFβ mimetic peptide can be selected from LTGKNFPMFHRN (SEQ ID NO:43), MHRMPSFLPTTL (SEQ ID NO:46).
(72) In one embodiment hereof, a microenvironment surface that combinatorially regulates the activity of both integrin and growth factor receptor to support self-renewal and pluripotency of an embryonic stem cell is provided. The microenvironment surface comprises a substrate protein presenting a combinatorial motif to activate α5β1 integrin and TGFβ receptor at the same time. The combinatorial motif is a combination of the substrate protein functionalized with a peptide such as fibronectin-derived peptide PHSRN-RGDSP (SEQ ID NO:17) to target α5β1 and TGFβ-derived peptide LTGKNFPMFHRN (SEQ ID NO:45), or MHRMPSFLPTTL (SEQ ID NO:46).
(73) Provided is a microenvironment surface that generates WNT/β-catenin signaling pathway by presenting WNT 1 peptide motif LCCGRGHRTRTQRVTERCNC (SEQ ID NO:47) or LGTQGRLCNKTSEGMDGCEL (SEQ ID NO:48). In one embodiment hereof, provided is a microenvironment surface that combinatorially regulates the activity of both integrin and frizzled receptor to support self-renewal and pluripotency of an embryonic stem cell. The microenvironment surface comprises a substrate protein presenting a combinatorial motif to activate α5β1 integrin and frizzled receptor at the same time. The combinatorial motif is a combination of the substrate protein functionalized with a peptide such as fibronectin-derived peptide PHSRN-RGDSP (SEQ ID NO:17) to target α5β1 and WNT-derived peptide LCCGRGHRTRTQRVTERCNC (SEQ ID NO:39) or LGTQGRLCNKTSEGMDGCEL (SEQ ID NO:40).
(74) Provided is a microenvironment surface that generating LIF/STAT3 signaling pathway by presenting LIF peptide motif IVPLLLLVLH (SEQ ID NO:49) or YTAQGEPFPNNVEKLCAP (SEQ ID NO:50).
(75) Various studies suggest that co-clustering or synergism occurs between downstream signaling molecules, once the basic requirements are met: growth factor receptor ligand-binding, integrin occupancy by a ligand and clustering of each type of receptor (see, M. A. Schwartz and V. Baron, Interactions between mitogenic stimuli, or, a thousand and one connections, Curr. Opin. Cell Biol. 11:197-202 (1999); K. M. Yamada and E. H. J. Danen, Integrin signaling, In Signaling Networks and Cell Cycle Control (ed. J. S. Gutkind) 1-25 (Humana Press, Totowa, N.J., 2000); S. Miyamoto et al., Integrins can collaborate with growth factors for phosphorylation of receptor tyrosine kinases and MAP kinase activation: roles of integrin aggregation and occupancy of receptors, J. Cell Biol. 135:1633-1642 (1996)).
(76) Provided is a microenvironment surface to activate at least two different receptors simultaneously by presenting a substrate protein having combinatorial motifs comprising at least two different peptide motifs that bind to adhesion receptors such as integrin, co-receptors such as growth factor- or cytokine receptor, or combination thereof. The suitable combinatorial motifs may include one or more spacers between two peptide motifs to optimize flexibility and/or solubility and so afford increased affinity and/or bioavailability. The combinatorial motifs may have a peptide spacer sequence of at least two amino acids, preferably 2-15 amino acids, appended to the C-termini of at least one of the two peptide motifs.
(77) A microenvironmentally defined 3D surface is provided. The 3D surface may be microenvironmentally defined over media found within a cell culture plate or other structure. A Substrate for the defined surface may include patterned or porous nanofiber being composed of various materials including polyvinylidene fluoride (PVDF), but not limited to cellulose, nylon, glass fiber; materials for bio-reactors used in batch or continuous cell culture or in bioreactors.
