Living sensor display

Abstract

To provide a material for producing a living sensor display that displays biomarkers of early inflammation.SOLUTION: Provided is an epidermal stem cell comprising a fusion gene of an NF-κB family response element and a GFP gene in a functionally active form.SELECTED DRAWING: None

Description

FIELD OF THEINVENTION

[0001] The present invention relates to a living sensor display and its applications. [Background technology]

[0002] Maintaining people's daily health is always a pressing issue. For this reason, it is required that each person continuously grasps the information inside the body, quickly knows the signs of poor health, and takes appropriate measures. However, except for cases where there is a symptom such as fever, people are only aware of vague sensations such as fatigue and stomach pain regarding changes in their daily health, and even if there is an abnormality, they cannot know the quantitative condition until they see the results of their health check, and they are slow to respond. Sensing of verifiable markers and biomarkers such as blood glucose levels, which are necessary for health management and healthcare, requires invasive tests such as blood tests, so there is a problem that individuals cannot measure in real time. Attempts have been made to analyze urine and measure sweat, saliva, and tears using wearable devices, but there are many issues such as the composition of these bodily fluids only indirectly related to internal information, large individual and environmental differences, the difficulty of continuous measurement and wearing for a long time, and the need for frequent sensor replacement.

[0003] In recent years, in response to the growing health consciousness of consumers, research into monitoring physiological conditions using wearable sensors that utilize the latest technologies such as power saving and miniaturization / flexibility of electronic devices has been actively conducted. Wearable devices for measuring physical information are commercially available, and wristwatch-like devices (e.g., Apple Watch) that mainly measure physical quantities such as body movement, body temperature, pulse rate / pulse wave, and blood oxygen concentration are widely used, but they do not obtain biochemical information (the same can be said for smart clothing).

[0004] At the research level, sensing of biochemical biomarkers using wearable devices has been proposed as a flexible device that can be attached to the epidermis. In this case, it is possible to attach sensors to the skin to analyze sweat, saliva, and tears, place them in the mouth, or use contact lenses, but it is difficult to wear them continuously for long periods of time, and real-time measurement is difficult due to limitations in power supply. In addition, there are problems such as individual differences, delays, and environmental differences that must be taken into account in order to associate the components of sweat and saliva with internal information. There is also research on devices that detect infrared spectroscopy and photoacoustic signals to measure blood glucose levels, but there is a problem that a close connection between the skin and the detector is required.

[0005] One of the means for solving the above problems has been reported to involve, for example, modifying regenerative cells derived from a subject so that a target polynucleotide and an indicator polynucleotide are simultaneously expressed, and administering the modified regenerative cells to the subject (Patent Document 1). This report claims that by monitoring an indicator signal generated by the expression of the indicator polynucleotide on the skin of a subject to which modified regenerative cells have been administered, the biological state of the cells defined by the expression of the target polynucleotide can be determined. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2017 / 109292 Summary of the Invention

[0007] In this specification, we aimed to overcome the above challenges by creating a living sensor display that intervenes in the pathways in which skin epidermal stem cells and their differentiated cells react to various biomarkers, and emits signals that can be read externally (e.g., fluorescent color, pigment changes) in response to changes in internal body information.

[0008] In this specification, in order to create a living sensor display that displays biomarkers of early inflammation, the researchers introduced a fusion gene (NF-κB-RE-GFP) of the NF-kB responsible element, an early inflammatory pathway, and GFP (reporter protein) into epidermal stem cells, and found that the epidermis of a target organism was obtained based on these epidermal stem cells, and an indicator of a biomarker of early inflammation was displayed on this epidermis.

[0009] Thus, the present invention provides a living sensor display that displays biomarkers of early inflammation, and materials for its manufacture.

[0010] According to one embodiment of the present invention, there is provided an epidermal stem cell comprising a fusion gene of an NF-κB family response element and a GFP gene in a functional form.

