Living sensor display implanted on skin for long-term biomarker monitoring

Domain: biology
Published: 2026-01-12
Editorial outcome: major_revision

Abstract
This study introduces an approach to biomarker monitoring through the development of a “Living Sensor Display” implanted on the skin, designed for long-term, continuous tracking of biomarkers, such as inflammatory cytokines. By leveraging the natural high selectivity and sensitivity of keratinocyte stem cells (KSCs) to environmental stimuli, we genetically engineered these cells to express fluorescent proteins in response to specific biomarker signals, specifically TNF-α signalling. This innovation allows for the real-time, non-invasive observation, with the only invasive step being the initial implantation, of biomarker levels directly from the skin surface. We constructed tissue-engineered skin with genetically modified KSCs and transplanted it onto mice, where it successfully engrafted long-term, matured to mimic human skin structures, and responsively indicated the presence of TNF-α through visible fluorescence. This living sensor could repeatedly detect inflammatory responses induced by TNF-α and lipopolysaccharides, highlighting its potential for monitoring various physiological and pathological processes over extended periods. This Living sensor display holds promise for advancing health monitoring and disease prevention strategies by enabling continuous, sensitive, and specific in vivo biomarker tracking.

Introduction
Measuring biomarkers, such as blood glucose levels and inflammatory cytokines is essential for identifying the chemical, physical, or biological processes within the body1,2. Such information plays a crucial role in the early detection and prevention of serious diseases and in the management of lifestyle-related diseases, providing insights into personal health status and disease progression. Therefore, the continuous monitoring of these biomarkers is highly important, leading to the development of various detection techniques. Conventionally, biomarker detection has primarily been achieved through intermittent blood sampling, which allows for accurate measurements. However, it is not suitable for long-term continuous monitoring owing to its invasive nature3. Recently, the development of wearable devices has intensified, enabling the long-term, continuous monitoring of health-related biomarkers using sweat, saliva, tears, and interstitial fluid4–8. However, no method is able to monitor biomarkers within the body with high selectivity and sensitivity over long durations.

The cells in living organisms sense environmental stimuli, such as biomarkers and chemicals, with a high sensitivity and selectivity relative to artificial sensors9. Subsequently, the cells respond appropriately, such as expressing proper genes, to these stimuli and maintain the homoeostasis of the organism. The human body is composed of approximately 60 trillion terminally differentiated cells of approximately 200 types, each of which has a unique lifespan. The adult stem cells residing in each tissue can self-renew, differentiate into various cell types, and serve as a source of lost cells10. The properties of stem cells and progeny cells are observed to remain constant. Mutations, a modification of the genome that occurs in stem cells, are maintained after self-renewal and passed on to their progenies through individual lifespans, resulting in changes in their properties. Thus, living cells have the advantage of being highly sensitive, selective, and maintenance-free when compared with mechanical parts.

The skin, the organ with the large area of the body, acts as a barrier against harmful stimuli, such as UVA and dehydration, and as a sensor for multiple biomarkers in vivo. The skin is composed of the epidermis, dermis, and skin appendages. The epidermal structure is maintained by the skin keratinocyte stem cells (KSCs) sparsely present in the basal layer of epidermis, which is only 100 µm away from the skin surface11,12. During the differentiation of keratinocytes, the KSCs divide vertically; the progeny cells push themselves upwards linearly as they proliferate and differentiate, and finally, the progeny die and fall off from the skin surface in approximately 10 and 50 days in mice and humans, respectively13,14. Therefore, when viewed from the body surface, differentiated cells derived from a single stem cell have a columnar structure. Because of the position of the KSCs and their columnar differentiation, changes in the KSCs and their progenies can be easily detected from the surface of the body, compared to that in other adult stem cells.

This study proposes a biomarker monitoring method that utilises living KSCs as sensors and displays simultaneously. They involve genetically modified living cells that can detect signals through membrane proteins and produce fluorescent proteins via cellular signal transduction. This approach allows for the targeting of specific biomarkers with high selectivity owing to the ability of membrane proteins to highly and strictly recognise the substances. The signal is subsequently amplified through multiple stages of transcription and translation within the cell, enabling the sensitive detection of these substances. By creating tissue-engineered skin from KSCs and transplanting this skin, it is possible to monitor changes in the target substances within the body. Furthermore, by transplanting the skin for integration into the body, continuous monitoring of biomarkers can be achieved over the long term, as the cells within the transplanted skin undergo natural turnover by engineered KSCs15. This sensor, capable of displaying internal information externally using living cells, has been named the “Living Sensor Display” (Fig. 1a). Here, we focus on the inflammatory cytokine tumour necrosis factor-α (TNF-α) as the target biomarker. To detect TNF-α, we first genetically engineered human epidermal keratinocytes (NHEK), including KSCs to express NF-κB-EGFP, which is activated by TNF-α stimulation16,17. Next, we used these cells to construct a tissue-engineered skin model and verify its concentration-dependent responsiveness to TNF-α stimulation. Furthermore, we transplanted the skin model into mice and observed changes in the fluorescence intensity of the transplanted skin after the administration of an inflammatory agent. Using these methods, we demonstrated that the Living Sensor Display is an effective technique for measuring the biomarkers within the body.

Results
Evaluation of NHEK cells responding to inflammatory cytokines
The nuclear factor-κB (NF-κB) pathway is involved in multiple biological processes, including innate immunity, adaptive immunity, inflammation, stress response, B cell development, and lymphoid tissue formation18. In this pathway, several ligands, including the inflammatory cytokine TNF-α, bind to its receptors resulting in the activation and nuclear transfer of NF-κB, a protein complex (Fig. 1b). Subsequently, NF-κB recognises specific nucleotide sequences, called the responsible element (RE) to transcribe downstream target genes.

To generate the Living Sensor Display, we aimed to generate genetically engineered KSCs that have transgene fused with RE and enhanced green fluorescent protein (EGFP) as a readout signal (NF-κB-EGFP NHEK cells). In general, the greater the number of RE and fluorescent protein genes, the higher the fluorescence intensity. Therefore, we investigated the numbers of RE and EGFP genes. To this end, we generated several vector constructs containing different numbers of RE and EGFP genes and transfected them into NHEKs using a lentiviral system. Next, we stimulated these transgenic cells with 20 ng/mL TNF-α for 16 h and analysed the fluorescence intensity by FACS, and found that the construct composed of three repeats of RE and two copies of EGFP gene expressed the highest fluorescence intensity (Figs. 1c and S1a). Therefore, this construct was used in this study.

To assess the expression of EGFP in response to the inflammatory cytokine TNF-α in NHEKs genetically modified with NF-κB-EGFP, we added TNF-α to the culture medium at concentrations of 0 ng/mL, 0.2 ng/mL, and 20 ng/mL, and examined the temporal changes. Bright-field images before the addition of TNF-α and fluorescence images of EGFP before addition and 12 and 24 h after addition are shown in Figs. 1d and S1b. At 0 ng/mL, almost no increase in fluorescence was observed after 12 h or 24 h. Conversely, in cells treated with TNF-α, an increase in the fluorescence intensity from EGFP was observed after 12 h, and further increased after 24 h when compared to that at 12 h. Additionally, the fluorescence was stronger and EGFP was more prominently expressed at both 12 and 24 h at 20 ng/mL compared to that observed at 0.2 ng/mL. The fluorescence intensities measured by a plate reader every hour for 48 h at TNF-α concentrations of 0 ng/mL, 0.2 ng/mL, and 20 ng/mL are shown in Fig. 1e, respectively. Similar to the fluorescence images, the fluorescence intensity increased significantly from 0 ng/mL to 20 ng/mL and from 0.2 ng/mL to 20 ng/mL at 24 h after TNF-α stimulation (Fig. S1b, c), with a steeper increase in the intensity at higher concentrations, indicating that genetically modified NF-κB-EGFP NHEK cells respond to the inflammatory cytokine TNF-α by producing EGFP, leading to an increase in the fluorescence intensity proportional to the TNF-α concentration, but detection below 0.02 ng/ml falls outside the dynamic range (Fig. S1d).

To investigate the cross-selectivity of NHEKs genetically modified with NF-κB-EGFP, we examined the response not only to TNF-α but also to IL-1β, which is involved in the NF-κB signalling pathway, and IL-2 and LPS, which are not directly involved in the NF-κB signalling pathway and lack membrane receptors. These factors were added to the culture medium at a concentration of 20 ng/mL, and changes in fluorescence intensity were observed (Fig. S2a–d). As a result, an increase in fluorescence intensity similar to that seen with TNF-α was confirmed for IL-1β. However, no increase in fluorescence intensity was observed for IL-2 or LPS. These results demonstrate that NHEKs genetically modified with NF-κB-EGFP exhibit high selectivity, responding specifically to target molecules associated with receptors involved in the NF-κB signalling pathway, while showing no cross-reactivity to IL-2 or LPS.

To verify whether NHEKs genetically modified with NF‑κB‑EGFP respond in a human immune environment, we performed co‑culture experiments using a culture insert system with NHEK cells and human‑derived Peripheral Blood Mononuclear Cells (PBMCs). A culture inserts containing 1 × 10⁶ PBMCs was placed over the NHEK cell layer. In another condition, a culture insert containing 1 × 10⁶ PBMCs activated by Lipopolysaccharide (LPS) (final concentration: 10 µg/mL in the well) was used. Changes in fluorescence intensity were then monitored (Fig. S3a, b). As a result, fluorescence intensity remained unchanged with PBMCs alone; however, in the LPS-stimulated condition, it increased after 60 h. These results suggest that NHEKs exhibit fluorescence responses in the presence of PBMCs, particularly under inflammatory conditions induced by LPS stimulation. Although this system does not fully represent a complete human immune system, it is considered to mimic the early stages of acute inflammatory responses, implying that in practical applications, fluorescence responses to cytokines can be observed in humans.