(78) As used herein “nanofiber” refers to the electroprocessed composition that may include particles being larger than a nanofiber as a result of the electroprocessed composition, where the surface of the nanofiber presents biochemical cues. Collectively, the nanofiber may provide in vivo like microenvironment to regulate the fate of cells of interest.
(79) As used herein, “WebTrix™” refers to a nanofibrous matrix to mimic three-dimensional microenvironment presenting biochemically defined and physically defined surface for precise regulation of cellular behavior such as cell adhesion, migration, growth, proliferation. WebTrix™ is the trade name for biochemically and physically defined nanofiber matrix and marketed by AMO LifeScience Co. Ltd.
(80) Provided is a three-dimensional microenvironment comprised of a nanofiber sheet presenting a plurality of adhesion receptor binding peptide motifs derived from extracellular matrix (ECM) or cadherin and co-receptor binding peptide motifs derived from growth factor (GF) or cytokine that precisely regulate cellular behavior such as cell adhesion, migration, growth or differentiation. The nanofiber may include dispersed particles being at least partially embedded into the nanofibers as a result of electroprocessed composition, the particle being larger than the average diameter of nanofibers.
(81) In one embodiment hereof, a combinatorial microenvironment surface comprising a nanofiber substrate having an average diameter of 100 nm to 20 microns, wherein the nanofiber surface presents extracellular components comprising extracellular matrix mimetic, growth factor mimetic, WNT mimetic, cytokine mimetic such as IL-3, LIF mimetic or combinations thereof.
(82) Provided is an electroprocessable biofunctional composition to engineer an extracellular microenvironment presenting controlled physical and/or biochemical cues. As used herein “biofunctional composition” refers to a composition that comprises a bioactive component and a structural component that is electroprocessable polymer solution. Electroprocess including electrospinning or electro-spraying is a means of producing fibers or particles with diameters generally between 10 to 2,000 nanometers. It has the ability to produce fibers or particles that are far smaller than those produced by conventional means such as wet spinning or melt spinning.
(83) Bioactive component is a, natural or synthetic, polymer or protein containing peptide motif. As used herein “peptide motif” refers to a short peptide, preferably three (3) to one hundred (100) amino acids in length that possesses a peptide derived from natural protein such as extracellular matrix (ECM), growth factors (GFs), or cytokines that mimic natural ECM, GF, or cytokine activity. Preferably, the bioactive peptide is a peptide that was originally identified in nature, produced by an animal, plant, fungus or bacterium as part of their natural mechanism.
(84) Provided is an electrospinnable biofunctional composition for a fibrous extracellular microenvironment comprised of two components, extracellular component and, and a structural component. In one embodiment, a structural component is a polymer to provide physical or mechanical cues such as pore size or elasticity whereas extracellular component provides biochemical cues.
(85) Any electrospinnable polymer, natural or synthetic, for use in this disclosure can be a structural component. Preferably, an electrospinnable polymer is a synthetic polymer which has the appropriate viscosity in solution. Any polymer meeting the above requirements is useful herein, and the selection of the specific polymer and acquisitions or preparation of such polymer would be conventionally practiced in the art (see, reference here). Preferred for such electrospinnable polymers are selected from groups comprising polyvinylidene fluoride (PVDF), polyacrylonitrile (PAN), polyethersulfone (PES), polylactic acid (PLA), polyglycolic acid (PGA), poly (lactide-glycolic) acid (PLGA), polycaprolactone, poly(alkylene oxides) particularly poly(ethylene glycols), poly(vinyl alcohols), polypeptides, poly(amino acids), such as poly(lysine), poly(allylamines) (PAM), poly(acrylates), polyesters, polyphosphazenes, pluronic polyols, polyoxamers, poly(uronic acids), and copolymers, including graft polymers thereof.
(86) This disclosure can be used in high throughput screening (HTS) to identify combinatorial peptide motifs to engineer optimal synthetic microenvironment that can specifically, selectively, simultaneously or sequentially generate signaling pathway to regulate self-renewal and pluripotency of pluripotent stem cells.