[0011] According to another aspect of the present invention, there is provided artificial skin comprising the above-mentioned epidermal stem cells in a position where they can function as epidermal stem cells.

[0012] According to another aspect of the present invention, there is provided a kit for producing an artificial skin graft to be transplanted onto a subject's skin to display an indication of the amount of a biomarker of early inflammation, the kit comprising a construct comprising a fusion gene of an NF-κB family response element and a GFP gene, or a vector for introducing the construct into epidermal stem cells.

[0013] This allows the cellular response to changes in biomarkers in the body to be read out as optical signals from the sensor cells, realizing a new method for direct, non-invasive, highly sensitive, quantitative and continuous measurement of biological information.From the perspective of the measurement system, there are many advantages, such as no need for sensor maintenance and the possibility of optical measurement using wearable devices.

[0014] In particular, if epidermal stem cells are placed in the right place in the epidermis (for example, the basal layer, which is the lowest layer), the cells that divide in the basal layer will migrate vertically toward the body surface and differentiate. The epidermis is renewed when the cells reach the keratinized layer and fall off, while the parts differentiated from specific stem cells remain in place and do not invade the surrounding area. By directly modifying epidermal stem cells, cells will continuously differentiate from the stem cells, and the modified functions will be continuously maintained even after cell differentiation. Unlike conventional methods that use the application of a patch on the epidermis or the injection of a luminescent compound, this method eliminates the problems of patch replacement, power supply to the patch, and loss of compounds due to metabolism and diffusion. In addition, since the epidermis is very thin, with an average thickness of about 0.2 mm, it can be observed from outside the body by converting the signal into an optical signal (such as fluorescent color) linked to a response system to target molecules. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a graph showing the production of light signal in cells transfected with the NF-κB-RE-EGFP construct as a function of the intensity of TNF-α stimulation. [Diagram 2] FIG. 2 is a graph showing the production of light signal in response to high concentrations of TNF-α stimulation in cells transfected with the NF-κB-RE-EGFP construct. [Diagram 3] FIG. 3 is a graph showing the production of optical signal as a function of the intensity of TNF-α stimulation in HEK293 cells (Tg-HEK) (left) and NHEKs (Tg-NHEK) (right) transfected with the NF-κB-RE-EGFP construct. [Figure 4] Figure 4 is a graph showing the production of optical signals according to the intensity of TNF-α stimulation in NHEKs transfected with a NF-κB-RE-EGFP construct modified by RE×3-EGFP×2 (NF-κB-RE-EGFP(RE×3-EGFP×2)). [Diagram 5] FIG. 5 is a schematic diagram of a transplantation method in which only the epidermal tissue is peeled off from the three-dimensional artificial skin and grafted onto the dorsal skin from which the epidermal layer has been peeled off of an SHO mouse. [Figure 6]FIG. 6 is a schematic diagram of a transplantation method when a three-dimensional artificial skin including a dermis layer is transplanted using the chamber method. [Figure 7] Figure 7 is a schematic diagram of a transplantation method in which the diameter of the three-dimensional artificial skin is expanded from 1 cm to 2.3 cm to ensure a sufficient transplant area, and the thickness of the dermis layer is reduced to reduce contraction. Description of the Invention

[0016] In this specification, by using epidermal stem cells and differentiated cells as biosensors and signal converters, we have demonstrated the possibility of long-term, real-time monitoring of biological information using more advanced biosensors that have not previously been possible, including: 1) high sensitivity through bioamplification of the signal, 2) maintenance-free long-term monitoring by constantly maintaining a living monitoring site and no deterioration of the sensor material, and 3) no need for external energy.