Evaluation of tissue-engineered skin responsive to TNF-α stimulation
To verify whether NF-κB-EGFP NHEK cells have the ability to construct proper skin structure and if so whether tissue-engineered skin is responsive to TNF-α concentrations, we generated tissue-engineered skin by culturing genetically modified NHEK cells and normal human dermal fibroblasts on a culture insert at an air–liquid interface (Fig. 2a). The tissue-engineered skin was circular with a diameter of approximately 20–25 mm. The cross-sectional haematoxylin and eosin (HE) images of the entire tissue-engineered skin are shown in Fig. 2b. The thickness of the tissue-engineered skin was approximately 100 µm to the centre, which thinner compared to actual human skin. Enlarged HE and immunostained fluorescence images of the tissue-engineered skin sections from the marked area are shown in Fig. 2c. The epidermal basal layer marker cytokeratin 14 (CK14)19 was observed on the epidermal part of the tissue-engineered skin, Vimentin20, a fibroblast marker, was found from the bottom to the centre of the tissue-engineered skin, and Type IV collagen, a marker for the basal layer21, was observed between CK14 positive cells and Vimentin-positive cells, indicating the formation of a layer of cells similar to that seen in natural skin across the entire thickness of the tissue-engineered skin. These results suggest that tissue-engineered skin can be constructed using genetically modified NHEK cells. Further, to check whether the TNF-α responsiveness observed in the NHEK was retained after forming tissue-engineered skin, TNF-α was added to the culture medium of the tissue-engineered skin, and the fluorescence response observed in 2D cell culture was also seen after forming 3D tissue-engineered skin. The images of the immunostained sections of tissue-engineered skin fixed at 24 h after the addition of TNF-α at concentrations of 0 and 20 ng/mL are shown in Fig. 2d, e, respectively. Although the difference was small, the EGFP fluorescence in the epidermal layer was stronger at a concentration of 20 ng/mL. Additionally, the fluorescence intensity measured by a plate reader every hour for 48 h at TNF-α concentrations of 0 ng/mL and 20 ng/mL are shown in Fig. 2f. The fluorescence intensity increased significantly from 0 ng/mL to 20 ng/mL at 17 h, peaking at approximately 17 h before gradually decreasing (Fig. S4a). The fluorescence intensity changes with various TNF‑α concentrations were observed in both cultured cells and tissue-engineered skin, indicating that both systems exhibit a dynamic range from 0 to 20 ng/mL (Figs. S1d, S4b). These results suggest that the tissue-engineered skin retained the TNF-α responsiveness observed in cells, indicating successful construction of tissue-engineered skin responsive to TNF-α stimulation.

Transplantation of TNF-α Responsive Tissue-engineered Skin onto Mice
Next, to confirm whether the TNF-α responsive tissue-engineered skin could stably engraft onto mice, we transplanted the tissue-engineered skin onto SCID Hairless Outbred (SHO) mice (Fig. 3a). Wound healing was observed (Fig. S5a–d). HE-stained images of the skin sections removed from around the transplantation site after four weeks are shown in Fig. 3b, with parts 1 and 2 indicating the remains of the transplant scar, and part 3 indicating the original skin of the mouse. Enlarged HE and immunostained images of each part of the removed skin are shown in Fig. 3c. Since the tissue-engineered skin used human-derived NHEK and fibroblasts, immunostaining for human-specific mitochondria in red revealed its expression in the epidermal part of location i, but not in location ii, indicating that the transplanted NHEK-derived tissue-engineered skin was engrafted in part i, whereas part ii healed with mouse tissue. No adipose tissue was observed beneath the transplanted scar; additionally, it was confirmed that the skin was engrafted on top of the fascia. EGFP expression was also confirmed in areas where human mitochondria were observed, which may have been due to an immune response after transplantation. Notably, a structure similar to the human epidermal papillary layer, which was not observed in mouse skin, was observed at the transplanted site. The TNF-α responsive tissue-engineered skin engrafted onto SHO mice was observed to differentiate into more mature human-like skin.

Furthermore, to investigate the inflammatory response state at three and four weeks post-transplantation, immunostaining for the neutrophil marker Ly6g, pan-macrophage marker F4/80, M1 macrophage marker CD86, and M2 macrophage marker CD163 was performed. The infiltration of inflammatory cell groups was observed to have subsided at 3 weeks after transplantation (Fig. S6) compared to 4 weeks after transplantation (Fig. S7), indicating that at least 4 weeks should be waited before fluorescence observation can be performed.

These findings confirm that, while EGFP cells remain at the transplantation site, the inflammatory response caused by transplantation subsides within approximately four weeks, suggesting that fluorescence observation should not be initiated until at least four weeks have passed. Additionally, to observe the size change of the transplanted tissue-engineered skin post-transplantation, EGFP autofluorescence was photographed over time, and the area of observed autofluorescence was calculated through image processing (Fig. S8). The observation system was constructed using a commercially available camera and a parallel light source to keep the optical path length constant, and bright-field and fluorescent images were captured (Fig. S9). The size of the transplantation site tended to decrease up to 30 days post-transplantation; however, there was hardly any change in size over 200 days thereafter, suggesting that once the tissue-engineered skin was engrafted onto the mouse, it maintained a constant size and location. These results suggest that transplanted tissue-engineered skin can be quantitatively assessed for changes in the fluorescence intensity normalised to the area of the transplant site.

TNF-α responsiveness of the transplanted tissue-engineered skin
To verify whether the engrafted tissue-engineered skin in mice could still respond to TNF-α and produce EGFP, a TNF-α solution was administered, and the change in the fluorescence intensity was examined. Bright-field and fluorescence images before administration in mice at more than two months post-transplantation are shown in Fig. 4a, b, respectively, confirming the near disappearance of EGFP fluorescence (Fig. S10). Next, 20 µL of 100 µg/mL TNF-α solution was subcutaneously injected near the transplant site. Day 0 was considered the first day of administration. On day 1 post-administration, an increase in the fluorescence intensity was observed (Fig. 4c), peaking on day 1 or 2 (Fig. 4f). Subsequently, the fluorescence intensity gradually decreased. Once the fluorescence was sufficiently reduced (Fig. 4d, g), 20 µL of TNF-α solution was again subcutaneously injected near the transplant site on day 28. One day after the second administration, on day 29, the fluorescence intensity increased again (Fig. 4e) and the fluorescence intensity was significantly higher than that before 2nd administration (Fig. 4i). The engrafted tissue-engineered skin similarly responded to TNF-α by producing EGFP, showing that the tissue-engineered skin can be repeatedly detected (Fig. 4h). Subsequently, to check the state of the mouse skin one day after TNF-α administration, an observation of the tissue section was conducted. An overall image of the immunostained skin tissue of the mice post-transplantation is shown in Fig. 4j. Strong green EGFP fluorescence was observed only in the transplanted part of the tissue-engineered skin with red-stained human mitochondria-positive papillary structures; neither human mitochondria nor EGFP were observed outside the transplant site (Fig. 4k). Additionally, an investigation of the inflammatory response state through immunostaining (Fig. S11) revealed numerous neutrophils, mature macrophages, M1 macrophages, and M2 macrophages at the transplant site and in normal tissue parts, indicating that the subcutaneous administration of TNF-α elicited an inflammatory response around the transplanted tissue-engineered skin. These results confirm that the engrafted tissue-engineered skin alone responded to TNF-α stimulation.

Detection of inflammatory responses in vivo
To examine whether tissue-engineered skin can monitor the inflammatory situation in addition to the presence of TNF-α, substances capable of inducing inflammation in the body were administered to transplanted mice, and changes in the fluorescence intensity were observed. LPS is the major component of the outer membrane of Gram-negative bacteria and is known to induce inflammatory responses, including the activation of macrophages22. In the 2D culture of NF-κB-EGFP NHEK cells, no increase in fluorescence was observed after 48 h post-stimulation by 20 ng/mL LPS, suggesting that LPS itself does not activate NF-κB signalling in these cells (Fig. S2d). To examine whether NF-κB-EGFP NHEK cell can monitor the inflammatory situation in vivo, 20 µL of LPS solution at a concentration of 1 mg/mL was administered intraperitoneally to the tissue-engineered skin-grafted mice (Fig. 5a–e). The response to LPS was slower compared to that of TNF-α, with 2 mice showing an increase in fluorescence intensity on day 2, while 3 mice showed a decrease (Fig. 5f). This could have occurred due to subsiding of the inflammatory reaction caused by the transplant. Next, to examine whether the tissue-engineered skin can repeatedly respond to LPS administration, we again subcutaneously injected 20 µL of 1 mg/mL LPS solution near the transplant site on day 7 after the first administration. After the second administration, a significant increase in the fluorescence intensity was observed in all individuals on day 2 (Fig. 5g, h). Furthermore, LPS solution (0.1 mg/mL) was administered via the tail vein at a dose of 0.1 mg/kg on Day 0 and Day 7. As a result, fluorescence intensity increased (Fig. S2a–e) from the day following administration, with a significant increase on the first day post-administration compared to baseline (Fig. S12f). By the seventh day post-administration, fluorescence had subsided; however, upon re-administration, fluorescence increased again 1 day later. On Day 8, fluorescence intensity was significantly higher than before the Day 7 administration (Fig. S2g). These results indicate that tissue-engineered skin can be used to monitor the inflammatory state of the body and increase the fluorescence intensity in response to inflammatory stimulation. To check the state of mouse skin on day 9 after the first LPS administration and 2 days after the second LPS administration, the tissue sections were observed. An image of an immunostained skin tissue section of a mouse is shown in Fig. 5i. Similar to TNF-α administration, a strong fluorescence of green EGFP overlapping with red-stained human mitochondria-positive papillary structures was observed only in the transplanted part of the tissue-engineered skin. These results indicate that the engrafted tissue-engineered skin responds to LPS-induced inflammation by producing EGFP. Additionally, an investigation of the inflammatory response state on day 9 through immunostaining (Figs. 5j and S13) showed that numerous neutrophils and mature macrophages were observed at the transplant site and in normal tissue parts, while fewer M1 and M2 macrophages were observed compared to during TNF-α administration, as consistent with the delayed and low fluorescence intensity after LPS administration compared to TNF-α injection. These results indicate that the NF-κB-EGFP NHEK cells in tissue-engineered skin can indirectly monitor the inflammatory situation via immune cells, such as macrophages.