(87) A “microenvironment array” is a combination of two or more chambers. Preferably, an array is comprised of chambers in addressable rows and columns. The layout of microenvironment arrays produced according to the disclosure can vary, dependent upon the particular cell lines of interest, for example, induced pluripotent stem cell or embryonic stem cell.
(88) A method for providing for a device of microenvironment array comprises: (a) preparing a biochemical cue composition; (b) placing the composition on the surface of a substrate for coating; and (c) obtaining the extracellular microenvironment array.
(89) In one embodiment hereof, a microenvironment array is provided. The array is a 12-well, microwell plate consisting of 4×3-well. Each well within a strip (4 wells total) is pre-coated with a different biofunctional composition to generate different extracellular microenvironment. Cells of interest can be seeded onto each well, whereby cells are cultured on different extracellular microenvironment surface. An extracellular microenvironment that induces a desirable cellular behavior can be identified and designed from the assay utilizing this extracellular microenvironment array.
(90) Provided is an antimicrobial environment where biological contamination is sufficiently prohibited without use of antibiotics. Microbial contamination is a major issue in cell culture, but there are a range of procedures which can be adopted to prevent or eliminate contamination. Contamination may arise from the operator and the laboratory environment, from other cells used in the laboratory, and from reagents. Antimicrobial surface can prevent this kind of microbial contamination by presenting antimicrobial peptide acting only on microbial membrane surface but not mammalian cytoplasmic membrane. The antimicrobial surface comprises mussel adhesive protein as a substrate protein, functionalized with one or two antimicrobial peptides which can be recombinantly incorporated into C-, N terminus or both C- and N-terminus of the substrate protein. The antimicrobial peptide can be selected from KLWKKWAKKWLKLWKA (SEQ ID NO:59), FALALKALKKL (SEQ ID NO:60), ILRWPWWPWRRK (SEQ ID NO:61), AKRHHGYKRKFH (SEQ ID NO:62), KWKLFKKIGAVLKVL (SEQ ID NO:63), LVKLVAGIKKFLKWK (SEQ ID NO:64), WSILAPLGTTLVKLVAGIGQQKRK (SEQ ID NO:65), GIGAVLKVLTTGLPALISWI (SEQ ID NO:66), SWLSKTAKKGAVLKVL (SEQ ID NO:67), KKLFKKILKYL (SEQ ID NO:68), GLKKLISWIKRAAQQG (SEQ ID NO:69), and GWLKKIGKKIERVGQHTRDATIQGLGIAQQAANVAATAR (SEQ ID NO:70).
(91) In one embodiment, antimicrobial nanofibrous surface was provided to inhibit bacterial growth and facilitate growth and proliferation of cells of interest.
(92) The following examples are provided to demonstrate preferred embodiments hereof and the disclosure is not intended to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.
EXAMPLES
Example 1
Preparation of Electrospinnable Biofunctional Composition to Engineer an Extracellular Microenvironment
(93) PVDF with an average molecular weight of 200 kDa from Sigma Aldrich (St. Louis, USA), PAN with an average molecular weight of 200 kDa, PES with an average molecular weight of 200 kDa purchased from Sigma Aldrich (St. Louis, USA), PLA with an average molecular weight of 200 kDa purchased from Sigma Aldrich were dissolved in DMAc to prepare 20 wt % solution. MAPTrix™ ECM (no peptide motif, PHSRN-RGDSP (SEQ ID NO:17) (combo) containing, and RGD motif containing) purchased from Kollodis BioSciences (North Augusta, S.C., USA) was dissolved in an aqueous solution composed of distilled water and DMAc. Each polymer solution was mixed well together with MAPTrix™ ECM solution by vortexing it for 10 minutes to make homogeneous 18 wt % solution.