[0017] In the living sensor display of the present invention, in the early inflammation pathway, a protein of the NF-κB family binds to its response element (NF-kB responsible element) on the expression control element of GFP (reporter protein), and this binding causes GFP to be expressed. This fusion gene (NF-κB-RE-GFP) of the NF-κB response element and GFP is present in epidermal stem cells, and when the epidermal stem cells expressing GFP differentiate, the fluorescence of GFP is eventually presented on the skin surface. Then, by measuring the intensity of the fluorescence on the skin surface from the outside, an index of the amount of biomarkers related to early inflammation can be obtained. According to this embodiment, by performing gene transfer after culture, gene transfer into skin stem cells present in the basal layer of the epidermal epithelial cell type in which skin stem cells exist was observed, and expression in lineage cells that differentiate from the skin stem cells was observed. It was demonstrated that by transplanting skin stem cells into which this gene was transferred, it was possible to detect biomarkers emitted by a living body that has experienced early inflammation, and a sufficient amount of fluorescence that can be observed on the skin was observed.

[0018] Therefore, according to the present invention, there are provided a gene construct used in the above-mentioned living sensor display, a vector containing this gene, epidermal stem cells containing this gene, artificial skin containing this epidermal stem cell, a kit for producing a graft of this artificial skin, and a method for displaying an indicator of the amount of a biomarker related to early inflammation on the surface of the skin.

[0019] The subject to which the living sensor display is applied may be any animal having skin, and the type is not particularly limited, but is preferably a mammal or avian animal, and more preferably a mammal (human or non-human animal).

[0020] The gene construct contains a fusion gene (NF-κB-RE-GFP) of a NF-κB responsible element, which is an early inflammatory pathway related to TNF-α and the like, and GFP (reporter protein). In this fusion gene, the NF-κB responsible element and the GFP gene are linked in a functional manner, i.e., so that the GFP gene is expressed due to binding to the NF-κB responsible element. In addition, the NF-κB responsible element and the GFP gene are preferably linked so that the GFP gene is located 3' from the NF-κB responsible element.

[0021] The NF-κB family response element (NF-κB-RE) is known in the art, and can be appropriately selected by a person skilled in the art. For example, the nucleotide sequence (SEQ ID NO: 2) shown in the following table can be used as the NF-κB-RE. In addition, the NF-κB-RE may contain some mutations as long as it functions as a response element. In summary, preferred examples of NF-κB-RE include the nucleotide sequence shown in SEQ ID NO: 2, or a nucleotide sequence that has 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more sequence homology to SEQ ID NO: 3, and can function as a response element of the NF-κB family. Here, the sequence homology of the nucleotide sequence can be specified by using an algorithm known in the art, such as BLASTN, with default settings.

[0022] GFP is known in the art, and can be appropriately selected by those skilled in the art. For use in living sensor display in skin, GFP that produces high-intensity fluorescence is preferred, and examples of such GFP include EGFP and vsfGFP-0.

[0023] As EGFP, for example, a protein having the amino acid sequence (SEQ ID NO: 3) shown in the following table can be used. EGFP may contain some mutations as long as it functions as GFP. In summary, preferred examples of EGFP include a protein having the amino acid sequence shown in SEQ ID NO: 3, or a protein having an amino acid sequence with 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more sequence homology to SEQ ID NO: 3, and capable of functioning as GFP. Here, the sequence homology of the amino acid sequence can be specified with default settings using an algorithm known in the art, such as BLASTP.

[0024] For example, a protein having the amino acid sequence (SEQ ID NO: 6) shown in the following table can be used as vsfGFP-0. Moreover, vsfGFP-0 may contain some mutations as long as it functions as GFP. In summary, preferred examples of vsfGFP-0 include a protein having the amino acid sequence shown in SEQ ID NO: 6, or a protein having an amino acid sequence with 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more sequence homology to SEQ ID NO: 6, and capable of functioning as GFP. Here, the sequence homology of the amino acid sequence can be specified with default settings using an algorithm known in the art, such as BLASTP.