Methods
Lentivirus vector construction
A sequence containing NF-κB-RE-miniP-EGFP was generated by PCR amplification from pNL3.2 NF-κB -RE plasmid (Promega, USA) and pEGFP-N1 plasmid (Addgene, USA). NF-κB -RE-miniP-EGFP sequence was amplified by PCR from pNL3.2 NF-κB EGFP plasmid (Promega, USA) and was inserted in the ClaI-NotI site of the pLVSIN-CMV-neo vector (Takara, Japan). The RE sequence between the ClaI and SfiI sites of this construct was replaced with a sequence that was repeated RE sequence three times (pNL3.2 NF-κB-REx3). Simultaneously, the EGFP sequence following IRES2 sequence was inserted after EGFP sequence of NF-κB -RE-miniP-EGFP plasmid (pNL3.2 NF-κB -EGFx2). Subsequently, two repeats of EGFP were extracted from pNL3.2 NF-κB -EGFx2 and inserted into pNL3.2 NF-κB -REx3 resulting in the plasmid containing three repeats of RE and two repeats of EGFP (pNL3.2 NF-κB -REx3EGFPx2).

Flow cytometry data were analyzed using FlowJo software (v10.10.0, FlowJo LLC). After doublet exclusion, SYTOX Blue-negative cells were defined as live cells. GFP fluorescence was quantified as median fluorescence intensity (MFI). For visual comparison across conditions, fluorescence histograms were normalised to mode, with the highest frequency set to 100% on the y-axis.

Cell culture and transfection
Normal human epidermal keratinocytes (NHEKs: KK-4009) were purchased from KURABO Industries LTD (Osaka, Japan). The NHEKs were maintained in HuMedia-KG2 (KURABO) at 37°C. Normal human dermal fibroblasts (NHDFs: KF-4109) were purchased from KURABO and were maintained in Dulbecco’s modified Eagle’s medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, JAPAN) that was supplemented with 10% foetal bovine serum (Biowest, Rue du Vieux Bour, Nuaillé, France), 100 units mL−1 penicillin, 100 µg mL−1 streptomycin (Thermo Fisher Scientific, MA, USA), and 10 ng ml−1 bFGF (PeproTech, NJ, USA) at 37°C. The virus solution was produced by co-transfection of the lentivirus vector and lentiviral high-titre packaging mix into Lenti-X 293T cells using TransIT-293 Transfection Reagent (TAKARA BIO Inc., Japan). Transfection was performed by incubating the NHEK cells in a 10 IFU/cell virus solution for 16 h.

In vitro fluorescence measurement
Normal Human Epidermal Keratinocytes (NHEKs) (KURABO, Japan) were first suspended in HuMedia (KURABO, Japan) at a concentration of 2.4 × 10⁵ cells/mL. The cell suspension was then aliquoted into a 12-well plate and 1 mL of the suspension was added to each well. The plate was incubated overnight at 37°C in an environment containing 5% CO2 and 20% O2, providing optimal conditions for cell growth and viability. Following to 16 h of incubation, the original medium in each well was replaced with 900 µL of fresh HuMedia supplemented with Polybrene at a final concentration of 8.9 µg/mL. After 24 h after the start of the culture, cells from the three wells were harvested to measure the cell counts and assess the proliferation and condition of the cells before proceeding with viral infection. For the infection process, the virus is diluted with HuMedia in the viral solution to a total volume of 50 µL, achieving a concentration of 10 IFU/cell. The diluted viral solution was added to each well. After 16 h of viral exposure, the medium containing Polybrene was discarded, and each well was replenished with 900 µL of Polybrene-free HuMedia. A day after replacing the medium, TNF-α was added at a concentration ten times higher than the final desired concentration, with 100 µL added to each well. The response of NHEK to the treatments was monitored using an imaging reader (BioTek Citation 5). The cells were observed using bright field microscopy, fluorescence microscopy, and fluorescence intensity measurements every hour under the conditions of 37°C, 5% CO2, and 20% O2.

Reconstruction of tissue-engineered skin
A total of 2 × 10⁶ normal human dermal fibroblasts (NHDFs) (2 × 10⁶) were resuspended in 0.6 mL of 4 mg/mL bovine dermis derived native collagen solution (KOKEN CO. Ltd., Japan) mixed with 1× DMEM (Thermo Fisher Scientific, USA), 10 mM HEPES (Dojindo, Japan), 10 mM NaHCO3 (FUJIFILM Wako Pure Chemical Corporation). The cell suspensions were plated in 6-well cell culture inserts (Corning, USA). After solidification of the collagen solution, transgenic NHEKs were seeded at a density of 2.0 × 10⁶ cells on dermal equivalents. The skin equivalents were cultured under submersion conditions for 4 days in a humidified atmosphere at 37°C with 12.5% O2 and then exposed to the air–liquid interface in a humidified atmosphere. On day 6 of the culture, the percentage of O2 increased to 20%. The medium for culturing skin equivalents consisted of Advanced DMEM/F12 (Thermo Fisher Scientific, USA) supplemented with 10% FBS, 1% penicillin/streptomycin (Thermo Fisher Scientific, USA), 1x GlutaMax Supplement (Thermo Fisher Scientific, USA), 1x HEPES (Thermo Fisher Scientific, USA), 1x N2 supplement (Thermo Fisher Scientific, USA), 1x B-27 supplement (Thermo Fisher Scientific, USA), 50 ng/mL Noggin (PEPROTECH, USA), 50 ng/mL FGF7 (R&D Systems, USA), 50 ng/mL FGF10 (R&D Systems, USA), and 50 ng/mL SAG (Enzo Life Science, USA).

Histochemical analysis and immunohistochemistry
The immunostaining procedure commenced with delineation of the tissue section perimeter on the glass slide using a Liquid Blocker, ensuring that any OCT compound residues on the slide edges were removed with a moistened Kimwipe. Subsequently, the slides were immersed in PBS for three 5-min washes to ensure thorough rinsing. The tissue sections were then treated with 0.1% Triton X-100 solution for 15 min in a moisture chamber to facilitate permeabilisation. After two 5-min PBS washes, the sections were incubated in a 3% BSA solution for 20 min to 2 h within the moisture chamber to block the non-specific binding sites.

Following the removal of the BSA solution, the tissue sections were incubated with the diluted primary antibody: Cytokeratin 14 Polyclonal antibody (Proteintech・10143-1-AP, CK14), Anti-Collagen IV antibody (abcam・ab6586, Col4), Vimentin (D21H3) XP Rabbit mAb (CST5741S, Vimentin), Anti-GFP (Rat IgG2a) Monoclonal (GF090R) CC (nacalali, 04404-84, GFP), Anti-GFP Antibody (abcam・ab290, GFP), Purified Rat Anti-Mouse CD31 (BD pharmingen・553370, CD31), Anti-Mitochondria antibody (Merck (Sigma)・MAB1273, Human Mitochondria), Rat Anti-Mouse F4/80 antibody (Bio-Rad・MCA497GA, F4/80), Anti-CD86 antibody (Cell Signalling Technology・ #19589S, CD86) Anti-CD163 antibody (abcam・ab182422, CD163), Anti-Ly6g antibody (abcam・ab25377, Ly6g), overnight at 4°C within the moisture chamber. The next day, the excess primary antibody was washed off with three 5-min PBS washes. Fluorescent-labelled secondary antibodies were subsequently applied, and the sections were incubated for approximately 1.5 h in a light-protected moisture chamber. After another series of three 5-min PBS washes to remove unbound secondary antibodies, a PBS-diluted nucleic acid staining solution, Hoechst 33342 at 5 µg/mL, was applied to the sections. A final incubation of 30 min in a light-protected moisture chamber was conducted for nuclear staining. Next, the slides were mounted, sealed with nail polish, and observed by upright microscope (BX51WI, Olympus).

In vivo fluorescence measurement
To measure the fluorescence intensity of the tissue-engineered skin transplanted into mice, an observation system was constructed by securing a tripod (Velbon 475213, 473264, 500274) with a camera (Canon EOS RP, RF35mm F1.8 MACRO IS STM) and a collimated light source (Lelmac, IBF-LX40AW), ensuring a constant optical path length. A short-cut filter (Fujifilm SC-52) was placed in front of the camera and a bandpass filter (Edmund #86-363) was positioned in front of the collimated light source (Fig. S9a, b). Edmund #86-363 cuts the wavelength range of the excitation light emitted from the light source that covers the fluorescence/detection light wavelength range, and SC-52 cuts the excitation light component from the detection light component incident on the detection camera. Fluorescence observations were conducted on anaesthetised mice that were positioned to maintain consistent body angles and locations. The fluorescence images were captured using an observation system and analysed using the ImageJ software to calculate the fluorescence intensity per unit area. Additionally, the values were normalised to the initial day’s measurements and presented in a graph.

Transplantation of tissue-engineered skin to the SHO mouse
The SHO mice were obtained from Oriental Yeast Co., Ltd. All mice were housed under a 12-h light-dark cycle. The flanks of the anaesthetised mice were disinfected using a cotton swab with povidone-iodine followed by ethanol. The inner circle of the culture insert was outlined on the flank skin using a pen. The skin within the marked area was excised using scissors. The tissue-engineered skin still on the membrane was then placed in the excised area. The membrane was carefully removed using a cotton swab moistened with culture medium and tweezers. The site was subsequently sutured with each stitch penetrating the tissue-engineered skin, muscle layer, and mouse skin. The muscle layer was kept moist with a saline solution as needed. The transplanted area was disinfected with povidone-iodine and ethanol, using cotton swabs. Protective tape was applied over the transplanted site using a short piece of tape directly over the site and a longer piece wrapped around the torso. Finally, the mice were placed in new cages at a density of approximately two mice per cage.