(94) The electrospinnable (e-spin) solution was placed in a plastic syringe fitted with a 27 G needle. A syringe pump (KD Scientific, USA) was used to feed the e-spin solution into the needle tip. A high voltage power supply was used to charge the needle tip. The nanofibers were collected onto grounded aluminum foil target located at a certain distance from the needle tip. The fiber meshes were then removed, placed in a vacuum chamber for two days to remove residual solvent, and then stored in a desiccator. Silver staining was used to detect MAPTrix™ protein on the surface of nanofiber membrane.
(95)
(96) The electrospinnable composition and electrospinning conditions are summarized in Tables 1 and 2, respectively.
(97) TABLE-US-00001 TABLE 1 Electrospinnable solution composition (structural component) Polymer Solvent MAPTrix ™ Solvent (1 mL) PVDF 100 mg DMAc 5 mL 7mg DW/DMAc (0.1/0.9) PES 100 mg DMAc 5 mL 7 mg DW/DMAc (0.1/0.9) PAN 100 mg DMAc 5 mL 7 mg DW/DMAc (0.1/0.9) PLGA 100 mg DMAc 5 mL 7 mg DW/DMAc (0.1/0.9) PVDF/PAN DMAc 5 mL 7 mg DW/DMAc (0.1/0.9) 50 mg/50 mg PVDF/PES DMAc 5 mL 7 mg DW/DMAc (0.1/0.9) 50 mg/50 mg
(98) TABLE-US-00002 TABLE 2 Electrospinning Parameters Rate E-Solution Concentration Voltage (kv) (mL/min) Distance (cm) PVDF 18% 21 0.1 11 PES 18% 21 0.1 10 PAN 18% 21 0.1 10 PLGA 18% 21 0.1 11 PVDF/PAN 18% 21 0.1 10 PVDF/PS 18% 21 0.1 10
(99) E-solution is the electrospinnable biofunctional composition prepared from the procedure described above in EXAMPLE 1.
(100) The nanofiber membranes obtained were stained.
Example 2
Preparation of Nanofiber Having Different Diameter
(101) Each Polyvinylidene fluoride (PVdF)-Kynar 761(Homopolymer, Mw: 400,000-500,000), and Polyvinylidene fluoride (PVdF)-Solef 21216(Co-polymer, Mw: 600,000) or Polyacrylonitril-Pulver (Co-PAN, Mw: 85,000) was dissolved in DMAC and blended. The blending ration of homopolymer to copolymer were 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9. Nanofibers having different diameter were formed by the same procedure mentioned in EXAMPLE 1.
(102) The diameter of each nanofiber sheet is measured by observation using a scanning electron microscope (SEM), a thin gold layer was deposited on the surface of each nanofiber sheet.
Example 3
Preparation of Nanofiber Having Particles
(103) MAPTrix™ ECM based particles were formed by reaction of the carboxyl group of MAPTrix™ activated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimides/N-hydroxysulfosuccinimide (EDC/s-NHS) on the C-terminus with the amino groups of the MAPTrix™.
(104) 1-Ethyl-3-[3-Dimethylaminopropyl]carbodiimide hydrochloride (EDC) solution is prepared by dissolving 10 mg of EDC in 1 ml of sodium bicarbonate buffer (10 mM, pH 6.5). 5 mg of solid sulfo-N-hydroxysulfosuccinimide (S—NHS) is added to the EDC solution. The EDC/S—NHS solution is added to the nanofiber surface to activate carboxyl group of MAPTrix™ surfaced on the nanofiber sheet for 30 minutes. After the C-terminus activation, 0.1 mg of MAPTrix™ having PHSRN-RGDSP (SEQ ID NO:17) motif dissolved in 1 mL distilled water was added to the nanofiber surface. Crosslinking is carried out at ambient temperature for 30 minutes to get crosslinked MAPTrix™ particle presenting PHSRN-RGDSP (SEQ ID NO:17) on the nanofiber surface.