[0025] The fusion gene may contain multiple copies of NF-κB family response sequences arranged in tandem in order to enhance the intensity of the fluorescence displayed on the skin surface. For the same purpose, the fusion gene may contain multiple copies of the GFP gene arranged in tandem. The specific copy number of these sequences or genes is not particularly limited, but may be, for example, 2 to 10 copies, preferably 2 to 5 copies, and more preferably 2 to 3 copies. For example, the fusion gene may contain 3 copies of the NF-κB family response sequence. The fusion gene may also contain 2 copies of the GFP gene. Furthermore, the fusion gene may contain 3 copies of the NF-κB family response sequence and 2 copies of the GFP gene.

[0026] The fusion gene may further contain a b-globin intron capable of promoting transcription efficiency, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) sequence capable of stabilizing mRNA, or a central polypurine tract (cPPT) sequence capable of increasing the efficiency of nuclear transport of the viral genome and recombination into the host chromosome.

[0027] The vector is a vector containing the fusion gene or the construct and is used to introduce the fusion gene or the construct into epidermal stem cells. Such a vector may be any vector capable of introducing the fusion gene into epidermal stem cells in a functional form, and the specific type of vector is not particularly limited, but for example, a viral vector such as a lentivirus can be preferably used.

[0028] The epidermal stem cells contain the fusion gene in a functional form. The epidermal stem cells before the introduction of the fusion gene may be any epidermal stem cells derived from an animal or may be an established cell line, but are preferably epidermal stem cells derived from the same animal as the subject of the living sensor display, that is, the subject of the epidermal stem cells containing the fusion gene. Furthermore, in order to minimize graft rejection when the epidermal stem cells are transplanted into a subject, it is preferable that the epidermal stem cells before the introduction of the fusion gene are obtained from the subject into which the epidermal stem cells containing the fusion gene are to be transplanted, that is, autologous cells. For example, when the subject is a human, it is preferable that the epidermal stem cells before the introduction of the fusion gene are obtained from an individual into which the epidermal stem cells containing the fusion gene are to be transplanted. Furthermore, the epidermal stem cells before the introduction of the fusion gene can be obtained from iPS cells, particularly iPS cells obtained from an individual into which the epidermal stem cells containing the fusion gene are to be transplanted.

[0029] The artificial skin contains the epidermal stem cells at a position where they can function as epidermal stem cells. The position where they can function as epidermal stem cells is not particularly limited, since it can be appropriately selected by a person skilled in the art depending on the type of animal to which the living sensor display is to be used, but for example, in normal skin, it is the position where epidermal stem cells are present among the layers forming the skin, typically the basal layer.

[0030] The kit for producing the artificial skin graft includes the construct or the vector containing the fusion gene. The production of the artificial skin graft using such a kit can be performed using epidermal stem cells into which the construct or the vector has been introduced. For example, the artificial skin graft can be produced by placing the epidermal stem cells into which the construct or the vector has been introduced in a position where they can function as epidermal stem cells in a method for producing a skin graft known in the art. The skin graft thus obtained is preferably transplanted so that the dermis layer is as thin as possible in the structure of the skin after the skin graft is transplanted, and it is particularly preferable that the skin graft is transplanted so that the thickness of the dermis layer is 1 mm or less.

[0031] The method for displaying an indicator of the amount of a biomarker related to early inflammation on the skin surface may be carried out so as to realize the above-mentioned mechanism of action of the living sensor display, and the specific steps are not particularly limited. For example, such a method includes (a) incorporating a fusion gene of an NF-κB family response element and a GFP gene into an epidermal stem cell in a functional form, (b) incorporating the obtained epidermal stem cell into the skin of a subject in a functional form as an epidermal stem cell, and (c) observing the amount of fluorescence caused by GFP from the skin of the subject. The measured amount of fluorescence can be used as an indicator of the amount of a biomarker related to early inflammation. In this method, it is preferable that the dermis layer is as thin as possible in the structure of the skin after the skin graft is incorporated, and it is particularly preferable that the thickness of the dermis layer is 1 mm or less. To make the dermis layer as thin as possible, for example, a site on the skin where the dermis layer is thin (for example, the thickness of the dermis layer is 1 mm or less) may be selected, and the epidermal stem cell may be incorporated therein. The incorporation of the epidermal stem cells can be carried out by a method known in the art, for example, by transplanting the artificial skin graft or by injecting the epidermal stem cells into the skin epidermis. In the method, if the indicator of the amount of biomarkers related to early inflammation indicates that inflammation is occurring in the body, it is possible to treat the inflammation at an early stage.