Ethics
All mouse experiments conducted in this study were approved by the Animal Care Committee at the University of Tokyo (approval number: 02-12) and the Institutional Animal Care and Use Committee of RIKEN Kobe Branch and were performed in accordance with the relevant guidelines and regulations. All animal care and experiments complied with the Guide for Animal Use and Care, published by the University of Tokyo.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The researchers have developed a system of genetically modified cells which can act as living sensors platform and detect the presence of a limited number of biomarkers. the noteworthy part is that manages to grow a tissue part from these cell and implanted it into the mice skin to monitor the levels of target biomarkers. However there are some serious concerns here\n-authors does not provide the full performance of the living tissue sensor, cross selectivity and sensitivity must be optimized\n- the current strategy to implant the living tissue sensors is not applicable for long term applications as the long term applications may pose some risk of reversing the genetic modifications.\n-skin implant is not the best was to keep the engineered cell alive, active and highly sensitive over long time periods and physiological conditions.", "reviewer_2": "Sawayama et al. have developed a tissue-engineered human skin equivalent that, upon transplantation to immunocompromised mice, retains the ability to detect proinflammatory stimuli when injected subcutaneously in the surrounding areas. To this end, they engineered keratinocyte cultures (containing stem cell subsets) with NFkB responsive element-controlled EGFP expression, generated tissue-engineered dermoepidermal constructs that were transplanted onto SCID hairless outbred mice, and noninvasively detected in vivo fluorescence emissions by the skin patches for >200 days. I found the presented data overall convincing and of high interest for scientists in diverse fields, since they explore quite a novel idea, but the manuscript needs major significant amendments to provide a better picture of how feasible this approach would be in real life.\nMajor comments:\n1. While the authors claim that “(…) Living Sensor Display is an effective technique for measuring the biomarkers within the body”, in truth, they only show convincing detection of repeated proinflammatory stimuli when injected in the vicinity of the skin transplant. This is key for the proposed approach to be of any practical use. Therefore, the detection of i.v. or i.p. delivered stimuli must be further explored to obtain a more convincing response in the absence of s.c. injection in the transplant area. Otherwise transplantation of skin patch into models of disease such as ob/ob mice might be of use to test applicability of the technology.\n2. Further on this line, the patches are quite big in terms of the relative proportion of total skin that has to be transduced (one has to imagine) to obtain a significant response. It would be necessary to establish the upper and lower limits of detection of a given metabolite with differently sized skin patches. Only with those data in hand, it would be possible to infer if the proposed technology would be transferable to humans or pets as they imply. Anything larger that a few square millimeters or centimeters would be difficult/impossible to put into practice in human or pet skin.\n3. NHEK cells were used in immunocompromised animals. However, to be of any future use in humans or other animals, the technology should be further validated. Specifically, the authors need to provide evidence that:\na) primary human epidermal keratinocyte cultures can be similarly transduced and the resulting skin patches crucially retain responsivity to proinflammatory stimuli long-term, indicating that the stem cell compartment can be targeted also in primary human keratinocyte cultures; and\nb)transduced mouse keratinocyte cultures can be grafted in immune competent mice and the system responsivity can still be demonstrated in an in vivo setting where the immune system, with all its checks and balances, is fully competent. Both experiments are key because non-autologous skin patches would quickly be rejected in immune-competent individuals of any species.\n4. On a similar vein, EGFP+ cells are immunogenic (See: Ansari et al. https://doi.org/10.1007/s12015-016-9670-8). It is to be expected that such skin patches would be immunogenic upon activation of EGFP expression in immune-competent animals. Therefore, it is key to demonstrate long term feasibility of this technology in animals that are naïve for GFP expression but with a fully competent immune system.\n5. All statements on immune stain data are qualitative because no quantitation of positive cells is provided. This must be corrected.\n6. Page 8: “These findings confirm that (…) the inflammatory response caused by transplantation subsides within approximately four weeks”. This can only be stated if cellular infiltration was assessed at earlier time points, which is not the case.\n7. The discussion needs some relevant amendments. Specifically:\na) “we unexpectedly observed maturation of tissue-engineered skin after transplantation in SHO mice”. I would argue that, on the contrary, this is to be expected as can be ascertained by checking the abundant literature on human dermoepidermal skin equivalents grafted onto SCID mice.\nb) Since the authors do not show any data that the technology is transferable outside mammals, any reference to plant leaves and other potential applications of this technology in the plant kingdom should be avoided.\nc) “Strategies to limit the spread of GMOs may be necessary”. This is the least concern in skin patch transplants, where migration of EGFP positive cells to germline is certainly not expected.\nOther comments:\n• Figure 1 would be more appropriate as graphical abstract for the manuscript instead of a main figure.\n• Introduction. Skin is not “the organ with the largest area of the body”. See: Sontheimer, https://doi.org/10.1038/jid.2013.335\n• Figure 2 panel b lacks scales and unit definitions. The statistical significance of the potential differences found among panels d, e, f are lacking.\n• I am concerned that the time allowed for in vitro stratification of the skin equivalents (four days) is too short. Accordingly, the pictures on Figure 3b-e show unconvincing epidermal stratification. 10d would be more appropriate. And more convincing staining of markers for basal and suprabasal keratinocytes should be shown.\n• The authors should provide an explanation (at least speculative) of why fluorescence intensity decreases over time in Figure 3g. Once more, is the difference between panels f and g statistically significant?\n• Reporting of construct-related data needs to be more detailed and transparent to enable reproducing the results\n• Appropriate statistical tests and accurate description of error bars and probability values are lacking", "reviewer_3": "In this manuscript, the authors developed the Living Sensor Display technique for long-term, in situ monitoring of biomarkers such as TNF-α and lipopolysaccharides. They engineered keratinocyte stem cells into living sensors, which were then transplanted into a tissue-engineered skin and engrafted onto mice. Thus, the tissue-engineered skin could continuously monitor the inflammatory situations in vivo. The concept is interesting, but major revisions, especially on the figures, must be made before considering publication in Nature Communications. The authors should carefully improve their figures, which are not clear, poorly aligned, and full of unprofessional mistakes.\nThe following detailed comments are given to strengthen their manuscript.\n1) Page 1, line 22. \" …for the real-time, non-invasive observation of biomarker…\". How do the authors define \"non-invasive\"? It seems that engrafting tissue-engineered skin will cause damage to the original skin surface.\n2) Page 4, line 14. The concentration-dependent responsiveness is not well supported as only three concentrations were evaluated (0, 0.2, 20 ng/ml). The authors need to provide a dose-response curve of their TNF-α sensors; that is, how the fluorescence levels change with various TNF-α concentrations. The dose-response curve will provide more information on the sensors' dynamic range, sensitivity, and detection limit.\n3) Page 26, Figure 2. A fluorescence histogram with error bars should be provided to show the fluorescence of three biological repeats. The current panel is hard to understand and only shows one biological repeat.\n4) Page 27, Figure 3. The fluorescence in Figures 3f and 3g peaks in 16h and then decreases. What causes this phenomenon, especially in the tissues without the TNF-α treatment (Figure 3f)? The tissues also exhibit a significantly higher background fluorescence (Figure 3f) and lower dynamic range (Figure 3g) than the NHEK cell-based sensors in Figure 2f. Discussion on this phenomenon is suggested.\n5) Page 28, Figure 4. It's weird that Figure 4c is not included in the result section, but in the discussion section. Please either describe it in the results or move it to supplementary figures. Similarly, the Figures 6a-f are not properly referred to in the texts.\n6) Some panels could use legends or captions to make them clearer. For instance, what do the lines and the symbols represent in Figures 5f, 6f, S3, and S8?\n7) Page 10, line 13. \" some mice …, while others…\". Precise mice numbers are needed here.\n8) Brief summaries of the results in the fluorescence images (e.g., Figures 4b, S2, S4, S5, and S7) could be added to the figure captions to help readers understand these figures.\n9) Page 32, Figure S1. The skin in Figure S1b should not have been treated with TNF-α; why does it show such obvious fluorescence?\nThere are also many minor mistakes in the texts and figures. The authors should carefully examine the manuscript and correct them. I listed some, but not all, below:\n1) Page 6, line 21. \"vimentin\" should be \"Vimentin\".\n2) Page 26, Figure 2b. Please separate \"Uninfectedunstimulated\".\n3) Page 26, Figure 2b. The unit, value, and title of the y-axis, and the unit and values of the x-axis are missing.\n4) Page 26, Figure 2b, right. The EGFP elements are not aligned correctly in the No.4 construct.\n5) Page 28, Figure 4. Three pictures in panel a are not properly aligned.\n6) The supplementary figures should be rearranged according to their order in the text. For example, Figure S9 should be changed to S4, as it appeared before the current S4.\n7) The scale bars in Figures 6i, 6j, and S4 need to be corrected."}, "author_response": "We appreciate the reviewerʼs valuable feedback on the performance of the Living Sensor Display. In this study, we have demonstrated that the system exhibits:\n1. High sensitivity to TNF-α at the ng/mL level, which is difficult to measure in vivo.\n2. Reproducible responses in a 3D tissue-engineered skin model.\n3. Long-term stability after transplantation, maintaining responsiveness for over 200 days.\n4. Real-time visualization of inflammatory states.\nRegarding cross-selectivity, as the reviewer pointed out, we have addressed this aspect in our study. Specifically, we examined the fluorescence response of NF-κB-EGFP modified NHEKs to various stimuli. Our findings (Fig. S2) demonstrate that:\n・TNF-α and IL-1β, both involved in NF-κB signaling, induced a fluorescence increase.\n・IL-2 and LPS, which lack direct receptors in the NF-κB pathway, did not induce a fluorescence response.\nThese results confirm that the Living Sensor Display exhibits high specificity for NF-κB pathway-related signals, reinforcing its reliability for biomarker monitoring.