Knockout of integrin β1 in induced pluripotent stem cells accelerates skin-wound healing by promoting cell migration in extracellular matrix

Background Induced pluripotent stem cells (iPSCs) have the potential to promote wound healing; however, their adhesion to the extracellular matrix (ECM) might decrease iPSC migration, thereby limiting their therapeutic potential. Integrin β1 (Itgb1) is the major integrin subunit that mediates iPSC-ECM adhesion, suggesting that knocking out Itgb1 might be an effective method for enhancing the therapeutic efficacy of iPSCs. Methods We knocked out Itgb1 in mouse iPSCs and evaluated its effects on the therapeutic potential of topically applied iPSCs, as well as their underlying in vivo and in vitro mechanisms. Results The Itgb1-knockout (Itgb1-KO) did not change iPSC pluripotency, function, or survival in the absence of embedding in an ECM gel but did accelerate wound healing, angiogenesis, blood perfusion, and survival in skin-wound lesions. However, embedding in an ECM gel inhibited the in vivo effects of wild-type iPSCs but not those of Itgb1-knockout iPSCs. Additionally, in vitro results showed that Itgb1-knockout decreased iPSC-ECM adhesion while increasing ECM-crossing migration. Moreover, ECM coating on the culture surface did not change cell survival, regardless of Itgb1 status; however, the in vivo and in vitro functions of both Itgb1-knockout and wild-type iPSCs were not affected by the presence of agarose gel, which does not contain integrin-binding sites. Knockout of Integrin α4 (Itga4) did not change the above-mentioned cellular and therapeutic functions of iPSCs. Conclusions Itgb1-knockout increased iPSCs migration and the wound-healing-promoting effect of topically applied iPSCs. These findings suggest the inhibition of Itgb1 expression is a possible strategy for increasing the efficacy of iPSC therapies. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-022-03085-7.


Background
Wound-healing delays and failures seriously affect patient recovery and appearance [1]. Stem cells can differentiate into cutaneous tissue cells, promote angiogenesis, and secrete beneficial substances [2,3], thereby helping to promote wound healing. However, due to the adhesion of stem cells to the extracellular matrix (ECM), their limited migration in wounds subsequently limits their therapeutic efficacy [4,5].
Although stem cells are migratory cells, their migration in the interstitial space is mediated by the type and number of cell-adhesion molecules expressed on their surface [6,7]. To achieve therapeutic efficacy, stem cells should home to the site where they are needed. The first step is extravasation, adhering to and migrating across the vascular wall. For mouse iPSCs, this step is facilitated by the interaction of integrin α4β1 on iPSCs with VCAM-1 on activated endothelial cells (ECs); the β1 subunit is the dominant β subunit in mouse iPSCs. In the second step, iPSCs migrate through capillary basement membranes and interstitial spaces [8]. Cell-ECM interactions are usually mediated by members of the integrin family of adhesion molecules and transmembrane receptors [9]. As cell-adhesion molecules that were initially found on leukocytes [10], integrins bind a variety of extracellular ligands, including cell receptors, ECM proteins, soluble proteins in multiple body fluids, and microbial proteins and carbohydrates [11]. The ECM ligands of the integrin β1 (Itgb1) subunit include proteins such as collagens, fibronectin, laminin, and vitronectin [12]. We previously found that Itgb1 is the most abundant β subunit expressed on the surface of induced pluripotent stem cells (iPSCs) [8]. Therefore, we hypothesized that Itgb1 mediates iPSC binding to the ECM to regulate cell adhesion and migration.
Integrins are composed of noncovalently linked α subunits and β subunits that combine to produce at least 24 different heterodimers [9]. VLA-4 (α4β1) is unique among integrins, because it is the only integrin heterodimer containing the β1 submit that binds to the ligand VCAM-1 to mediate leukocyte rolling, transendothelial cell migration, and binding to the ECM. To determine whether the amount of α unit affects the function of β1-mediated iPSC-ECM interaction, iPSCs with Itgα4knockout (Itga4-KO) were also tested.
As angiogenesis and recovery of blood flow precede wound healing [13,14], promoting angiogenesis is one of the mechanisms by which stem cells facilitate tissue repair [15]. In the present study, we established Itgb1knockout iPSCs to investigate the role of Itgb1 in iPSCmediated wound healing and its underlying mechanisms.