[0032] Furthermore, the present invention provides a construct comprising a fusion gene of an NF-κB family response element and a GFP gene for displaying an indicator of the amount of a biomarker of early inflammation on the skin of a subject.

[0033] Furthermore, the present invention provides epidermal stem cells that functionally contain a fusion gene of an NF-κB family response element and a GFP gene, for displaying an indicator of the amount of a biomarker of early inflammation on the skin of a subject.

[0034] Furthermore, the present invention provides the use of a construct comprising a fusion gene of an NF-κB family response element and a GFP gene for displaying an indicator of the amount of a biomarker of early inflammation on the skin of a subject.

[0035] Furthermore, the present invention provides the use of epidermal stem cells comprising a functional fusion gene of an NF-κB family response element and a GFP gene for displaying an indicator of the amount of a biomarker of early inflammation on the skin of a subject.

[0036] Furthermore, the present invention provides the use of a construct comprising a fusion gene of an NF-κB family response element and a GFP gene in the production of epidermal stem cells for displaying an indicator of the amount of a biomarker of early inflammation on the skin of a subject.

[0037] Furthermore, the present invention provides the use of epidermal stem cells comprising a functional fusion gene of an NF-κB family response element and a GFP gene in the production of a skin graft for displaying an indicator of the amount of a biomarker of early inflammation on the skin of a subject.

[0038] The following table summarizes the nucleotide and amino acid sequences used in the examples below. [Table 1] TIFF2025034835000002.tif238170TIFF2025034835000003.tif105170

[0039] The present invention has the following features and advantages: - By optically reading out the signal and transmitting it wirelessly, unconstrained, continuous measurement is possible. Not only healthcare, but also monitoring of drug response and metabolism is possible. In the future, it will be possible to continuously monitor signs of deterioration such as heatstroke in elderly people and COVID-19 infected people undergoing home treatment, and contribute to the realization of a medical system that can provide appropriate and prompt treatment by understanding the pathological condition through the network. The skin is the largest organ in the body and is directly connected to the biological information system through capillaries and nerves that run throughout the body. Since it covers the surface of the body, it has the advantage of being easily accessible from the outside. - Because epidermal basal layer stem cells and their differentiated epidermal cells proliferate and differentiate vertically, the genetically modified cells remain at the transplant site and there is no risk of them infiltrating other areas. -Because the signals emitted by cells are read optically, it is non-invasive, unlike methods that involve collecting liquid samples, and since it does not require chemical sensors, it is easier to wear than existing wearable devices (a device similar to a smartwatch can be envisioned). Also, unlike infrared spectroscopy and photoacoustic signal detection, it does not require close coupling between the skin and the detector.

[0040] Furthermore, the present invention is expected to have a wide range of applications other than healthcare. For example, by forming skin displays of early inflammation markers on chicken combs or livestock, it can be used for animal health management in poultry farms and livestock farms, especially for early detection of avian influenza and animal infectious diseases. The present invention is also effective for health management of pets that cannot complain about their illness. EXAMPLES

[0041] The present invention will be specifically described based on the following examples, but the present invention is not limited to these examples.

[0042] In this example, we established a system to genetically modify epidermal stem cells, sense target molecules, and detect the response as an optical signal, and demonstrated its functionality using a three-dimensional skin model and immunodeficient mice.