\nFor further clarity, we have highlighted this discussion in the revised manuscript (Fig.S2) : “To investigate the cross-selectivity of NHEKs genetically modified with NF-κB-EGFP, we examined the response not only to TNF-α but also to IL-1β, which is involved in the NF-κB signaling pathway, and IL-2 and LPS, which are not directly involved in the NF-κB signaling pathway and lack membrane receptors. These factors were added to the culture medium at a concentration of 20 ng/mL, and changes in fluorescence intensity were observed (Fig. S2a, b, c and d). As a result, an increase in fluorescence intensity similar to that seen with TNF-α was confirmed for IL-1β. However, no increase in fluorescence intensity was observed for IL-2 or LPS. These results demonstrate that NHEKs genetically modified with NF-κB-EGFP exhibit high selectivity, responding specifically to target molecules associated with receptors involved in the NF-κB signaling pathway, while showing no cross-reactivity to IL-2 or LPS.”\n- the current strategy to implant the living tissue sensors is not applicable for long term applications as the long term applications may pose some risk of reversing the genetic modifications.\nWe appreciate the reviewerʼs concern regarding the stability of the genetic modifications in long-term applications. However, our results indicate that the Living Sensor Display remains stable and functional for over 200 days post-transplantation in SHO mice. Given that the median survival time of immunodeficient mice is approximately 37 weeks (6‒12 months), the ability of the Living Sensor Display to maintain fluorescence response, turnover, and structural integrity without any maintenance suggests its feasibility for long-term applications.\nFurthermore, we observed consistent fluorescence intensity and stable tissue size throughout the experiment, indicating that the engineered skin undergoes natural turnover while preserving its function. This is explicitly stated in the manuscript:\n\"Moreover, the area of the skin showing a fluorescent response hardly changed and was maintained for over 200 days after engraftment.\"\nThese findings suggest that the risk of genetic modification reversal is minimal within the observed timeframe. While further studies in different models would be valuable for future clinical applications, our results support the Living Sensor Display as a long-term, stable biomarker monitoring system.\n-skin implant is not the best was to keep the engineered cell alive, active and highly sensitive over long time periods and physiological conditions.\nWe appreciate the reviewerʼs comment on the long-term viability of engineered cells in a skin implant. However, we would like to clarify that this study focuses on demonstrating the proof-of-concept (POC) of the Living Sensor Display rather than optimizing the best method for maintaining engineered cells under all physiological conditions.\nAs detailed in the manuscript, we systematically evaluated multiple approaches to tissue-engineered skin transplantation, ultimately selecting a method that enabled stable engraftment and long-term responsiveness. Our results show that the Living Sensor Display remained functional for over 200 days post-transplantation, maintaining stable fluorescence intensity and cellular turnover, which supports its feasibility for long-term applications.\nThese findings are explicitly discussed in the Discussion section, particularly regarding the stability and performance of the Living Sensor Display. Given this, we believe the current methodology is appropriate for the scope of this study and no modifications to the manuscript are necessary.\nReviewer #2 (Remarks to the Author):\nSawayama et al. have developed a tissue-engineered human skin equivalent that, upon transplantation to immunocompromised mice, retains the ability to detect proinflammatory stimuli when injected subcutaneously in the surrounding areas. To this end, they engineered keratinocyte cultures (containing stem cell subsets) with NFkB responsive element-controlled EGFP expression, generated tissue-engineered dermoepidermal constructs that were transplanted onto SCID hairless outbred mice, and noninvasively detected in vivo fluorescence emissions by the skin patches for >200 days.\nI found the presented data overall convincing and of high interest for scientists in diverse fields, since they explore quite a novel idea, but the manuscript needs major significant amendments to provide a better picture of how feasible this approach would be in real life.\nMajor comments:\n1. While the authors claim that “(…) Living Sensor Display is an effective technique for measuring the biomarkers within the body”, in truth, they only show convincing detection of repeated proinflammatory stimuli when injected in the vicinity of the skin transplant. This is key for the proposed approach to be of any practical use. Therefore, the detection of i.v. or i.p. delivered stimuli must be further explored to obtain a more convincing response in the absence of s.c. injection in the transplant area. Otherwise transplantation of skin patch into models of disease such as ob/ob mice might be of use to test applicability of the technology.\nWe appreciate your valuable feedback. As you noted, our previously published data only included subcutaneous (s.c.) administration of TNF-α and LPS. In response to your suggestion, we conducted additional experiments using intravenous (i.v.) LPS administration. Although the number of animals was limited, the results showed that LPS administered via the tail vein in mice carrying the Living Sensor Display led to an increase in fluorescence intensity. This suggests that target molecules secreted during in vivo inflammation leak from the vasculature and can be detected by the Living Sensor Display̶a significant step toward practical application.\nWe have added these results in Fig. S12. In the Results section, we have inserted the following text:\n“Furthermore, LPS solution (0.1 mg/mL) was administered via the tail vein at a dose of 0.1 mg/kg on Day 0 and Day 7. As a result, fluorescence intensity increased (Fig. S12a‒e) from the day following administration, with a significant increase on the second day post-administration compared to baseline (Fig. S12f). By the seventh day post-administration, fluorescence had subsided; however, upon re-administration, fluorescence increased again two days later. On Day 9, fluorescence intensity was significantly higher than before the Day 7 administration (Fig. S12g).”\nAdditionally, we have updated the Discussion section by adding “We demonstrated the ability to continuously monitor inflammatory cytokines in vivo by transplanting tissue-engineered skin into mice. This skin was constructed from NHEK cells that produce EGFP upon stimulation by inflammatory cytokines, such as TNF-α and IL-1β, secreted from human immune cells.”\n2. Further on this line, the patches are quite big in terms of the relative proportion of total skin that has to be transduced (one has to imagine) to obtain a significant response. It would be necessary to establish the upper and lower limits of detection of a given metabolite with differently sized skin patches. Only with those data in hand, it would be possible to infer if the proposed technology would be transferable to humans or pets as they imply. Anything larger that a few square millimeters or centimeters would be difficult/impossible to put into practice in human or pet skin.\nWe appreciate the reviewerʼs comment regarding the size of the transplanted Living Sensor Display and its implications for practical applications. In the manuscript, we primarily present images of relatively large engrafted patches (10 mm × 20 mm) in Fig. S5a,b, as these cases provided the clearest visualization of fluorescence responses. However, we would like to clarify that successful fluorescence detection and biomarker response were also observed in much smaller patches (~3 mm × 3 mm) as shown in Fig. S5c,d, demonstrating that the system functions effectively even at reduced sizes.\nGiven this, we believe that the Living Sensor Display could be adapted for practical use in animals, including pets, without significant size constraints. While a precise determination of the upper and lower detection limits relative to patch size would be an important aspect for future clinical translation, our current results indicate that effective responses are achievable in small patches, which supports the feasibility of this approach.\n3. NHEK cells were used in immunocompromised animals. However, to be of any future use in humans or other animals, the technology should be further validated. Specifically, the authors need to provide evidence that:\na) primary human epidermal keratinocyte cultures can be similarly transduced and the resulting skin patches crucially retain responsivity to proinflammatory stimuli long-term, indicating that the stem cell compartment can be targeted also in primary human keratinocyte cultures; and\nWe appreciate the reviewerʼs concern regarding the validation of the Living Sensor Display in primary human epidermal keratinocytes. However, we would like to clarify that the NHEKs used in this study are, in fact, primary human neonatal epidermal keratinocytes, which we obtained from KURABO Industries LTD. These cells were carefully screened prior to purchase and selected based on their ability to form skin equivalents, undergo natural turnover, and retain functional properties necessary for tissue engineering applications.\nFurthermore, it has been reported that NHEKs contain a certain proportion of stem cells1, and our results indicate that the engineered skin retains turnover ability after transplantation, supporting the conclusion that stem cells within the NHEK population contribute to long-term function. Since our study already demonstrates stable engraftment, biomarker responsiveness, and turnover in vivo, we believe that the evidence requested by the reviewer has already been provided in the manuscript.\n1. Wong, Y. L., Okubo, T., Uno, E., Suda, K. & Ishii, T. Role of CD99 in regulating homeostasis and differentiation in normal human epidermal keratinocytes. Biochem. Biophys. Res. Commun. 606, 108‒113 (2022).\nb)transduced mouse keratinocyte cultures can be grafted in immune competent mice and the system responsivity can still be demonstrated in an in vivo setting where the immune system, with all its checks and balances, is fully competent. Both experiments are key because non-autologous skin patches would quickly be rejected in immune-competent individuals of any species.\nWe appreciate the reviewer for the valuable comment regarding the immune system, which is critical for the practical implementation of the Living Sensor Display. The purpose of this study is to obtain proof-of-concept data demonstrating the utility of the Living Sensor Display with human applications in mind. Therefore, we developed human-derived tissue-engineered skin and conducted experiments in immune-competent mice. We acknowledge that this concern is understandable̶for instance, when human-derived tissue-engineered skin was transplanted onto C57BL/6 mice, it was rejected and failed to engraft. On the other hand, transplantation into humans would obviate this issue; however, that would require a highly advanced clinical study, which is beyond the scope of our current work.\nTo address whether the system can respond in the presence of a human immune environment, we performed proof-of-concept experiments using a co-culture system with genetically modified NHEK cells and human-derived Peripheral Blood Mononuclear Cells (PBMCs) in a culture insert. In these experiments, we placed a culture insert containing a solution of 1×106 cells of PBMCs over the NHEK cell layer, and in another condition, we added LPS (to a final concentration of 10 µg/mL in the well) to activate 1×106 cells of PBMCs within the insert. We then monitored changes in fluorescence intensity. No change was observed with the addition of PBMCs alone, but in the LPS-stimulated PBMC condition, an increase in fluorescence intensity was detected after 60 hours.