Kit for in vitro screening of active compounds, method and use of the kit

Abstract

The present invention relates to a kit comprising a co-culture microdevice containing peripheral sensory neurons (PSN) and human epidermal keratinocytes (HEK) in cell culture suitable for these two cell types. The present invention also describes a method for screening active compounds using the kit according to the present invention, and its use in in vitro drug testing and in the production of cosmetic products for various dermatological applications such as atopic dermatitis, sensitive skin, photoaging, wound healing and epidermal thickness in aging skin.

Description

Field of the Invention

[0001] The present invention relates to a kit comprising a co-culture microdevice containing peripheral sensory neurons (PSN) and human epidermal keratinocytes (HEK) in a cell culture suitable for these two cell types. The present invention also describes a method for in vitro screening of active compounds using the kit according to the present invention, and its use in in vitro drug testing and in the production of cosmetic products for various dermatological applications such as atopic dermatitis, sensitive skin, photoaging, wound healing and epidermal thickness in aging skin. Background Art

[0002] Keratinocytes have extensive interactions with peripheral sensory neurons (PSNs) during development and in mature skin. For example, keratinocytes release neurotrophic factors that induce free nerve endings to branch and neurites to grow outward toward the skin surface (Albers & Davis, The skin as a neurotrophic organ. Neuroscientist. 2007; 13: 371–82). They also release receptors involved in responding to tissue damage and hypersensitivity, as well as responding to cold and hot inflammatory mediators through the TRP family of cation channels (Chung et al., TRPV3 and TRPV4 mediate warmth-evoked currents in primary mouse keratinocytes. J Biol Chem. 2004; 279: 21569–75).

[0003] On the other hand, sensory nerve endings not only transduce sensory signals, but also play an important role in the metabolism and homeostasis of the skin by secreting proinflammatory neuropeptides and inflammatory mediators that control angiogenesis and tissue renewal (Roosterman et al., Neuronal control of skin function: the skin as aneuroimmunoendocrine organ. Physiol Rev. 2006; 86: 1309–79). In particular, TRPV1-positive nociceptors also regulate skin lifespan and metabolism, as well as immune responses after aging (Riera et al., TRPV1 pain receptors regulate longevity and metabolism by neuropeptide signaling. Cell 2014; 157, 1023–1036).

[0004] Neuropeptides produced by sensory neurons innervating the skin regulate cell proliferation, wound healing, pigmentation, and keratinocyte innate immune responses. Neuropeptides are known to stimulate inflammatory mediators produced by keratinocytes, but little information is available on the mechanisms by which neuropeptide activation of keratinocyte cell surface receptors ultimately leads to upregulation of those mediators.

[0005] The discovery of the interaction between keratinocytes and peripheral sensory neurons (PSNs) can help treat and / or prevent a variety of dermatological conditions and disorders, such as wound healing and skin aging. During skin aging, a decrease in the number and rate of neurotrophy, proliferation, differentiation, and neuritis leads to a decrease in skin sensation and skin thickness. Therefore, the restoration of sensitive free nerve endings should restore skin nerve sensation and epidermal thickness, while increasing trophic function toward the epidermis, increasing neurite generation, and thus restoring skin innervation.

[0006] In light of the above, the object of the present invention is to provide a neuroskin in the form of a kit comprising a co-culture microdevice containing peripheral sensory neurons (PSNs) and human epidermal keratinocytes (HEKs) in a suitable cell culture medium that allows the assessment of the interaction between sensory free ends and epidermal keratinocytes. Such a kit is intended to study the biology and pharmacology of the interaction of sensory free ends and epidermal keratinocytes and to screen for potential new drugs of interest for the beauty industry. Summary of the invention

[0007] The present invention discloses a novel and effective kit comprising a co-culture microdevice, peripheral sensory neurons (PSNs), human epidermal keratinocytes and a suitable cell culture medium, which simulates the connection between free nerve endings and epidermal keratinocytes in human skin.

[0008] Such cutaneous nerves are advantageously used in an in vitro method for screening active compounds, the method comprising the steps of: (a) providing a kit according to the present invention and a test compound, and (b) contacting the test compound with the kit and measuring peripheral sensory function, wherein measuring the function consists of measuring the activity of at least one neuronal marker.

[0009] Furthermore, the present invention also discloses the use of the kit defined herein in performing in vitro tests and producing cosmetic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1Immunohistochemistry of (A) nestin, (B) β-tubulin III (TUJ1), (C) peripherin, (D) TRPV1, (E) Nav1, and (F) CGRP in neural crest progenitor cells (NCPCs) and human neural stem cells (NSCs) obtained from human induced pluripotent stem cells (hiPSCs) is shown. Cells were positive for all markers under both conditions.

[0011] Figure 2 Shown are quantifications of neuronal markers (A) nestin, (B) β-tubulin III (TUJ1), (C) peripherin, (D) TRPV1, (E) Nav1, and (F) CGRP expressed by neural crest progenitor cells (NCPCs) obtained from human induced pluripotent stem cells (hiPSCs) and human neural stem cells (NSCs).