[0043] 1. Genetic modification of epidermal stem cells and proof of concept with artificial skin 1-1. Examination of biomarker candidates for living sensor display There are a wide variety of biomarkers related to health management and disease, and depending on the type, they can show a transient immediate reaction such as an inflammatory reaction, or a chronic reaction such as diabetes. Therefore, it is very important for this project to clarify which biomarkers are suitable or not suitable for detection by the living sensor display using genetically modified epidermal stem cells. For this reason, as a starting point, we decided to examine blood glucose concentration, intracellular calcium concentration, TNF-α (tumor necrosis factor-α), histamine, ultraviolet light, and oxidative stress, which are physiological reactions that occur systemically or locally and are immediate or chronic.

[0044] 1-2. Study of methods for converting biological reactions into optical signals There are several methods to convert bioreactions into optical signals: 1) using the Foerster resonance energy transfer (FRET) phenomenon, in which energy is transferred nonradiatively from an excited donor molecule to a nearby acceptor molecule; 2) using the conformational change of fluorescent proteins; 3) producing fluorescent proteins in response to the target bioreaction; and 4) producing enzymes in response to the target reaction and emitting light in the presence of a substrate. Methods 1) and 2) have a short reaction time of a few milliseconds from the occurrence of a bioreaction to the production of an optical signal, but the signal strength is weak. Method 3) takes a long time to produce an optical signal, but has the advantage that the optical signal continues to be produced while the bioreaction is ongoing, and high fluorescence intensity can be expected. Method 4) requires a substrate to produce an optical signal, and does not fit the purpose of this project. In order to determine the optimal method for converting biological reactions into optical signals for the purpose of this project, 1) Nano Lantern, a calcium sensor, 2) Green Glifon, a glucose sensor, and 3) a fusion gene of the NF-κB responsible element, an early inflammatory pathway including TNF-α, and EGFP (NF-κB-RE-EGFP) (sequence number 1) were transiently introduced into HEK293 cells, a human embryonic kidney-derived cell line, and the responsiveness and optical signal production were examined.

[0045] 24 hours after the start of cell culture, each gene construct was transiently introduced into HEK293 cells by lipofection, a gene introduction method that utilizes lipid membrane fusion. After another 24 hours, glucose, ionomycin, which increases intracellular calcium concentration, or TNF-α were added to the medium, and the change in optical signal intensity was measured. As a result, no significant increase in optical signal in response to stimulation was observed in cells introduced with the Green Glifon construct. In addition, in cells introduced with the Nano Lantern construct, no increase in optical signal intensity was observed in analysis by fluorescence microscopy, but a transient increase in intensity was observed immediately after stimulation when measured with a plate reader. On the other hand, in cells introduced with the NF-κB-RE-EGFP construct, optical signals were produced in response to the intensity of the stimulus, and it was revealed that strong optical signal intensity could be maintained for at least 54 hours as long as stimulation was continued (Figure 1). Furthermore, when stimulated with high concentrations of TNF-α, an increase in optical signal intensity equivalent to that observed with a short stimulation period of 1 hour was observed as compared to a 30-hour stimulation period, indicating that this construct is capable of producing a relatively strong optical signal in response to both immediate inflammatory responses caused by abrasions, etc., and chronic increases in TNF-α caused by cancer, etc. (Figure 2).

[0046] From these results, it is believed that the optimal method for converting biological reactions into optical signals in this project is to produce fluorescent proteins according to the target biological reaction. Among the biomarker candidates listed in 1-1 above, the biological reactions that can be converted into optical signals using method 3) are TNF-α and oxidative stress pathways, so from this point on, the project focused on these biological reactions.

[0047] 1-3. Functional evaluation of permanently genetically modified cells To use epidermal stem cells as living sensor displays, permanent genetic modification is required. Therefore, we used lentivirus-based genome editing to introduce an NF-κB-RE-EGFP construct into HEK293 cells and normal human dermal keratinocytes (NHEKs), where RE is a response element (a region in the regulatory region of a gene that is involved in the binding of transcription factors).