\nThese results indicate that human tissue can function in a PBMC environment. Although this co-culture system does not fully recapitulate the complete human immune system, it is considered to mimic the early stages of acute inflammatory responses in humans. This suggests that when the Living Sensor Display is applied in humans, fluorescence responses induced by inflammatory cytokines such as TNF-α and IL-1β can also be observed.\nWe have added these findings in Fig. S3. In the Results section, the following text has been included:\n“To verify whether NHEKs genetically modified with NF-κB-EGFP respond in a human immune environment, we performed co-culture experiments using a culture insert system with NHEK cells and human-derived Peripheral Blood Mononuclear Cells (PBMCs). A culture insert containing 1×10⁶ PBMCs was placed over the NHEK cell layer. In another condition, a culture insert containing 1×10⁶ PBMCs activated by LPS (final concentration: 10 µg/mL in the well) was used. Changes in fluorescence intensity were then monitored (Fig. S3a, b). As a result, fluorescence intensity remained unchanged with PBMCs alone; however, in the LPS-stimulated condition, it increased after 60 hours. These results suggest that NHEKs exhibit fluorescence responses in the presence of PBMCs, particularly under inflammatory conditions induced by LPS stimulation. Although this system does not fully represent a complete human immune system, it is considered to mimic the early stages of acute inflammatory responses, implying that in practical applications, fluorescence responses to cytokines can be observed in humans.”\nFurthermore, in the Discussion section, we have inserted the following text: “We demonstrated the ability to continuously monitor inflammatory cytokines in vivo by transplanting tissue-engineered skin into mice. This skin was constructed from NHEK cells that produce EGFP upon stimulation by inflammatory cytokines, such as TNF-α and IL-1β, secreted from human immune cells into mice.”\n4. On a similar vein, EGFP+ cells are immunogenic (See: Ansari et al. https://doi.org/10.1007/s12015-016-9670-8). It is to be expected that such skin patches would be immunogenic upon activation of EGFP expression in immune-competent animals. Therefore, it is key to demonstrate long term feasibility of this technology in animals that are naïve for GFP expression but with a fully competent immune system.\nWe appreciate the reviewerʼs insightful comment regarding the potential immunogenicity of EGFP-expressing cells in immune-competent animals. While EGFP is expressed intracellularly and is not typically secreted into the extracellular space, minimizing the likelihood of directly triggering a local immune response, we acknowledge that EGFP peptides could still be recognized by the immune system if released due to cell death or damage. This could lead to antigen presentation by immune cells, as noted in previous studies (Ansari et al., 2016).\nHowever, as part of our preliminary investigations, we attempted transplantation into fully immune-competent C57BL/6 (B6) mice, but the tissue-engineered skin did not engraft, likely due to immune rejection. This suggests that further optimization, such as the use of autologous cells or immunomodulation, would be necessary for translation to fully immune-competent models. Although evaluating this technology in humans would be the ultimate goal, such experiments require advanced clinical trials and are beyond the scope of this study.\nTo address this point, we have added the following statement to the Discussion section：\n”Unlike conventional wearable sensor technologies, this method enables the long-term, real-time acquisition of minute biochemical signals, with potential applications ranging from health management and early disease detection to veterinary medicine and livestock monitoring. The target biological reactions include inflammatory markers, hypoxic and oxidative stress, and hormones, among others. This system is expected to have broad applications beyond human healthcare, such as improving the health management of pets that cannot communicate their medical conditions. However, several challenges remain before widespread implementation. While SHO mice lack mature B and T lymphocytes, the observed innate immune responses (Fig. 6j) suggest that the transplanted tissue remains biologically active. Given reports of EGFP immunogenicity25, future studies using fully immune-competent models will be essential. Potential strategies include using autologous cells or alternative reporter proteins with lower immunogenicity to ensure broader applicability. In addition to biological limitations, societal acceptance may also pose a challenge. Strategies to control the spread of genetically modified organisms may be necessary; however, applications targeting non-human subjects, such as veterinary medicine and livestock health, may be more readily accepted.”\n5. All statements on immune stain data are qualitative because no quantitation of positive cells is provided. This must be corrected.\nWe appreciate the reviewerʼs suggestion and have now quantified the number of positively stained cells in the immunohistochemical images. Specifically, we counted the number of macrophages (F4/80+) and neutrophils (Ly6g+) in the transplanted tissue at different time points.\nOur results indicate that macrophage count decreased by approximately 90% from week 3 to week 4 post-transplantation. Neutrophil count decreased by approximately 75% in the same period. These quantitative findings have been added to the caption of Fig.S7 to provide a more precise interpretation of the immune response over time.\n6. Page 8: “These findings confirm that (…) the inflammatory response caused by transplantation subsides within approximately four weeks”. This can only be stated if cellular infiltration was assessed at earlier time points, which is not the case.\nWe appreciate the reviewerʼs suggestion and have now included data from an earlier time point (week 3 post-transplantation) to further support our conclusion.\n1. At week 3, inflammatory cells were still observed near the transplant site, confirming that an acute inflammatory response was occurring, even in SHO mice.\n2. By week 4, the infiltration of inflammatory cells had significantly decreased, suggesting that the immune response had subsided.\nTo provide a clearer comparison, we have added these findings to Fig. S6. In the Results section, we have inserted the following text:\n” The infiltration of inflammatory cell groups was observed to have subsided at 3 weeks after transplantation (Fig. S6) compared to 4 weeks after transplantation (Fig. S7), indicating that at least 4 weeks should be waited before fluorescence observation can be performed.”\nWe believe this additional data strengthens our conclusion that the inflammatory response caused by transplantation resolves within approximately four weeks.\n7. The discussion needs some relevant amendments. Specifically:\na) “we unexpectedly observed maturation of tissue-engineered skin after transplantation in SHO mice”. I would argue that, on the contrary, this is to be expected as can be ascertained by checking the abundant literature on human dermoepidermal skin equivalents grafted onto SCID mice.\nWe appreciate the reviewerʼs comment and acknowledge that the maturation of tissue-engineered skin after transplantation into immunodeficient mice has been previously reported in the literature. However, the key novel finding of this study is that by transplanting an immature, less-differentiated skin equivalent, we observed an increase in engraftment success, which has not been previously emphasized in the context of skin grafting.\nImportantly, our study does not aim to induce maturation but rather demonstrates that a less mature construct enhances engraftment efficiency, which may have implications for optimizing tissue-engineered skin transplantation. This aspect is explicitly discussed in the Discussion section, where we highlight that maturation is not a necessary requirement for successful transplantation in this model.\nb) Since the authors do not show any data that the technology is transferable outside mammals, any reference to plant leaves and other potential applications of this technology in the plant kingdom should be avoided.\nWe appreciate the reviewerʼs suggestion and agree that the discussion on plant applications extends beyond the scope of this study. To maintain the focus on the core findings, we have removed references to plant-related applications from the manuscript.\nWe believe this revision enhances the clarity of the discussion and ensures that the manuscript remains aligned with the primary objectives of our study.\nc) “Strategies to limit the spread of GMOs may be necessary”. This is the least concern in skin patch transplants, where migration of EGFP positive cells to germline is certainly not expected.\nWe appreciate the reviewerʼs insightful comment. We agree that the risk of EGFP-positive cells migrating to the germline in skin patch transplants is extremely low, making GMO containment a less critical concern in this context. Nonetheless, we remain committed to following current safety and regulatory guidelines, ensuring that all necessary precautions are taken as the technology advances.\nOther comments:\n• Figure 1 would be more appropriate as graphical abstract for the manuscript instead of a main figure.\nWe appreciate the reviewerʼs perspective. Figure 1 was intentionally designed to convey the concept of the Living Sensor Display clearly at a glance, helping readers grasp the key idea efficiently. We believe that maintaining Figure 1 as a main figure enhances the manuscriptʼs readability and does not compromise its scientific rigor. Given its role in setting the foundation for the study, we would prefer to keep it in its current placement.\n• Introduction. Skin is not “the organ with the largest area of the body”. See: Sontheimer, https://doi.org/10.1038/jid.2013.335\nWe appreciate the reviewerʼs correction. As suggested, we have revised the text to replace \"the organ with the largest area\" with \"an organ with a large surface area\" to ensure accuracy. This change has been implemented in the Introduction section. Thank you for bringing this to our attention.\n• Figure 2 panel b lacks scales and unit definitions. The statistical significance of the potential differences found among panels d, e, f are lacking.\nWe appreciate the reviewerʼs suggestion and have addressed these concerns as follows:\nFor Figure 2 panel b, we have added appropriate scales and unit definitions to improve clarity.\nFor panels d, e, and f, we have included a new graph displaying fluorescence intensity at 24 hours, along with statistical significance markers to clearly indicate the differences among conditions in Fig.S1a.\nAdditionally, we have revised the manuscript in P.6, line 4, by adding:\n\"…significantly from 0 ng/mL to 20 ng/mL and from 0.2 ng/mL to 20 ng/mL at 24h after TNF-α stimulation (Fig.S1a,b),\"\nThese revisions have been incorporated into Figure 2 and the corresponding figure legend, ensuring a more precise and statistically supported presentation of our results.\n• I am concerned that the time allowed for in vitro stratification of the skin equivalents (four days) is too short. Accordingly, the pictures on Figure 3b-e show unconvincing epidermal stratification. 10d would be more appropriate. And more convincing staining of markers for basal and suprabasal keratinocytes should be shown.\nWe appreciate the reviewerʼs concern regarding the in vitro stratification period. However, based on our preliminary experiments, we found that over-maturation of the tissue-engineered skin negatively affects engraftment success. Specifically, prolonging the in vitro culture period before transplantation led to a significant reduction in engraftment efficiency, which is why we selected a four-day stratification period. This aspect is discussed in the Discussion section, where we emphasize that transplanting an immature skin equivalent enhances engraftment efficiency. Given that successful engraftment, turnover, and long-term function were observed in vivo, we believe our approach is appropriate for this study.