[0012] Figure 3 Shown are neural cells cultured with 20 μg / mL laminin (A) and 5 μg / mL laminin (B) 5 days after plating in the co-culture microdevice.

[0013] Figure 4 Neuronal aggregation in the lower (A) and upper (B) co-culture microdevices is shown.

[0014] Figure 5 Shown is a micrograph of a peripheral sensory neuron (PSN) showing healthy axons and the presence of growth cones.

[0015] Figure 6 Shown are (A) β-tubulin III (TUJ1), (B) peripherin, (C) nuclei stained with DAPI and (D) merged immunohistochemistry.

[0016] Figure 7 Peripheral sensory neurons (PSNs) co-cultured with keratinocytes in a co-culture microdevice are shown. Neurites migrate through tubules into the keratinocyte compartment. Red arrows indicate varicosities.

[0017] Figure 8 Shown are micrographs showing the cellular heterogeneity observed following the differentiation protocol. F: fibroblast-like, P: pyramidal neuron-like, ?: unidentified morphology.

[0018] Fig. 9 Shown are neurospheres obtained from neural crest progenitor cells (NCPCs).

[0019] Fig.10 Shown are neurospheres plated on a co-culture device, showing reduced cellular heterogeneity.

[0020] Fig.11Showing the tropism of neurites from neurospheres toward human epidermal keratinocytes (HEK). A: Co-culture microdevice. B: Migration of neurites from neurospheres toward the human epidermal keratinocyte (HEK) chamber. C: Contact between neurites and human epidermal keratinocytes (HEK). D: Presence of varicosities.

[0021] Fig.12 Shown are immunohistochemistry for β-tubulin III (TUJ1, green), peripherin (red), and nuclei stained with DAPI.

[0022] Fig.13 A co-culture microdevice with an added central punched hole is shown. The red arrow in A indicates the punch hole. The blue arrow shows the line drawn to measure neurite outgrowth.

[0023] Fig.14 It was shown that quantification of neurite outgrowth depends on the culture conditions in the co-culture microdevice.

[0024] Fig.15 Matrigel is shown to block the migration of human epidermal keratinocytes (HEK). A: HEK cells cultured on Matrigel are confined to the hole area (red arrows), B: HEK cells cultured without Matrigel can be observed invading the tubules of the co-culture microdevice (blue arrows).

[0025] definition

[0026] Stem cells are undifferentiated cells, defined by their ability to self-renew and differentiate at the single cell level. Stem cells can produce offspring cells, including self-renewal progenitor cells, non-renewal progenitor cells, and terminally differentiated cells. Stem cells are also characterized by their ability to differentiate into the ability of functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm, and ectoderm) in vitro. Stem cells also result in the tissue of multiple germ layers after transplantation, and contribute substantially to most (if not all) tissues after being injected into blastocysts. Stem cells are classified according to their developmental potential.

[0027] As used herein, the term "pluripotency" refers to the ability to develop into the three developmental germ layers of an organism, including endoderm, mesoderm, and ectoderm.

[0028] The characteristics of pluripotent stem cells are well known to those skilled in the art, and additional characteristics of pluripotent stem cells continue to be identified. Pluripotent stem cell markers include, for example, the expression of one or more of the following: ABCG2, cripto, FOXD3, connexin 43, connexin 45, OCT4, SOX2, NANOG, hTERT, UTF1, ZFP42, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81. In one embodiment, the pluripotent stem cells suitable for the present invention express one or more of NANOG, SOX2, TRA-1-60 and TRA-1-81, and lack the expression of markers for differentiation neural markers Islet1, BRN3A, peripherin and TRPV1.

[0029] As used herein, the term "human induced pluripotent stem cells (hiPSC)" refers to stem cells induced from somatic cells, such as differentiated somatic cells, and the stem cells have a higher potency than the somatic cells. Human induced pluripotent stem cells (hiPSC) are capable of self-renewal and differentiation into mature cells, such as neural crest progenitor cells (NCPC).

[0030] As used herein, the term "human peripheral sensory neuron (PSN)" refers to the main type of neuron present in the skin layers (such as dermis and epidermis), interacting with skin cells and structures, such as epidermal keratinocytes, fibroblasts, melanocytes, sweat glands, hair follicles, etc.

[0031] "Cell culture" or "culture" generally refers to cells taken from a living organism and grown under controlled conditions ("in culture" or "cultured"). Primary cell culture is the culture of cells, tissues, or organs taken directly from an organism before the first subculture. When cells are placed in a growth medium under conditions that are favorable to one or both of cell growth and division, they expand in culture, resulting in a larger cell population. When cells expand in culture, the rate of cell proliferation is sometimes measured by the amount of time required for cells to double in number (called the doubling time).