[0048] In genetically modified HEK293 cells (Tg-HEK), an increase in optical signal intensity was observed depending on the TNF-α concentration, but in genetically modified NHEK cells (Tg-NHEK), no clear increase in optical signal intensity was observed (Figure 3). The reason for this was thought to be that the activity of the signal transduction pathway (phosphorylation of P65 and transcription) after TNF-α stimulation enters the cells was weaker in NHEK cells compared to HEK293 cells, so the fluorescent protein was made brighter. In general, it is known that the intron of b-globin promotes transcription efficiency, and vsfGFP-0 has high brightness among fluorescent proteins. When the NF-κB-RE-bg-vsfGFP-0 construct (sequence number 4) was created, the fluorescent intensity increased 1.2-fold compared to NF-κB-RE-EGFP. Furthermore, by tandeming this fluorescent protein, we succeeded in creating a high-brightness fluorescent protein construct (RE×3-EGFP×2) (SEQ ID NO: 7) with a fluorescence intensity 2.9 times higher than before tandemization and a dynamic range 3.4 times higher than unstimulated. The RE×3-EGFP×2 construct-introduced genetically modified NHEK showed an increase in fluorescence intensity dependent on the TNF-α concentration, and no cell death was observed due to the introduction, completing the creation of a construct that can verify living sensor display (Figure 4).

[0049] 1-4. Evaluation of a three-dimensional artificial skin model using genetically modified epidermal stem cells To verify the feasibility of the living sensor display, we performed verification using a three-dimensional artificial skin model. To evaluate the effect of gene transfer on cells, we constructed a three-dimensional artificial skin model using Tg-NHEK cells transfected with an always fluorescent construct. As with artificial skin using cells without gene transfer, it was shown that a normal epidermal structure with a four-layer structure was formed. In addition, TNF-α stimulation slightly enhanced the expression of fluorescent proteins. This result also confirmed the introduction of the NF-κB-RE-GFP fusion gene into epidermal stem cells. Furthermore, verification was performed using a four-color always fluorescent construct as the POC of the living sensor display that expresses different fluorescent proteins in response to multiple stimuli, and it was revealed that it was possible to distinguish at the single cell level within the three-dimensional artificial skin. These results demonstrated that a living sensor display is possible by combining high-brightness and multiple fluorescent proteins with stimuli.

[0050] 2. In vivo proof of concept 2-1. Establishment of a method for transplanting genetically modified epidermal stem cells To verify the living sensor display in an in vivo environment, we investigated the method of transplantation into immunodeficient SHO mice. Because Tg-NHEK is important, we peeled off only the epidermal tissue from the three-dimensional artificial skin and transplanted it onto the dorsal skin of an SHO mouse from which the epidermal layer had been peeled off (Figure 5). Histological analysis of the skin on the 35th day after transplantation showed that the epidermal layer structure had been formed in the transplanted area, but no expression of human cell markers was observed. Therefore, we performed a three-dimensional artificial skin transplant including the dermal layer using the chamber method (Figure 6), and it was observed that the transplanted tissue had taken root and remained on the 22nd day after transplantation. Histological analysis showed the expression of human cell markers at the transplant site, and furthermore, a papilla-like structure characteristic of human skin had been formed, so we determined that the transplantation was successful. However, in preliminary experiments, a certain area or more was required to detect the optical signal, so we further optimized the transplantation method. To ensure a sufficient graft area, the diameter of the 3D artificial skin was expanded from 1 cm to 2.3 cm, and the thickness of the dermis layer was thinned (to less than 1 mm) to reduce contraction (Figure 7). As a result of these results, a relatively large area of ​​grafted skin was maintained. Based on these results, a grafting method for engrafting 3D artificial skin using Tg-NHEK cells was established.