(105) As presented in
Example 4
Cell Adhesion and Self-Renewal Assay
(106) Cell fate is regulated by soluble factors such as FGFs and interactions involving cell-cell and cell-extracellular matrix (ECM) contacts. For example, cell survival or self-renewal of stem cells is required for cells to be anchored in their native microenvironment via cell adhesion molecules. Integrins are cell surface receptors that mediate cell-ECM contacts. The extracellular domains of integrins bind directly to ECM proteins such as collagen, fibronectin, and laminin. Many cells including pluripotent stem cells highly express several integrins including α5β1, α6β1, αvβ3.
(107) In order to identify peptide motifs that activate these integrins, arrays of 31 different peptide motifs that support cell-ECM interaction were prepared as represented in
(108) For arraying, stock solutions of each ECM and cytokine such as WNT and LIF (leukemia inhibitory factor) mimetic were suspended and dissolved in distilled water at a concentration of 0.06 mg/mL. ECM and/or cytokine mimetic solutions were then coated in a 12 microwell plate via EDC/S—NHS mediated crosslinking reaction as set forth in EXAMPLE 3. The layout for each array was represented in
(109) For array preparation, the following MAPTrix™ were used for cell adhesion surface in each well.
(110) TABLE-US-00003 TABLE 3 Peptide motif sequence Peptide motif SEQ ID NO: Source RGD 15 Fibronectin PHSRN-RGDSP 17 Fibronectin SPPRRARVT 18 Fibronectin WQPPRARI 19 Fibronectin KNNQKSEPLLIGRKKT 20 Fibronectin KKQRFRHRNRKGYRSG 56 Vitronectin NRWHSIYITRFG 34 Laminin TWYKIAFQRNRK 33 Laminin RKRLQVQLSIRT 21 Laminin RYVVLPR 57 Laminin RNIAEIKDI 51 Laminin GKNTGDHFVLYM 22 Laminin VVSLYNFEQTFML 23 Laminin GIIFFL 58 Laminin RFDQELRLVSYN 24 Laminin RLVSYSGVLFFLK 25 Laminin ASKAIQVFLLGG 26 Laminin VLVRVERATVFS 27 Laminin TVFSVDQDNMLE 28 Laminin RLRGPQRVFDLH 29 Laminin FDLHQNMGSVN 30 Laminin QQNLGSVNVSTG 31 Laminin SRATAQKVSRRS 32 Laminin LCCGRGHRTRTQRVTERCNC 47 WNT5 LGTQGRLCNKTSEGMDGCEL 48 WNT1 IVPLLLLVLH 49 LIF 1 YTAQGEPFPNNVEKLCAP 50 LIF 2 LFSHAVSSNG 52 E-cadherin ADTPPV 53 E-cadherin DQNDN 54 E-cadherin LRAHAVDING 55 E-cadherin
Example 5
Culture and Self-Renewal of ESCs on Adhesion Surface
(111) The ability of integrin- or cytokine receptor activating surface to support self-renewal of mESCs was evaluated by serial passaging of murine ES cells on the microenvironment surface as prepared in EXAMPLE 4. These murine ES cells were obtained from cultures of early blastocysts.
(112) The array was incubated with media containing serum replacement media and murine embryonic stem cells were grown on the array for 5 days. For the maintenance of murine embryonic stem cell cultured on poly-D-lysine (PDL, Sigma-Aldrich) coated surface, DMEM Glutamax (GIBCO, Life Technology) containing high glucose 4.5 g/L, Na-pyruvate (0.11 g/L) and L-glutamine was used with 1% non-essential amino acid (Sigma-Aldrich), 50 U/mL Penicillin/streptomycin (GIBCO) and 0.1 mM 2-Mercaptoethanol (GIBCO) as the basal medium, which was added with 20% fetal bovine serum (FBS, Hyclone) and leukemia inhibitory factor (LIF, 1,000 units/mL, Millipore) at 37° C., 5% CO2 incubator.