[0032] The culture container for culturing stem cells may include, but is not particularly limited to, a culture flask, a culture flask for tissue culture, a dish, a Petri dish, a dish for tissue culture, a multi dish, a micro culture container, a microplate, a multi plate, a multi-well plate, a micro slide, a chamber slide, a schale, a test tube, a tray, a culture bag and a roller bottle, as long as it can culture the stem cells therein.

[0033] The culture vessel can be cell-adhesive or non-adhesive, depending on its purpose. Cell-adhesive culture vessels can be coated with any matrix for cell adhesion, such as an extracellular matrix (ECM), to improve the adhesion of the vessel surface to the cells. The matrix for cell adhesion can be any material intended to attach stem cells or feeder cells (if used).

[0034] The culture conditions can be appropriately determined. For example, the culture temperature may be about 30 to 40° C. and preferably about 37° C., but is not particularly limited thereto. 2 The concentration may be about 1 to 10% and preferably about 2 to 5%. The oxygen tension may be 1-10%.

[0035] Differentiation is the process by which an unspecialized ("uncommitted") or less specialized cell acquires the characteristics of a specialized cell (e.g., a nerve cell or a muscle cell). A differentiated cell, or a cell induced to differentiate, is a cell that has assumed a more specialized ("committed") position within a lineage of cells. The term "committed," when applied to the process of differentiation, refers to a cell that has progressed to a point in the differentiation pathway where it would normally continue to differentiate into a specific cell type or subset of cell types and would normally be unable to differentiate into a different cell type or revert to a less differentiated cell type.

[0036] As used herein, the term "inhibitor" refers to a compound that reduces or eliminates the biological function or activity of the signal transduction pathway by interfering with a specific target that is part of the signal transduction pathway, or by interfering with the interaction between two or more targets. In order to reduce or eliminate the biological function or activity of the protein to be inhibited, the inhibitor may perform any one or more of the following effects: (i) reduce the transcription of the gene encoding the protein to be inhibited, that is, the mRNA level is reduced, (ii) reduce the translation of the mRNA encoding the protein to be inhibited, (iii) in the presence of the inhibitor, the protein performs its biochemical function with reduced efficiency, and (iv) in the presence of the inhibitor, the protein performs its cellular function with reduced efficiency.

[0037] Such compounds may include, but are not limited to, small molecules, peptides, peptidomimetics, natural compounds, siRNA, antisense nucleic acids, aptamers, or antibodies.

[0038] In other words, an inhibitor is any compound or molecule that alters any activity of a specified protein (signaling molecule, any molecule involved in a specified signaling molecule, a specified related molecule, such as glycogen synthase kinase 3β (GSK3β), for example, by directly interacting with SMAD signaling, interacting with SMAD mRNA, causing a conformational change in SMAD, reducing SMAD protein levels, or interfering with the interaction of SMAD with a signaling partner and affecting the expression of SMAD target genes.

[0039] Inhibitors also include molecules that indirectly modulate SMAD biological activity by intercepting upstream signaling molecules (e.g., examples of signaling molecules and actions within the extracellular domain include: Noggin, which sequesters bone morphogenetic protein, inhibits activation of ALK receptors 1, 2, 3, and 6, thereby preventing downstream SMAD activation. Likewise, Chordin, Cerberus, follistatin, similarly sequester extracellular activators of SMAD signaling. Bambi, a transmembrane protein, also acts as a pseudo-receptor that sequesters extracellular TGFβ signaling molecules. Antibodies that block activin, nodal, TGFβ, and BMP are contemplated for neutralizing extracellular activators of SMAD signaling, etc.).

[0040] As used herein, the term "mitogen" refers to those compounds that are members of the fibroblast growth factor family, such as FGF-2 (basic FGF) and FGF-4. Epidermal growth factor (EGF), functional homologues, and other factors that bind to the EGF receptor are also exemplary. Other candidate growth factors are platelet-derived growth factor (PDGF), insulin-like growth factor (IGF). These mitogens are used to increase the number of lineage cells, causing them to further proliferate in culture.

[0041] "Neurotrophic factors" are endogenous peptides found in the nervous system or in non-neural tissues innervated by the nervous system, whose function is to promote the survival and maintenance of phenotypic differentiation of neural and / or glial cells. The family of trophic factors known as neurotrophic factors currently includes: brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3), NT-4 / 5, and NT-6. All neurotrophic factors can be used alone or in combination.

[0042] The preferred amount of each neurotrophic factor used is between about 1 ng / mL and about 25 ng / mL, more preferably between about 5 ng / mL and about 15 ng / mL, and more preferably about 10 ng / mL.

[0043] As used herein, the expression "differentiation inducing agent" refers to ascorbic acid (AA).

[0044] The preferred amount of differentiation inducing agent used is between about 50 μM and about 500 μM, more preferably between about 100 μM and about 300 μM, more preferably about 200 μM.

[0045] As used herein, the expression "cell transduction inducer" refers to a compound that mediates signal transduction, such as, for example, cAMP.