Construction of Itgb1-and Itga4-knockout iPSCs
An Itgb1 CRISPR was designed using an online tool (https:// zlab. bio/ guide-design-resou rces) as described previously [8]. Oligonucleotides corresponding to Itgb1 were obtained from Integrated DNA Technologies (Coralville, IA, USA) and included the sense 5'-ATG GTG TGT AGC TAG GCT AATG-3' and anti-sense 5'-TGG ACT GAC ACT CTG CTT TG -3' . Single-guide (sg) RNA oligos were annealed and ligated to the Cas9 Lenti CRISPR version 2.0 vector that had been digested with BbsI to generate the individual guide plasmid, which coexpressed Cas9 and the sgRNA. The CRISPR vectors were prepared and packaged according to the manufacturer's instructions. iPSCs were infected with the CRISPR retrovirus vector and cultured in iPSC medium containing 10 μg/mL puromycin. Single-cell clones were generated from these transfected iPSCs and were expanded after 10-14 days of transduction. Mismatch loss of Itgb1 was confirmed by DNA sequencing and pairwise dot matrix comparison. Successful Itgb1-knockout iPSCs were confirmed by PCR and western blot.

Alkaline phosphatase (AP) assay
Staining of iPSC colonies was performed using the AP detection kit (#SCR004; Sigma-Aldrich) according to the manufacturer's instructions; AP activity can be identified by red staining.

Total RNA isolation and reverse transcription (RT)-PCR
RNA from skin wounds was isolated using TRIzol reagent (#15596026; Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. RNA concentrations and quality were determined by absorbance using a NanoDrop spectrophotometer (Implen GmbH, Munich, Germany) at 260/280 nm. RT-PCR was performed using an input of 1 µg total RNA using the First Strand cDNA synthesis kit (#FSK-100; TOYOBO Bio-Technology, Osaka, Japan). Quantitative PCR was performed using cDNA and real-time PCR master mix (#QPK-201; TOYOBO Bio-Technology) in an ABI 7300 real-time PCR system (Applied Biosystems, Foster City, CA, USA) with the profile: 95 °C for 5 min, followed by 40 cycles of amplification at 95 °C for 20 s, 60 °C for 20 s, and 72 °C for 20 s. RT-PCR was performed according to the manufacturer's instructions, with Actb used as the reference gene, and each sample was run in triplicate. Data were normalized to Actb expression, and relative expression was calculated using the 2 −ΔΔCt method. PCR primers were designed and confirmed by Integrated DNA Technologies

Teratoma-formation assay
Aliquots of 5 × 10 6 iPSCs in 50 µL of Matrigel (#356234; BD Biosciences, San Jose, CA, USA) per site were injected subcutaneously into C57BL/6 mice. Four weeks after injection, the mice were euthanized and tumors confirmed using hematoxylin/eosin staining and RT-PCR to determine whether teratomas contained tissues from all three germ layers.

Establishment of the full-thickness excisional cutaneous-wound model and iPSC treatments
Before creation of a full-thickness excision-wound model, mice were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg/kg body weight) after induction of sedation by an intramuscular injection of ketamine hydrochloride (10 mg/kg body weight). The dorsal skin was shaved and removed with a depilatory cream (Veet; Reckitt Benckiser, Massy, France), the central axis of the dorsal skin was lifted, and two symmetrical full-thickness cutaneous wounds were produced using 4-mm-diameter biopsy punchers (BD Pharmingen, San Diego, CA, USA). The mice received topical applications of PBS, wild-type (WT)-iPSCs, Itgα4 −/− -iPSCs immediately after creation of the wound (n = 10 mice/topical application). We used 1 × 10 4 iPSCs in 20 μL PBS, 20 μL 1% low-melting-point agarose, or 20 μL 1% Matrigel for topical application to the skin wound, with PBS (20 μL) without cells used as the control. The mice were euthanized on days 6 and 12 after wound creation, and wound tissue from dorsal skin was excised. The wound tissue was divided equally: half was stored at − 80 ℃ for the use of RNA and DNA solution, and the other half was fixed with 4% paraformaldehyde for frozen sectioning.

Survival of fluorescent iPSCs
To track iPSC fates in vivo, additional experiments were performed with the lesions having topical applications of PBS, wild-type (WT)-iPSCs, . All cells were labeled with PKH26 as described below. Lesions were monitored and scored as described for the experiment with nonlabeled cells. Furthermore, the same experiments were performed using iPSCs expressing GFP instead of PKH26-fluorescence labeled iPSCs. n = 3.

Cell labeling with PKH26 Red
iPSCs were labeled using the PKH26 Red fluorescent cell linker kit (#PKH26GL; Sigma-Aldrich) according to the manufacturer's instructions prior to their use. Cells (1 × 10 7 ) were dissociated using 0.25% trypsin-EDTA, washed with DMEM without FBS, and resuspended in 500 μL diluent C from the cell linker kit to prepare a 2 × cell suspension. PKH26 cell linker reagent (2 μL) was diluted in another volume of 500 μL diluent C as working solution, mixed thoroughly, then incubated at room temperature for 5 min. Staining was stopped by adding an equal volume of FBS and then incubation for 1 min to allow binding of excess reagent. After washing with PBS, cells were resuspended for subsequent processing.