(113) The mESCs was cultured in KnockOut™ DMEM medium (Invitrogen) supplemented with 20% KnockOut™ Serum Replacement (KSR; Invitrogen), 0.1 mM of 2-mercaptoethanol (Invitrogen), MEM Non-essential Amino Acids (Invitrogen), GlutaMAX™ Supplement (Invitrogen), leukemia inhibitory factor (LIF, 1,000 units/mL, Millipore), and 60 ng/mL of MAPTrix™ PHSRN-RGDSP (SEQ ID NO:17) (Kollodis BioSciences).
(114) Cells were washed in PBS and fixed in formaldehyde (37%) for 30 seconds, washed and stained for 15 minutes in 100 μL of FBB Alkaline solution (Sigma-Aldrich) in sodium nitrile solution. Stained cells were analyzed on an Olympus microscope.
(115) We confirmed that the murine embryonic stem cells were maintained in an undifferentiated stage on the surface presenting several integrin-, cadherin-, and cytokine receptor binding peptide motifs as shown in
Example 6
Concentration Effect of Fibronectin Mimetic on Self-Renewal
(116) Among the peptide motifs identified in Example 5, α5β1 integrin binding motif PHSRN-RGDSP (SEQ ID NO:17) enabled mESCs to self-renewal and proliferate in an undifferentiated, comparable with those cultured on Matrigel™. To evaluate the concentration effect of PHSRN-RGDSP (SEQ ID NO:17) on human induced pluripotent stem cells (hiPS), the concentration effect of MAPTrix™ (PHSRN-RGDSP (SEQ ID NO:17) motif containing) on cell morphology, colony size and shape was analyzed. A wide range of concentration of MAPTrix™ coating solution was prepared and coated on the substrate according to the procedure as set forth in Example 4. The hiPS cells were cultured on the microenvironment surface prepared from the 0.01 mg/mL, 0.06 mg/mL, 0.1 mg/mL, 0.5 mg/mL, and 1 mg/mL coating solution.
(117)
Example 7
Microenvironment Surface Presenting Combinatorial Signaling from Crosstalk Between Integrin-Growth Factor Receptor
(118) FGF signaling appears to be of central importance to human pluripotent stem cells self-renewal. To evaluate the synergic effect of FGF signaling together with integrin signaling, a microenvironment surface presenting α5β1 integrin binding motif and FGF and TGFβ receptor binding motif was prepared in accordance with the procedure as set forth in Example 4. The concentration of MAPTrix™ containing FGF mimetic peptide ANRYLAMKEDGRLLAS (SEQ ID NO:37), ERGVVSIKGV (SEQ ID NO:38), WYVALKRTGQYKLG (SEQ ID NO:39), HFKDPKRLYCK (SEQ ID NO:40), FLPMSAKS (SEQ ID NO:41), KTGPGQKAIL (SEQ ID NO:42), and TGFβ mimetic peptide LTGKNFPMFHRN (SEQ ID NO:43), MHRMPSFLPTTL (SEQ ID NO:46) used to create microenvironment was 50 ng/mL. The concentration of MAPTrix™ containing PHSRN-RGDSP (SEQ ID NO:17) α5β1 integrin binding motif was 0.5 mg/mL. In contrast to the microenvironment surface, the same MAPTrix™ containing FGF mimetic peptide and TGFβ mimetic peptide solution were added to E6 medium in order to evaluate the MAPTrix™ FGF or TGFβ mimetic as supplements.
(119) Human embryonic stem cells (H9) were maintained in serum replacement media conditions before switching from the serum-free media to the microenvironment surface. The cell clusters were seeded on the microenvironment surface in E6, E8, E6 supplemented with basic FGF (60 nM), E6 supplemented with TGFβ (5 nM), respectively. Medium was changed daily. After four days, cells were washed and fixed for AP staining according to the procedure as set forth in EXAMPLE 5.