[0046] The preferred amount of cell transduction inducer used is between about 0.01 mM and about 1 mM, more preferably between about 0.1 mM and about 0.8 mM, more preferably about 0.5 mM.

[0047] As used herein, a "marker" is a nucleic acid or polypeptide molecule that is differentially expressed in a cell of interest. In the present context, differential expression refers to an increase in the level of a positive marker and a decrease in the level of a negative marker compared to an undifferentiated cell. The detectable level of the marker nucleic acid or polypeptide in the cell of interest is sufficiently higher or lower than that in other cells so that the cell of interest can be identified and distinguished from other cells using any of a variety of methods known in the art.

[0048] As used herein, a cell is "positive" or "positive" for a specific marker when the specific marker is sufficiently detected in the cell. Similarly, a cell is "negative" or "negative" for a specific marker when the specific marker is not sufficiently detected in the cell.

[0049] As used herein, the terms "activator" and "activation" refer to compounds that are used to activate molecules that lead to the directed differentiation of cells of the present invention. Exemplary activators include, but are not limited to, harmful heat / cold, mechanical stimulation, chemical stimuli (menthol, piperine, acute capsaicin, cinnamaldehyde, resin toxins, bradykinin, ATP, prostaglandins, inflammatory cytokines, acidic saline, fibroblast growth factor (FGF), etc.).

[0050] As referred to herein, active compounds refer to known cosmetic ingredients, dermatological and biological actives such as neuromodulators, anti-aging compounds, neuroaging regulators, which are used to control the growth rate and density of free nerve endings, the conductivity along the skin layers, trigger action potentials in peripheral sensory neurons, increase the interaction between these neurons and other skin cell types (keratinocytes, fibroblasts and melanocytes, adipocytes, hair follicle cells, glands, cartilage, stem cells, etc.).

[0051] As referred to herein, TRP (transient receptor potential) channels include a variety of ligand-gated, primarily non-selective cation channels that are robustly expressed in sensory systems across species (Nilius & Szallasi, Transient receptor potential channels as drug targets from the science of basic research to the art of medicine. Pharmacol Rev 2014; 66(3): 676–814). Of these, TRPV1 is the most well-studied and is considered to be a typical TRP channel present in somatic sensory neurons (Basbaum et al., Cellular and molecular mechanisms of pain. Cell.; 2009; 139: 267-284). TRPV1 can be directly gated by external molecules (such as capsaicin, resiniferatoxins, and piperine), and is also positively or negatively regulated by activation of other receptors and second messenger systems (such as PIP2 hydrolysis and PKC phosphorylation) (Julius, TRP channels and pain. Annu Rev Cell Dev Biol. 2013; 29: 355-84). One of the receptors that appears to inhibit TRPV1 activation is the cannabinoid 1 receptor (CB1), which is also present in somatosensory neurons (Julius & Basbaum, Molecular mechanisms of nociception. Nature. 2001; 413: 203-210). However, anandamide, an endogenous agonist of CB1, is also an agonist of TRPV1, although it has an EC50 that is one order of magnitude higher in the latter (Zygmunt et al., Vanilloid receptors on sensory nerves mediate the vasodilator action of anandamide. Nature. 1999; 400: 452-457).

[0052] Substance P (SP) is a neuropeptide member of the tachykinin family, synthesized by sensory neurons, which radiate their extensions from the DRG to the more superficial layers of the skin, mediating communication between peripheral neurons and epidermal keratinocytes (Ribeiro-da-Silva & Hokfelt, Neuroanatomical localization of Substance P in the CNS and sensory neurons. Neuropeptides. 2000; 34: 256-271). Most neurons that release substance P are sensitive to capsaicin, highlighting the importance of TRPV1 expression and sensory neuron-keratinocyte interactions. DETAILED DESCRIPTION

[0053] The present invention is directed to a novel and specific kit comprising:

[0054] (i) Co-culture microdevice;

[0055] (ii) peripheral sensory neurons (PSNs);

[0056] (iii) human epidermal keratinocytes (HEK); and

[0057] (iv) Cell culture medium.

[0058] According to the present invention, the peripheral sensory neurons (PSN) are derived from human neural stem cells (NSC) or human induced pluripotent stem cells (hiPSC). Preferably, the peripheral sensory neurons (PSN) are derived from human induced pluripotent stem cells (hiPSC).

[0059] Peripheral sensory neurons (PSN) derived from hiPSC can be obtained by any method known in the art. In a preferred embodiment, the method of inducing differentiation into peripheral sensory neurons (PSN) comprises the following steps: contacting human stem cells, such as human induced pluripotent stem cells (hiPSC) with at least one SMAD pathway inhibitor in a neural induction medium to produce primary differentiated cells such as neural crest progenitor cells (NCPCs), and culturing the primary differentiated cells with at least one mitogen, a neurotrophic factor, a differentiation inducer and a cell transduction inducer to obtain human peripheral sensory neurons (PSN).