\nWhile we acknowledge that longer in vitro maturation may improve stratification in vitro, our focus is on achieving optimal transplantation outcomes rather than maximizing pre-transplant stratification.\n• The authors should provide an explanation (at least speculative) of why fluorescence intensity decreases over time in Figure 3g. Once more, is the difference between panels f and g statistically significant?\nWe appreciate the reviewerʼs suggestion. The decrease in fluorescence intensity over time in Figure 3g is likely due to EGFP photobleaching, which occurs progressively during prolonged fluorescence measurements. This is a common phenomenon in live-cell imaging and fluorescence-based assays.\nTo address the reviewerʼs concern regarding statistical significance, we have now added a new graph in Figure S4a, showing the fluorescence intensity at 17 hours post-peak, along with statistical significance markers. These data confirm that the differences observed in Figure 3f and 3g are statistically significant.\nIn the Results section, we have inserted the following text: “The fluorescence intensity increased significantly from 0 ng/mL to 20 ng/mL at 17 h, peaking at approximately 17 h before gradually decreasing (Fig.S4a).”\nWe believe these additions provide a clearer understanding of the fluorescence intensity changes over time and further support the robustness of our findings.\n• Reporting of construct-related data needs to be more detailed and transparent to enable reproducing the results\nWe appreciate the reviewerʼs suggestion and have added detailed information on the cell constructs in the Methods section (Page 17, lines 4‒5) to enhance reproducibility. These additions clarify the specific characteristics of the engineered cells used in our study.\n• Appropriate statistical tests and accurate description of error bars and probability values are lacking\nWe appreciate the reviewerʼs feedback and have now included statistical analyses for both TNF-α-stimulated cultured cells and tissue-engineered constructs. These results, along with appropriate statistical tests, error bar descriptions, and p-values, have been added to Fig.S1a and Fig.S4a. The new data confirm that the observed differences are statistically significant.\nWe believe these revisions provide greater clarity and ensure the rigor of our analyses.\nReviewer #3 (Remarks to the Author):\nIn this manuscript, the authors developed the Living Sensor Display technique for long-term, in situ monitoring of biomarkers such as TNF-α and lipopolysaccharides. They engineered keratinocyte stem cells into living sensors, which were then transplanted into a tissue-engineered skin and engrafted onto mice. Thus, the tissue-engineered skin could continuously monitor the inflammatory situations in vivo. The concept is interesting, but major revisions, especially on the figures, must be made before considering publication in Nature Communications. The authors should carefully improve their figures, which are not clear, poorly aligned, and full of unprofessional mistakes.\nThe following detailed comments are given to strengthen their manuscript.\n1) Page 1, line 22. \" …for the real-time, non-invasive observation of biomarker…\". How do the authors define \"non-invasive\"? It seems that engrafting tissue-engineered skin will cause damage to the original skin surface.\nWe appreciate the reviewerʼs comment and acknowledge that the initial implantation step involves an invasive procedure. To clarify this, we have revised the sentence in Page 1, line 22, by adding:\n\"…with the only invasive step being the initial implantation, \"\nThis ensures a more precise definition of \"non-invasive\" in the context of long-term biomarker monitoring.\n2) Page 4, line 14. The concentration-dependent responsiveness is not well supported as only three concentrations were evaluated (0, 0.2, 20 ng/ml). The authors need to provide a dose-response curve of their TNF-α sensors; that is, how the fluorescence levels change with various TNF-α concentrations. The dose-response curve will provide more information on the sensors' dynamic range, sensitivity, and detection limit.\nWe appreciate the reviewerʼs suggestion and have now included a dose-response curve showing the concentration-dependent changes in fluorescence intensity for both cultured cells and tissue-engineered skin. These data have been added to Fig.S1b and Fig.S4b.\nIn the Results section, we have inserted the following text:\n“The fluorescence intensity changes with various TNF-α concentrations were observed in both cultured cells and tissue-engineered skin, indicating that both systems exhibit a dynamic range from 0 to 20 ng/mL (Fig.S1b and S4b)”\nFurthermore, we note that in vivo, TNF-α concentrations rarely exceed 20 ng/mL, meaning that the Living Sensor Display has a sufficient dynamic range for physiological conditions. This point is already discussed in the Discussion section.\n3) Page 26, Figure 2. A fluorescence histogram with error bars should be provided to show the fluorescence of three biological repeats. The current panel is hard to understand and only shows one biological repeat.\nWe appreciate the reviewerʼs suggestion and have revised Figure 2d and 2e to improve clarity. Since the standard deviation is very small, the error bars overlapped in the original figure, making them difficult to visualize. To address this, we have added a new graph at the 24-hour time point in Figure S1a, where the fluorescence intensity for different concentrations is displayed with error bars.\nAdditionally, we have revised the manuscript in P.6, line 4, by adding:\n\"…significantly from 0.2ng/mL to 20 ng/mL at 24h after TNF-α stimulation (Fig.S1a)\"\nThese modifications ensure that the statistical significance of fluorescence intensity differences is clearly conveyed.\n4) Page 27, Figure 3. The fluorescence in Figures 3f and 3g peaks in 16h and then decreases. What causes this phenomenon, especially in the tissues without the TNF-α treatment (Figure 3f)? The tissues also exhibit a significantly higher background fluorescence (Figure 3f) and lower dynamic range (Figure 3g) than the NHEK cell-based sensors in Figure 2f. Discussion on this phenomenon is suggested.\nWe appreciate your valuable feedback. In particular, the slight increase in fluorescence intensity observed in tissues not treated with TNF-α is likely due to the inevitable peak seen in all cases when using tissue-engineered skin. This suggests that the genetically modified cells may respond to various stimuli, and medium changes might also introduce some stimulation.\nFurthermore, regarding the high background and low dynamic range: although fluorescence measurements were performed using the same apparatus as that for cultured cells, the tissue-engineered skin tends to shrink due to its own contractile force when removed from the culture insert, making quantitative measurement difficult. Therefore, we conducted fluorescence observations directly on the tissue-engineered skin fabricated within the culture insert. Because the transparency of both the culture insert and the tissue-engineered skin is lower than that of cultured cells, scattering is increased, which results in a higher background and a smaller dynamic range.\nIn the Results section, we have inserted the following text:\n“Compared to the data obtained from cultured cells, the dynamic range relative to the background in tissue-engineered skin is smaller. This is likely because the fluorescence measurements were conducted through a culture insert and scattering from the culture insert and the tissue-engineered skin may have influenced the results.”\n5) Page 28, Figure 4. It's weird that Figure 4c is not included in the result section, but in the discussion section. Please either describe it in the results or move it to supplementary figures. Similarly, the Figures 6a-f are not properly referred to in the texts.\nThank you for your valuable feedback. We have added Figure 4c and Figures 6a‒f into the result.\n6) Some panels could use legends or captions to make them clearer. For instance, what do the lines and the symbols represent in Figures 5f, 6f, S3, and S8?\nWe appreciate your valuable feedback and have made the necessary revisions. The dotted lines indicate the timing of fluorescence observation, while the triangles denote the timing of administration. Additional details have been included in the figure caption.\n7) Page 10, line 13. \" some mice …, while others…\". Precise mice numbers are needed here.\nThank you for your valuable feedback. We have included the number of mice and made the necessary revisions.\n8) Brief summaries of the results in the fluorescence images (e.g., Figures 4b, S2, S4, S5, and S7) could be added to the figure captions to help readers understand these figures.\nWe appreciate your suggestion. We have added brief summaries of the fluorescence image results to the figure captions.\n9) Page 32, Figure S1. The skin in Figure S1b should not have been treated with TNF-α; why does it show such obvious fluorescence?\nThis figure shows a mouse at 4 weeks post-transplantation, when the fluorescence intensity is elevated due to inflammation from the transplant. Subsequently, after waiting 2‒3 weeks for the fluorescence intensity to decrease, administration experiments were performed using mice. Further details are provided in the caption in Fig. S5. We have revised the sentence in Page 9, line 26, by adding:\n\"…suggesting that fluorescence observation should not be initiated until at least four weeks have passed. \"\nThere are also many minor mistakes in the texts and figures. The authors should carefully examine the manuscript and correct them. I listed some, but not all, below:\n1) Page 6, line 21. \"vimentin\" should be \"Vimentin\".\nWe appreciate your valuable feedback and have made the necessary revisions.\n2) Page 26, Figure 2b. Please separate \"Uninfectedunstimulated\".\nWe appreciate your valuable feedback and have made the necessary revisions\n3) Page 26, Figure 2b. The unit, value, and title of the y-axis, and the unit and values of the x-axis are missing.\nWe appreciate your valuable feedback and have made the necessary revisions\n4) Page 26, Figure 2b, right. The EGFP elements are not aligned correctly in the No.4 construct.\nWe appreciate your valuable feedback and have made the necessary revisions\n5) Page 28, Figure 4. Three pictures in panel a are not properly aligned.\nWe appreciate your valuable feedback and have made the necessary revisions\n6) The supplementary figures should be rearranged according to their order in the text. For example, Figure S9 should be changed to S4, as it appeared before the current S4.\nWe appreciate your valuable feedback and have made the necessary revisions\n7) The scale bars in Figures 6i, 6j, and S4 need to be corrected.\nWe appreciate your valuable feedback and have made the necessary revisions"}, {"round": 2, "reviewer_comments": {"reviewer_1": "I do not have additional comments", "reviewer_2": "Samayama et al. have now satisfactorily responded to most of my original concerns, but some aspects need further clarification. To facilitate the conversation, I will order the still unresolved needs according to my original list:\n1- I am satisfied by the new results shown on Fig S12, where they now demonstrate significant detection of i.v. stimuli 2 days post-injection. This is a very relevant result.However, the fact that there is a 2-day delay from i.v. stimulus to patch detection should be acknowledged in the Discussion section, because it has implications on the use of this technology that cannot be overseen.