[0051] 2-2. Evaluation of engraftment: Inflammation analysis at the transplant site To monitor inflammation throughout the body, it is necessary to start monitoring after the inflammatory response caused by transplantation has settled down. Therefore, we investigated how many days after transplantation it takes for the inflammatory response caused by transplantation to settle down. Using mice transplanted using the chamber method, the subcutaneous tissue around the transplant site was excised 7, 14, 28, and 63 days after transplantation, and the presence or absence of inflammatory responses and accumulation of immune cells was observed over time by immunostaining. As inflammatory markers, F4 / 80, a marker for mouse mature macrophage tissue, CD86, a marker for inflammatory macrophages, CD163, a marker for anti-inflammatory / tissue repair monocytes and macrophages, and Ly-6G, a marker for peripheral neutrophils, were used for observation.

[0052] On the 7th day after transplantation, Ly-6G was expressed in the skin at the transplant site, and migration and accumulation of neutrophils was observed, confirming the biological response to the transplantation. On the 14th day after transplantation, accumulation of neutrophils was observed in some areas, but it was found that the overall inflammatory response had subsided. On the 28th and 63rd days after transplantation, accumulation of immune cells such as inflammatory macrophages and neutrophils was not observed at the transplant site. In addition, NHEKs expressing mPlum on the 7th and 14th days after transplantation, EGFP on the 28th day after transplantation, and mTagBFP2 on the 63rd day after transplantation were found to express NHEKs. It was found that there was little difference in engraftment and inflammatory tendency even if the cells expressing different fluorescent proteins or the number of cells transplanted were different. From the above, it was revealed that in transplantation using the chamber method, the influence of local inflammatory reactions due to transplantation disappears after 28 days or more after transplantation, and that the artificial skin can be used for inflammation monitoring.

Claims

1. An epidermal stem cell comprising a fusion gene of an NF-κB family response element and a GFP gene in a functional form.

2. The epidermal stem cell according to claim 1, wherein the fusion gene comprises multiple copies of tandemly arranged NF-κB family response elements.

3. The epidermal stem cell according to claim 1, wherein the fusion gene comprises three tandem copies of an NF-κB family response element.

4. The epidermal stem cell of claim 1 , wherein the fusion gene comprises multiple copies of the GFP gene arranged in tandem.

5. The epidermal stem cell of claim 1 , wherein the fusion gene comprises two copies of a tandemly arranged GFP gene.

6. The epidermal stem cell according to claim 1, wherein the fusion gene comprises three copies of an NF-κB family response element arranged in tandem and, on the 3' side thereof, two copies of a GFP gene arranged in tandem.

7. An artificial skin comprising the epidermal stem cell according to any one of claims 1 to 6 in a position where it can function as an epidermal stem cell.

8. The artificial skin described in claim 7, wherein the location capable of functioning as an epidermal stem cell is the stratum basale.

9. A kit for producing an artificial skin graft to be transplanted onto a subject's skin to display an indicator of the amount of a biomarker of early inflammation, the kit comprising a construct containing a fusion gene between an NF-κB family response element and a GFP gene, or a vector for introducing the construct into epidermal stem cells.

10. The kit according to claim 9, wherein the fusion gene comprises multiple copies of tandemly arranged NF-κB family response elements.

11. The kit according to claim 9, wherein the fusion gene comprises three copies of tandemly arranged NF-κB family response elements.

12. The kit of claim 9 , wherein the fusion gene comprises multiple copies of the GFP gene arranged in tandem.

13. The kit of claim 9 , wherein the fusion gene comprises two copies of the GFP gene arranged in tandem.

14. The kit according to claim 9, wherein the fusion gene comprises three copies of an NF-κB family response element arranged in tandem and, on the 3' side thereof, two copies of a GFP gene arranged in tandem.

15. The kit of claim 9 , wherein the vector is a viral vector.

16. The kit according to claim 9 , wherein the artificial skin graft is produced using epidermal stem cells into which the construct or the vector has been introduced.

17. The kit according to claim 16 , wherein the epidermal stem cells before the introduction of the construct or vector are obtained from the subject.

18. The kit according to claim 9, wherein in the structure of the skin after the skin graft is transplanted, the thickness of the dermis layer is 1 mm or less.

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