[0060] In a preferred embodiment, peripheral sensory neurons (PSNs) are induced to spontaneously form neurospheres during the maturation of the neurons, thereby reducing the heterogeneity of the culture.

[0061] The cell culture medium of the present invention is any suitable culture medium suitable for the two cell types (i.e., peripheral sensory neurons (PSN) and human epidermal keratinocytes). In a preferred embodiment, the cell culture medium is a 3N culture medium, in particular a 3N culture medium comprising a 1:1 mixture of an N2 culture medium containing B27 and a culture medium containing B27.

[0062] In a preferred embodiment, the medium containing N2 comprises DMEM / F12 supplemented with N2 supplement (GIBCO), insulin, L-glutamine, non-essential amino acids (NEAA), β-mercaptoethanol, penicillin and streptomycin. In a preferred embodiment, the medium containing B27 comprises Neurobasal medium (Invitrogen) supplemented with B27 supplement (GIBCO), L-glutamine, penicillin and streptomycin.

[0063] Preferably, 3N medium is supplemented with NGF, TGFβ and / or a BMP signaling inhibitor to produce a neural induction medium.

[0064] In a preferred embodiment, the cell culture medium has a culture medium gradient between the two cell types.

[0065] According to the present invention, the co-culture microdevice is a microfluidic device containing several microchannels within which peripheral sensory neurons (PSNs) are grown in an orderly manner, allowing good connection with human epidermal keratinocytes (HEKs). Advantageously, the co-culture microdevice is used in a horizontal setup, thereby providing improved culture conditions for connecting neurons to keratinocytes.

[0066] In a preferred embodiment, the co-culture microdevice is a microchip for cell culture made of biocompatible silicone and comprising four to twenty independent chambers, wherein skin cell types, such as peripheral sensory neurons (PSNs) and human epidermal keratinocytes (HEKs), are alternately plated on each side of the independent chambers, with channel diameters that allow axon elongation and neurite growth in the microchannels between chambers in the chip and prevent cell bodies from migrating into other chambers and invading the co-cultured cell compartment space.

[0067] Preferably, the co-culture microdevice comprises a hole, wherein human epidermal keratinocytes (HEK) are plated in the hole.

[0068] Still in a preferred embodiment, the co-culture microdevice is covered with matrigel or laminin or poly-ornithine or collagen to create a microenvironment appropriate for each cell type and prevent human cells from migrating into other compartments, as well as to allow neuronal growth, thereby giving mechanical cues to induce the neuronal fate of neural crest progenitor cells (NCPCs) or neuronal progenitor cells (NPCs) and epidermal keratinocytes (HEKs) through the device chambers.

[0069] Another object of the present invention relates to a method for in vitro screening of active compounds, comprising:

[0070] (a) Provide:

[0071] (i) a kit as defined herein;

[0072] (ii) a test compound; and

[0073] (b) contacting the test compound with the kit and measuring peripheral sensory neuron function, wherein measuring the function comprises measuring the activity of at least one neuronal marker.

[0074] Preferably, the active compounds screened by the method of the present invention act on the regulation of neuron growth, the number of nerve endings, neuron activity and epidermal regeneration, for example. More preferably, the regulation of neuron activity is mediated by the release of growth factors induced by human epidermal keratinocytes (HEK), and epidermal regeneration is mediated by regulating the release of factors from neurons.

[0075] In a preferred embodiment, the active compounds screened by the method of the present invention are used to treat and / or prevent, for example, atopic dermatitis, sensitive skin, photoaging and light pollution effects, neuroaging, wound healing, neuron-controlled skin barrier function, itch, skin mechanical perception and epidermal thickness in aged skin.

[0076] A further object of the invention is the use of a kit as defined herein for performing an in vitro test, preferably the test is for screening active compounds, the active compounds acting for example on the regulation of neuronal growth, number of nerve endings, neuronal activity and epidermal regeneration. More preferably, the regulation of neuronal activity is mediated by inducing the release of growth factors from human epidermal keratinocytes (HEK), and epidermal regeneration is mediated by regulating the release of factors from neurons.

[0077] In a preferred embodiment, the kit of the present invention is used for screening active compounds for treating and / or preventing, for example, atopic dermatitis, sensitive skin, photoaging and light pollution effects, neuroaging, wound healing, neuron-controlled skin barrier function, itch, skin mechanical perception and epidermal thickness in aged skin.

[0078] It was unexpectedly and surprisingly found that the novel and specific kit of the present invention comprising a co-culture microdevice, peripheral sensory neurons (PSNs), human epidermal keratinocytes (HEKs) and a cell culture medium allows the orderly growth of peripheral sensory neurons (PSNs) and connection with human epidermal keratinocytes (HEKs) so as to mimic the connection between sensory free nerve endings and epidermal keratinocytes (HEKs) in human skin.

[0079] The following examples are intended to illustrate the invention with reference to some preferred embodiments, but are not limited to the details shown. On the contrary, various modifications can be made in the details without departing from the scope of the invention.