\n2- I am also satisfied with the response that both patches of 9 and 200 sq mm worked similarly well. While I am not asking for further experimentation here, I believe that the discussion paragraph where they speak of detection limits (“Through this work, we demonstrated the effectiveness of detecting substances…”) should also include some words on the patch size and how it influences detection limits (I am assuming that, in equal conditions, larger patches will have a lower detection limit).\n3a- Thanks for the clarification, my mistake.\n3b- The PBMC co-culture surrogate assay is acceptable.\n4- Limitation of non-testing in immune-competent animals is now correctly acknowledged.\n5- Quantitative statements cannot be \"approximately 90%\" or \"approximately 75%\". I am afraid we need to see a proper quantitative study where they show graphs with means and standard deviations in an appropriate number of replicates as per journal guidelines.\n6- Same here as comment 5.\n7a- The authors are free to maintain this interpretation if they wish, but I insist that maturation of monolayer human epidermal equivalents upon transplantation onto mice is a well-known fact and not to be unexpected. On the contrary, the epidermis would be unexpected to remain immature if skin correctly engrafted.\n7b & 7c- OK\nOther (minor) comments. OK.", "reviewer_3": "Comments to authors\nThe authors have made some revisions, mainly to the texts. However, I am not convinced by their explanations in the response letter, and I insist that their figures are far from the publishable level, with detailed comments given below.\n1） (Fig. 1) The authors only added one concentration (2 ng/ml) to their previous results, and in total, they only tested four concentrations (0, 0.2, 2, and 20 ng/ml). For biosensor characterization, these concentrations are too few to reflect the crucial properties of biosensors, e.g., the limit of detection and sensitivity. The authors should add fluorescence data at more concentrations below 0.2 ng/ml.\n2） (Fig. 2) The authors claim that their error bars are too small to see clearly. However, it is wired that obvious error bars are in Fig. S1a-b, while no deviation can be seen in Figs. 2d-f, 3f-g, S1d, S2, and S3.\n3） (Fig. 2b) The authors should change the flow cytometry histograms in Figure 2b into bar graphs. The current version is messy and not clear enough to display the relative fluorescence intensities and dynamic ranges of each construct, both unstimulated and stimulated.\n4） (Fig. 5) I noticed that the error bars in Fig. 5g-i were not centered. The lines showing statistical significance in Fig. 5g, 5i, and 6g seem drawn manually and not aligned well. Were these error bars and statistical significance directly generated by the plotting software or manually marked by the author?\n5） The quality of the figures is still poor and far from the publishable standards. For instance:\na) (All figures) The authors use \"ng/ml\" in some figures (Fig. S1c) but \"ng/mL\" in others (Fig. S1a). Please make them consistent.\nb) (Fig. S1a-c) Panels are not properly aligned.\nc) (Fig. 3b) An extra line (blue) and arrow (black) are inserted without any annotations. Remove them.\nd) (Fig. S1c) The Y-axis title is missing.\ne) (Fig. 5k) The text and scale bars overlap.\nf) (Fig. S4b) There is no scale on the X-axis. Remove \")\" in the legend.\ng) (Fig. S14) It's weird that Fig.S14 is not included in the result section.\nh) (Fig. S5) Delete \";\" in the legend.\ni) (Fig. S8) Delete \".\" in the legend.\nj) (Fig. S6, 7 & 10). The scale bars in HE panels are not consistent. Some are labeled with 100 μm, but the others were not.\nk) (Figs. 1-6) Remove the \".\" following \"Figure\"."}, "author_response": "REVIEWER COMMENTS\nReviewer #2\n1- I am satisfied by the new results shown on Fig S12, where they now demonstrate significant detection of i.v. stimuli 2 days post-injection. This is a very relevant result. However, the fact that there is a 2-day delay from i.v. stimulus to patch detection should be acknowledged in the Discussion section, because it has implications on the use of this technology that cannot be overseen.\nThank you for your comment. The graph in question was originally plotted at the 2-day time point to match the timing used for i.p. and s.c. stimulations. As a result, it may appear that there is a 2-day delay for i.v. detection. However, as shown in the time-course profile in Fig. S12e, a clear increase in signal following i.v. injection is already evident at 1-day post-stimulation. Therefore, we believe that the patch can detect i.v. stimuli as early as 1 day after injection.\nTo avoid confusion, we have revised panels (f) and (g) to present the 1-day data instead of the 2-day time point.\n2- I am also satisfied with the response that both patches of 9 and 200 sq mm worked similarly well. While I am not asking for further experimentation here, I believe that the discussion paragraph where they speak of detection limits (“Through this work, we demonstrated the effectiveness of detecting substances…”) should also include some words on the patch size and how it influences detection limits (I am assuming that, in equal conditions, larger patches will have a lower detection limit).\nAs suggested, we have revised the Discussion section. Following the sentence “Through this work, we demonstrated the effectiveness of detecting substances…”, we added the sentence as :\n“We also found that even small-sized patches can exhibit responsiveness; however, as the transplantation area decreases, the total fluorescence signal becomes weaker, leading to a reduced detection limit.”\nThis addition, which addresses the influence of patch size on detection sensitivity as pointed out by the reviewer, has been inserted on page 15, line 16 of the revised manuscript.\n3a- Thanks for the clarification, my mistake.\n3b- The PBMC co-culture surrogate assay is acceptable.\n4- Limitation of non-testing in immune-competent animals is now correctly acknowledged.\nThank you for your understanding.\n5- Quantitative statements cannot be \"approximately 90%\" or \"approximately 75%\". I am afraid we need to see a proper quantitative study where they show graphs with means and standard deviations in an appropriate number of replicates as per journal guidelines.\n6- Same here as comment 5.\nWe appreciate the reviewerʼs concern regarding the use of approximate percentages. In this case, our intention was simply to convey whether red fluorescent signals were present or absent in the immunostained tissue images. In response to the previous revision request, we performed cell counting to provide numerical data; however, given that nonspecific staining can occasionally occur in in vivo immunohistochemistry, we acknowledge that the resulting numbers may have limited quantitative precision. Therefore, we would like to respectfully request that the use of approximate percentages (e.g., “approximately 90%”) be allowed in this context, as we do not intend to make strong claims based on these figures.\n7a- The authors are free to maintain this interpretation if they wish, but I insist that maturation of monolayer human epidermal equivalents upon transplantation onto mice is a well-known fact and not to be unexpected. On the contrary, the epidermis would be unexpected to remain immature if skin correctly engrafted.\nWe respectfully acknowledge the reviewerʼs perspective. However, to the best of our knowledge, there have been no previous reports in which tissue-engineered skin transplanted onto mice exhibited both well-developed rete ridge-like structures and sustained epidermal turnover over an extended period. In our experiments, tissue-engineered constructs that included pre-formed dermal and epidermal layers did not engraft successfully. Likewise, subcutaneous co-culture of keratinocytes and fibroblasts resulted in minimal engraftment.\nTherefore, from our experimental experience, the observed epidermal maturation and long-term turnover were truly unexpected, and we used the term “unexpected” in that context.\n7b & 7c- OK\nOther (minor) comments. OK.\nThank you for your understanding.\nReviewer #3\n1） (Fig. 1) The authors only added one concentration (2 ng/ml) to their previous results, and in total, they only tested four concentrations (0, 0.2, 2, and 20 ng/ml). For biosensor characterization, these concentrations are too few to reflect the crucial properties of biosensors, e.g., the limit of detection and sensitivity. The authors should add fluorescence data at more concentrations below 0.2 ng/ml.\nAs suggested, we have added fluorescence data for two additional lower concentrations (0.002 ng/ml and 0.02 ng/ml) in Fig. S1d. Based on these results, we have noted in the manuscript that “but detection below 0.02 ng/ml falls outside the dynamic range.”\n2） (Fig. 2) The authors claim that their error bars are too small to see clearly. However, it is wired that obvious error bars are in Fig. S1a-b, while no deviation can be seen in Figs. 2d-f, 3f-g, S1d, S2, and S3.\nAll graphs have been recreated using GraphPad Prism to ensure consistent formatting, and the error bars have been adjusted to be clearly visible in all relevant figures.\n3） (Fig. 2b) The authors should change the flow cytometry histograms in Figure 2b into bar graphs. The current version is messy and not clear enough to display the relative fluorescence intensities and dynamic ranges of each construct, both unstimulated and stimulated.\nAs suggested, we have replaced the original flow cytometry histograms in formerly Figure 2b with bar graphs showing the median fluorescence intensity (MFI) for each construct under both unstimulated and stimulated conditions. The original histograms have been moved to Supplementary Figure S1 for reference.\nAdditionally, we have updated the Lentivirus vector construction section in the Materials and Methods (page 17, line 6) to include the following sentence:\n“Flow cytometry data were analyzed using FlowJo software (v10.10.0, FlowJo LLC). After doublet exclusion, SYTOX Blue-negative cells were defined as live cells. GFP fluorescence was quantified as median fluorescence intensity (MFI). For visual comparison across conditions, fluorescence histograms were normalized to mode, with the highest frequency set to 100% on the y-axis.”\n4） (Fig. 5) I noticed that the error bars in Fig. 5g-i were not centered. The lines showing statistical significance in Fig. 5g, 5i, and 6g seem drawn manually and not aligned well. Were these error bars and statistical significance directly generated by the plotting software or manually marked by the author?\nAs the reviewer pointed out, the error bars in Figure 4g-i(formerly Fig. 5g‒i) were slightly misaligned. The previous figures were created in Excel and exported as images, and we assure you that no parts were hand-drawn or manipulated. The statistical significance markers were manually added using shapes, as Excel lacks built-in tools for this purpose.\nTo avoid any misunderstanding or doubt, we have now recreated all relevant graphs using GraphPad Prism, which ensures accurate placement of error bars and significance markers.\n5） The quality of the figures is still poor and far from the publishable standards. For instance:\na) (All figures) The authors use \"ng/ml\" in some figures (Fig. S1c) but \"ng/mL\" in others (Fig. S1a). Please make them consistent.\nb) (Fig. S1a-c) Panels are not properly aligned.\nc) (Fig. 3b) An extra line (blue) and arrow (black) are inserted without any annotations. Remove them.\nd) (Fig. S1c) The Y-axis title is missing.\ne) (Fig. 5k) The text and scale bars overlap.\nf) (Fig. S4b) There is no scale on the X-axis. Remove \")\" in the legend.\ng) (Fig. S14) It's weird that Fig.S14 is not included in the result section.\nh) (Fig. S5) Delete \";\" in the legend.\ni) (Fig. S8) Delete \".\" in the legend.\nj) (Fig. S6, 7 & 10). The scale bars in HE panels are not consistent. Some are labeled with 100 μm, but the others were not.\nk) (Figs. 1-6) Remove the \".\" following \"Figure\".\nWe sincerely thank the reviewer for the careful and detailed feedback. All the points mentioned have been addressed."}]

Decision letter