Lentiviral GFP transfection of iPSCs
The GFP plasmids (pCMV-N-EGFP, with CMV promoter) were amplified and cloned into lentivirus constructs to generate a GFP plasmid vector. Then, the lentiviral expression constructs and the packaging plasmid mixture, including pGag/Pol, pRev, and pVSVG, were cotransfected into human 293 T cells. The GFP plasmid vectors were obtained from the supernatant at 48 and 72 h, respectively. Mouse iPSCs were transfected with these vectors and then cultured for 2 weeks in iPSC medium supplemented with 0.5 mg/ml puromycin to select for GFP-expressing iPSCs.

Wound-closure evaluation
The day of wound creation was designated as day 0. Wounds were monitored for 14 days, with images captured on days 0, 2, 4, 6, 8, 10, and 12. Wound areas were measured using ImageJ software (v.1.51 k; National Institutes of Health, Bethesda, MD, USA), and the percentage of wound closure is calculated using the equation:

Blood perfusion measurement
Perfusion in skin tissues was measured using serial scanning with a laser Laser Doppler signals were quantified using regions of interest (same size between measurements) and are presented as relative perfusion units (PUs; a relative unit defined against a controlled motility standard).

Production of frozen sections
Tissues were fixed with 4% paraformaldehyde for 24 h and then dehydrated through 15%, 20% and 30% continuous sucrose gradients. The dehydrated tissues were embedded in Tissue-Tek O.C.T. Compound (#4583, SAKURA, Finetek, USA) to ease handling and then continuously sectioned at 5 μm at − 20 °C using a Tissue-Tek cryostat (SAKURA, Tokyo, Japan).

Immunohistochemistry (IHC)
Frozen sections were subjected to antigen retrieval using 10-mM sodium citrate buffer (pH 6

Quantification of iPSCs in wound tissues
Tet-on is a noncoding gene that can be carried into iPSCs by retroviral vectors and used for iPSC induction but not expressed in recipient cells, making it useful as a marker for counting iPSC quantification. We isolated genomic DNA from wound tissues, using DNAzol reagent (#10503027; Thermo Fisher Scientific) according to the manufacturer's instructions. Each sample was subjected to PCR in triplicate. PCR products were separated and detected using 2.5% agarose gel electrophoresis. Intensities of PCR bands were measured using ImageJ software (v.1.51 k; National Institutes of Health), with 18S rDNA PCR products were used as internal controls to quantify Tet-on expression. The following Tet-on-and 18S rDNAspecific PCR primers were designed and confirmed by

Cell adhesion assay
Matrigel was slowly thawed at 4 °C and added to a 96-well plate at a concentration of 50 µL/cm 2 using precooled pipette tips. We then added 1% low-melting-point agarose (#75510019; Thermo Fisher Scientific) to another 96-well plate at a concentration of 50 µL/cm 2 to coat the plate as a nonprotein matrix control for Matrigel. The 96-well plates were incubated at 37 °C for 30 min to allow polymerization, after which a suspension of PKH26labeled iPSCs (1 × 10 3 ) was seeded onto uncoated, Matrigel, and agarose plates, followed by incubation for another 2 h. Nonadherent cells were washed away with PBS; then, adherent iPSCs were counted using fluorescence microscopy. The experiment was performed using five replicates and repeated three times.

Cell-migration assay
Matrigel (50 µL/cm 2 ) was added to the upper side of the basement membrane of a Transwell chamber (#353097; Falcon cell culture inserts for 12-well plates with 8-μm membrane pores; Corning, Corning, NY, USA) using pre-cooled pipette tips; in addition, 1% low-meltingpoint agarose (50 μL/cm 2 ) was added in the same way as a nonprotein matrix control for Matrigel. The chambers were then incubated at 37 °C for 30 min to allow polymerization, and the basement membrane was hydrated before use. A PKH26-labeled iPSC suspension (1 × 10 4 cells) was used to seed the upper side of the basement membrane, followed by incubation for 36 h. Nonmigrating cells on the upper side of the basement membrane in the Transwell chambers were washed away with PBS; then, migrated iPSCs were counted using fluorescence microscopy. The experiment was performed using three replicates and repeated three times.

Cell proliferation assay
Matrigel (50 µL/cm 2 ) was added to a 96-well-plate using pre-cooled pipette tips, and 1% low-melting-point agarose (50 µL/cm 2 ) was added to another 96-well plate as a nonprotein matrix control for the Matrigel, followed by incubation at 37 °C for 30 min to allow polymerization. Plates were seeded with iPSCs (5 × 10 3 cells/well) in culture medium. Proliferation was measured using Cell Counting Kit-8 (CCK-8) reagent (#CKD4; Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer's instructions. Absorbance at 450 nm was measured using a microplate spectrophotometer (BioTek, Winooski, VT, USA) at 0, 24, 48, 72, and 96 h after seeding. The experiment was performed using six replicates and repeated three times.