[0080] Example

[0081] Example 1

[0082] Methods for inducing differentiation into peripheral sensory neurons (PSNs)

[0083] Differentiation of human induced pluripotent stem cells (hiPSCs) into neural crest progenitor cells (NCPCs)

[0084] Standard culture conditions (37°C, 5% CO 2), human induced pluripotent stem cells (hiPSC) were cultured in Essential 8 medium (Thermo Fisher Scientific, USA) on a dish coated with matrigel (BD Biosciences). Colonies were separated using 0.5mM EDTA (Thermo Fisher Scientific, USA) every 4-5 days. Human iPS cell cultures at 40-70% confluence were used for NCPC induction. hiPSCs were exposed to a chemically defined 3N induction medium (DMEM+neural basal medium 50:50v / v, 1% Glutamax, 0.5% N2, 1% B27, 0.5% NEAA, 55mM β-mercaptoethanol and 1% penicillin / streptomycin, all from Thermo Fisher Scientific, USA) freshly supplemented with three small molecule compounds for 10 days. These compounds were added as follows: Day 1: 500 nM LDN (Stemgen, USA) + 10 μM SB (Sigma Aldrich, USA); Day 2: 500 nM LDN + 10 μM SB + 3 μM CHIR (Tocris Bioscience, USA); Day 3: 10 μM SB + 3 μM CHIR. On days 4, 6 and 8, the medium was supplemented with only 3 μM CHIR. After 10 days of differentiation, NCPCs were further cultured in expansion medium (3N medium freshly supplemented with 10 ng / mL βFGF and 10 ng / mL EGF, both βFGF and EGF were from Thermo Fisher Scientific, USA). On day 11, NCPCs were enzymatically passaged (passage 0) at 37°C for 2-3 min using Accutase (Merck Millipore, USA) and aliquoted 1:3 onto poly-L-ornithine (100 ug / mL, Sigma Aldrich, USA) / laminine (20 μg / mL, Thermo Fisher Scientific, USA) coated dishes and cultured to confluence. The culture medium was replaced every other day. When 70-100% confluence was reached (usually 24-48 h after passage 0), the cells were passaged again and cultured at a specific density: 1 x 10 per 60 mm dish. 6 Cells or 3x 10 per 100 mm dish 6 The cells were cultured in a culture vessel. 10 μM ROCK inhibitor (Merck Millipore, USA) was added on the day of passage and removed after 24 h.

[0085] Generation of peripheral sensory neurons (PSNs) from NCPCs

[0086] Neural crest progenitor cell (NCPC) cultures at approximately 80% confluence (usually on day 13) were used for neuronal differentiation. Briefly, cells were maintained for approximately 23 days in a neural induction medium containing the following differentiation factors: 0.5 mM AMPc (Sigma Aldrich, USA), 200 μM AA (Sigma Aldrich, USA), 10 ng / mL NT-3 (R&D Systems, USA), 10 ng / mL NGF (R&D Systems, USA), 10 ng / mL BDNF (R&D Systems, USA) and 10 ng / mL GDNF (R&D Systems, USA). The culture medium was replaced every 3-4 days. Neurons were enzymatically detached (if necessary) at 37°C for 3-5 min using Accutase (Merck Millipore, USA) and detached onto a freshly prepared poly-L-ornithine / laminine dish. 10 μM ROCK inhibitor (Merck Millipore, USA) was added at each passage to increase the survival and attachment of neurons. Around day 35, the neurons were harvested and cultured in culture vessels for analysis and / or further experiments.

[0087] Human epidermal keratinocyte (HEK) culture

[0088] Neonatal human epidermal keratinocytes (HEKn) were obtained from Cascade Biologicals (Portland, OR) and cultured in EpiLife serum-free medium (ThermoFischer). Cells were cultured at 10,000 cells per well in culture vessels. Culture vessels pre-treated with gelatin (Sigma) and medium EpiLife (Thermo Fisher Scientific) were separated when 70% to 75% confluent for 48 h, then collected and freshly added to neuronal culture medium and centrifuged to remove debris and dead cells.

[0089] Co-culture of human PSNs and HEKs and treatment of conditioned media

[0090] On day 35 of neural differentiation, peripheral sensory neurons were harvested and cultured at 30,000 cells per well in 96-well (Perkin-Elmer, USA) culture vessels coated with poly-L-ornithine / laminine, and cultured for two, five and ten days under the following conditions: co-culture with HEKn cells in standard neural induction medium; and without HEKn cells but with HEKn conditioned medium added at three different ratios (25, 50 and 75%). The conditioned medium was changed every 3 days.