(120)
Example 8
Microenvironment Surface Presenting Combinatorial Signaling from Crosstalk Between Integrin and Growth Factor/Cytokine Receptor
(121) WNT signaling has been involved in the control over various types of stem cells including pluripotent stem cells and may act as a niche factor to maintain stem cells in a undifferentiated state. Addition of WNT mimetic peptide to the microenvironment surface may inhibit human embryonic stem cell (H9) from partially differentiation during the proliferation as shown in EXAMPLE 7.
(122) A microenvironment surface to inhibit the partial differentiation was prepared by combining integrin-, growth factor receptor- and frizzled receptor binding motif. Two types substrate to create a microenvironment surface, namely, two-dimensional (2D) and three-dimensional (3D) substrate, but each has the same biochemical surface to induce the same combinatorial signaling.
(123) A series of WebTrix™ matrices, 3D nanofibrous substrates, was prepared according to the procedure as set forth in EXAMPLE 1. Six-well plates from Thermo Fischer Scientific were used as 2D substrate. The composition to create combinatorial signaling from fibronectin/FGF2 and WNT5a is summarized in the Table 4.
(124) TABLE-US-00004 TABLE 4 Composition to create WNT signaling microenvironment surface 3D substrate Molecule SEQUENCE 2D substrate (WebTrix™) Fibronectin PHSRN-RGDSP (SEQ ID NO: 17) 0.1 mg/mL 0.05 mg/mL WNT5a LGTQGRLCNKTSEGMDGCEL (SEQ ID 0.1 mg/mL 0.1 mg/mL NO: 48) FGF2 WYVALKRTGQKLG (SEQ ID NO: 71) 0.1 mg/mL 0.1 mg/mL WNT5a/FGF LGTQGRLCNKTSEGMDGCEL (SEQ ID 0.1 mg/mL 0.1 mg/mL NO: 48) - WYVALKRTGQKLG (SEQ ID NO: 71) TGFβ LTGKNFPMFHRN (SEQ ID NO: 45) 0.1 mg/mL 0.1 mg/mL LIF IVPLLLLVLH (SEQ ID NO: 49) 0.1 mg/mL 0.1 mg/mL FGF2/ TGFβ WYVALKRTGQKLG (SEQ ID NO: 71) - 0.1 mg/mL 0.1 mg/mL LTGKNFPMFHRN (SEQ ID NO: 45) TGFβ/WNT5a LTGKNFPMFHRN (SEQ ID NO: 45) - 0.1 mg/mL 0.1 mg/mL LGTQGRLCNKTSEGMDGCEL (SEQ ID NO: 48) FGF2/ WYVALKRTGQKLG (SEQ ID NO: 71) - 0.1 mg/mL 0.1 mg/mL TGFβ/WNT5a LTGKNFPMFHRN (SEQ ID NO: 45) - LGTQGRLCNKTSEGMDGCEL (SEQ ID NO: 48)
(125) Using the composition listed above, 18 different microenvironment surfaces were prepared using EDC/S—NHS as a crosslinking agent as set forth in EXAMPLE 4.
(126) Additionally, to investigate the effect of composition ratio of PHSRN-RGDSP (SEQ ID NO:17)/Wnt or LIF peptide in order to activate integrin/frizzled receptor signaling and integrin/LIF receptor signaling on the self-renewal of human induced pluripotent stem cells, a series of WebTrix™ with four different composition ratios (100/0, 80/20, 50/50, 20/80) were prepared.
(127) Human induced pluripotent stem cells were maintained in serum replacement medium before seeding on to the 18 different microenvironment surfaces. Instead of E6 supplemented growth factor or E8 medium, mTesR (Stem Cell Technologies, Inc.) was used and changed daily. After four days, cells were washed and stained with AP according to the procedure in EXAMPLE 5.