[0091] Example 2

[0092] Comparison between NCPCs and neural stem cells (NSCs) derived from iPS

[0093] Immunocytochemistry

[0094] Neural crest progenitor cells (NCPCs) and NSCs obtained from hiPSCs were cultured in 96-well culture vessels and fixed with 4% paraformaldehyde, permeabilized with Triton X-100 and blocked with 3% bovine serum albumin (BSA). Cells were incubated with primary antibodies diluted in 3% BSA for 2 hours. After washing with PBS, conjugated secondary antibodies were added for 40 minutes in the dark, washed thoroughly with PBS, and then incubated with DAPI (4',6-diamidino-2-phenylindole) for 5 minutes for nuclear staining. After rinsing with PBS and water, 50 μl of glycerol was added as a mounting medium, and the culture vessel was sealed with an aluminum sticker before analysis. The primary antibodies used were: nestin (1:100, Sigma-Aldrich, USA), anti-β-tubulin III (1:200, Merck-Millipore, Germany), anti-peripherin (1:250, Santa Cruz Biotechnology), anti-TRPV1 (1:1000, Abcam), anti-Nav1 (1:1000, Abcam), anti-CGRP (1:250, Santa Cruz Biotechnology). The secondary antibodies conjugated with Alexa Fluor 488 and Alexa Fluor 594 (1:400, Life Technologies, USA) were incubated in the dark for 40 minutes. Nuclear staining was performed with 0.5 μg / mL 4′-6-diamino-2-phenylindole (DAPI) for 5 min. Images were obtained using a high-content screening microscope Operetta (PerkinElmer, USA) and analyzed using high-content image analysis software Harmony 5.1 (PerkinElmer, USA).

[0095] test

[0096] Neural stem cells (NSCs) are generated from iPS cells and can differentiate into neurons and glial cells. They are easy to manipulate and can undergo multiple freeze / thaw cycles without losing the ability to differentiate. Obtaining NCPCs from NSCs can avoid the loss of efficiency that may occur in the differentiation of NCPCs after thawing.

[0097] The expression of neural markers: nestin, beta-tubulin III (TUJ1), peripherin, TRPV1, Nav1, and CGRP in NCPCs derived from iPS and NSCs was compared. First, the results showed that both presented similar expression patterns confirming their neural stem cell fate ( Figure 1 ). However, some differences were observed when the immunolabeling was quantified ( Figure 2 ).

[0098] Example 3

[0099] Strategies to increase PSN maturation in vitro and in co-culture

[0100] To find the optimal configuration for PSN growth and subsequent co-culture with HEKs, several co-culture microdevices were tested and various parameters described below were adjusted.

[0101] Neuronal differentiation requires the use of an extracellular matrix that allows their development and migration, such as laminin. Conventional protocols typically use 20 μg / mL laminin. However, this concentration causes clogging of the microdevice's tubules and disrupted neurite outgrowth ( Figure 3 A). After adjusting the laminin concentration to 5 μg / mL, neurites grew along a straight path of 11 μm in 5 days ( Figure 3 B).

[0102] The second step is to adapt the microdevice to the specific cell model of the present invention. The differentiation protocol promotes the formation of aggregates from which neurons migrate. The first microdevice does not allow the entry of aggregates, reducing the final number of neurons ( Figure 4 A). Height variation allows for the entry of aggregates and the uniform distribution of neurons within the microdevice ( Figure 4 B).

[0103] Subsequently, co-culture of PSNs with keratinocytes was performed. However, these cells do not adapt well to neuronal culture medium. Therefore, PSNs were cultured with keratinocyte-conditioned medium, which promotes an increase in the number of neuronal processes and a more abundant expression of markers such as TRPV1.

[0104] In such tests, different ratios of neuron culture medium were mixed with keratinocyte culture medium. A combination consisting of 75% keratinocyte culture medium and 25% neuron culture medium appeared to be sufficient to keep neurons alive and healthy ( Figure 5 ).

[0105] When cultured under these conditions, neurons were also positive for β-tubulin III (TUJ1) and peripherin ( Figure 6 ).

[0106] Following this, plating each cell type at different times (e.g., PSNs first and then keratinocytes, or vice versa) was tested, as well as modifying the flow between the chambers. Neurons were observed to fire their processes into the keratinocyte chamber, suggesting that interactions between the cells ( Figure 7). It is interesting to note that the neurons present varicosities (red arrows), a feature found in skin biopsies (Cauna, 1980; McCarthy, BG et al., 1995; Talagas et al., 2018). This is due to the intense trafficking of vesicles, specifically through these cells.

[0107] Example 4

[0108] Variability in differentiation induction

[0109] Recently, Schwartzentruber et al. (2017) conducted a large-scale study with 123 differentiation steps from iPS cells to sensory neurons. They observed cellular heterogeneity for each differentiation. Indeed, although iPS cells were able to generate the same cell types in the same amount of time, they showed a greater degree of variability than embryonic stem cells (Huby et al., 2010).

[0110] In the scheme of the present invention, it was also observed that the variability in cell morphology changed in proportion to each differentiation ( Figure 8 ).

[0111] In an attempt to reduce the heterogeneity of the cultures, a strategy used by other protocols for neuronal differentiation was adopted. After NCPC generation, neurosphere formation was induced, and such neurospheres spontaneously formed as the neurons matured, albeit heterogeneously. Therefore, it was concluded that by controlling neurosphere formation, heterogeneity could be reduced, potentially promoting differentiation. Cell density is a critical factor for the proper formation of neurospheres. Thus, 9x10 3 and 18x10 3 Two amounts of cells were used and it was found that the smallest amount formed healthier and more homogeneous neurospheres with very similar yields ( Fig. 9 ).

[0112] Example 5

[0113] Differentiation of neurospheres in microfluidic devices

[0114] After successful differentiation of neurospheres, they were plated as described and, as expected, the number of contaminating cells was reduced and there was extensive migration of neurites ( Fig.10 ). Moreover, there was a 2-week reduction in differentiation time.

[0115] Thereafter, a co-culture microdevice with 4 independent chambers was used. Neurospheres (NS) and keratinocytes (K) were alternately plated ( Fig.11 A). Neurites from neurospheres were observed to migrate toward the keratinocyte compartment ( Fig.11B). After removal of the microdevice, contact areas and the presence of varicosities between neurites and keratinocytes were observed, indicating interaction between the cells ( Fig.11 CD).

[0116] Neurons obtained from NCPC neurospheres were peripherin positive, had varicosities, and showed tropism for keratinocytes ( Fig.12 ).

[0117] Keratinocytes move faster than dendrites, and, since removing the microdevice sometimes means pulling the neurospheres out of the substrate, a hole was punched between identically paired wells and keratinocytes were plated into the hole ( Fig.13 A, red arrows). The area where the migrating neurites were mainly present was observed, and the highest migration radius ( Fig.13 B, blue arrow).

[0118] To further test this modified co-culture microdevice, the culture medium (3N vs. 75% CM of HEKn) and the presence (or absence) of Matrigel in the medium were compared by measuring the maximum extension of neurites outward from the neurospheres ( Fig.14 A). No statistically significant differences were observed. Then, neurite extension was compared in the presence or absence of keratinocytes in the holes. Again no differences were observed ( Fig.14 B). Finally, another variable was tested: the addition or absence of NGF to the Matrigel in the central punch hole. Although no statistical significance was achieved when comparing the treatments, a trend was clear that NGF increased migration, particularly between the 3N medium (first and second bars in the figure) and the 75% CM plus HEKn (fifth and last bar) groups ( Fig.14 C).

[0119] Also note that Matrigel prevents HEK from coming out of the hole ( Fig.15 A). In the absence of Matrigel, HEKs tend to migrate through the device ( Fig.15 B).

Claims

1. A kit comprising: (i) A co-culture microdevice, wherein: The co-culture microdevice is a microfluidic device containing a microchannel, (ii) peripheral sensory neurons (PSNs), wherein the peripheral sensory neurons (PSNs) grow horizontally within a microchannel of a co-culture microdevice, (iii) human epidermal keratinocytes (HEK), and (iv) cell culture medium, The co-culture microdevice is a microchip for cell culture made of biocompatible silicone and comprising four to twenty independent chambers, wherein the peripheral sensory neurons (PSNs) and human epidermal keratinocytes (HEKs) are alternately plated on each side of the independent chambers. wherein the co-culture microdevice comprises holes punched between identical paired wells, and wherein the human epidermal keratinocytes (HEK) are plated in the holes, Wherein, the peripheral sensory neurons (PSN) are derived from human induced pluripotent stem cells (hiPSC), Therein, the peripheral sensory neurons (PSNs) are induced to spontaneously form neurospheres during the maturation of the neurons.

2. The kit according to claim 1, wherein The cell culture medium is a 3N culture medium, which comprises a 1:1 mixture of a culture medium containing N2 and a culture medium containing B27.

3. The kit according to claim 2, wherein The N2-containing medium includes DMEM / F12 supplemented with N2 supplement, insulin, L-glutamine, non-essential amino acids (NEAA), β-mercaptoethanol, penicillin and streptomycin.

4. The kit according to any one of claims 2 to 3, wherein The B27-containing medium includes neurobasal medium supplemented with B27 supplement, L-glutamine, penicillin and streptomycin.

5. The kit according to any one of claims 2 to 4, wherein The cell culture medium is supplemented with TGFβ and BMP signaling inhibitors to produce a neural induction medium.

6. The kit according to any one of claims 2 to 5, wherein The cell culture medium has a medium gradient between the two cell types.

7. The kit according to any one of claims 1 to 6, wherein The co-culture microdevices are covered with Matrigel, or laminin, or poly-ornithine, or collagen to create a microenvironment appropriate for each cell type and prevent human cells from migrating to other compartments.

8. The kit according to any one of claims 1 to 7, wherein Peripheral sensory neurons (PSNs) were connected to co-cultured human epidermal keratinocytes (HEKs), mimicking the connections between free nerve endings and HEKs in human skin.

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