(128)
(129)
(130) For the analysis of stemness markers OCT4 and Sox2 expressed when cultured on different microenvironment surface, cells were fixed using 4% paraformaldehyde (Sigma-Aldrich) in 0.5% TRITON® X-100 solution for 30 minutes, accompanied by three times PBS washing, and 10% normal goat serum (Sigma-Aldrich) was added. The monoclonal antibodies against Oct4 (Santa Cruz Biotechnology) diluted at a concentration of 1:1,000 and Sox2 (Santa Cruz Biotechnology) diluted at a concentration of 1:1,000 was added to the above solution. The antibody was reacted for 24 hours at 4° C. and washed with PBS three times with 0.5% TRITON® X-100 added PBS. As the secondary antibody, goat anti-mouse ALEXA FLUOR® 546 (Invitrogen) was diluted with 0.5% TRITON® X-100 added PBS diluted at a concentration of 1:1,000, and reacted for 1.5 hours. Thereafter, the cells were reacted with 10 μg/mL of TO-PRO3 (Invitrogen) for the cell nucleus staining, and observed with fluorescent scanning microscopy (Zeiss).
(131)
(132) During the culture period, some of the wells were trypsinized and the cell number was determined on the daily basis using a hemocytometer. The average number for each single day was calculated and the values were used to plot a growth curve for each stem cell.
(133)
Example 8
Cell Migration Assay
(134) In wound healing or tissue regeneration, cells movement is essential process as a tightly or loosely associated cohesive group, and integrin-based adhesion has served as a model for studying the central role of adhesion in cell migration. Cell migration requires the dynamic interaction between a cell and the substratum on which it is attached and over which it migrates. Optimum cell speed occurs at intermediate levels of expression of α5β1 or α2β1 integrins or intermediate concentrations of ligand, including fibronectin or collagen. In general, in most cell types, there is an intermediate cell-substratum adhesiveness that supports maximum migration.
(135) Generally, integrin α2β1, α3β1 and α5β1 are the key adhesion receptors to regulate cell migration, and α2β1 integrin functions as the major receptor for collagen type I on a large number of different cell types including keratinocytes and fibroblasts.
(136) To identify optimal integrin binding peptide motif, a screening for keratinocyte adhesion and migration was performed. Briefly, eight different integrin α2β1 and α3β1 at a concentration of 0.01 mg/mL were coated on a 24-well plate.
Example 9
Effect of Particle Size on Stem Cell Culture
(137) Surface topographical cue, one of important physical cues to determine the cell fate, has been considered being a key feature to regulate cell behaviors. In fact, stem cells can interact with underlying material through nanosized integrin receptors. Therefore, the manipulation of topographical cues at a nanoscale level may be employed to regulate the cell fate.
(138) Different particle size embedded in nanofiber sheet, prepared according to the procedure as set forth in EXAMPLE 3, was used to investigate the effect of nanoscale (500 nm) and microscale (500 μm) particle size on human induced pluripotent stem cell behaviors.
(139) Human-induced pluripotent stem cells were maintained in serum replacement media conditions before switching from the serum-free media to the microenvironment surface. The cell clusters were seeded on the microenvironment surface in mTeSR™ (Stem Cell Technologies). Medium was changed daily. After four days, cells were washed and fixed for AP staining according to the procedure as set forth in EXAMPLE 5.
(140) The results are shown in
Example 10
Antimicrobial Microenvironment
(141) A series of antimicrobial nanofiber sheet with varying surface density of antimicrobial peptide motif, KLWKKWAKKWLKLWKA (SEQ ID NO:59) was prepared according to EXAMPLE 1. To control the surface density of antimicrobial peptide motif, A gram negative bacterium (E. coli) and a gram-positive bacterium Staphylococcus aureus (SA) were used to evaluate the antimicrobial activity of the nanofiber sheet. The antimicrobial activity of the nanosheet was determined by JIS Z 2801:2000, described by Haldar et al., Nature Protocols 2007, 2(19):2412.
(142) After the nanofiber sheet is inoculated by two bacteria (E. coli and SA), the colony-forming unit (CFU) of both bacteria was counted. As seen in
(143)