Apoptosis assay
Cells were scored using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and an annexin V/FITC apoptosis detection kit (#AD10-10; Dojindo Laboratories), with 10,000 events acquired and analyzed using Cell Quest Software (BD Biosciences). iPSCs were cultured on plates that were uncoated or precoated with Matrigel or agarose for 48 h, followed by detachment of the iPSCs using trypsin to prepare a single-cell suspension and transfer of 2 × 10 5 cells to a flow tube for washing with 2 mL PBS. Cells were resuspended in 200 µL of 1 × Annexin-V binding buffer to prepare a suspension at 1 × 10 6 cells/mL. We then added 5 μL of Annexin-V/ FITC solution and 10 μL propidium iodide-staining solution to the suspension, which was incubated in the dark for 15 min. Cells were analyzed within 1 h by flow cytometry. The experiment was repeated three times. Data were analyzed using FlowJo software (v.10.0; FlowJo LLC, Ashland, OR, USA). Annexin V-FITC − /PI + (Q1), annexin V-FITC + /PI + (Q2), annexin V-FITC + /PI − (Q3) and annexin V-FITC − /PI − (Q4) were necrotic, late apoptotic, early apoptotic and living cells, respectively. Their percent rates out of total cells (Q1 + Q2 + Q3 + Q4) were calculated, respectively.

Statistical analysis
Data are presented as mean ± standard deviation, with an unpaired t-test used for comparisons between two correspondent groups. p < 0.05 was considered significant.

Absence of ITGß1 accelerates wound healing by iPSCs
We created a full-thickness excisional cutaneous-wound model on the dorsal skin of mice, followed by topical transplantation of iPSCs or an equal volume of PBS as a control. Macroscopic evaluation was performed at two-day intervals after wounding. We found that the wound healing with WT-iPSC treatment was significantly more rapid than that with the PBS control on days 2 ( (Fig. 2a).
We also performed serial laser Doppler imaging of the dorsal skin at 2 days post-wounding. Numbers of blood PUs with WT-iPSC treatment were significantly higher than those in PBS controls on days 2 (423. 19 (Fig. 2d).

Absence of ITGß1 increases iPSC survival in wounds
To assess in vivo iPSC survival, we quantified the expression of the Tet-on gene carried into iPSCs by the retrovirus used for iPSC induction in wound tissues on days 6 and 12 post-treatment. Tet-on expression after Itgb1 −/− -iPSC treatment was significantly higher than that after WT-iPSC treatment on days 6 (1.37 ± 0.19 vs 0.88 ± 0.29) and 12 (1.32 ± 0.50 vs 0.80 ± 0.22) post-treatment (Fig. 2e).

Absence of ITGß1 does not affect proliferation, apoptosis, adhesion, or migration in vitro in the absence of Matrigel
We then assessed adhesion and -migration capabilities of WT and Itgb1-knockout iPSCs. We found no significant differences in either capability (Fig. 3a, b). To assess in vitro iPSC survival, we measured apoptosis and proliferation of WT-and ITGβ1 −/− -iPSCs in the absence of Matrigel. We found no significant difference in either activity between Itgb1 −/− -and WT-iPSCs (Fig. 3c, d).

iPSC adhesion to Matrigel and migration are ITGß1-dependent
Collagen is the primary ligand for integrin β1 in the ECM. We tested the hypothesis that adhesion and migration in the ECM are the key factors responsible for the treatment effect. The numbers of Itgb1 −/− -iPSCs that adhered to Matrigel in 2 h were significantly lower than the numbers of WT-iPSCs (9.00 ± 2.53 cells/mm 2 vs. 15.40 ± 3.07 cells/mm 2 ). The numbers of Itgb1 −/− -iPSCs that adhered to collagen in 2 h were significantly lower than the numbers of WT-iPSCs (9.30 ± 2.02 cells/mm 2 vs. 14.57 ± 2.39 cells/mm 2 ). By contrast, using agarose as a nonprotein control matrix for Matrigel revealed no significant difference between genotypes. However, the numbers of WT-iPSCs that adhered to Matrigel in 2 h were significantly higher than the numbers that adhered to agarose (15.40 ± 3.07 cells/mm 2 vs. 11.20 ± 1.16 cells/ mm 2 ), whereas there was no significant difference for Itgb1 −/− -iPSCs between Matrigel and agarose (Fig. 4a).

Absence of ITGß1 rescues Matrigel-mediated inhibition of accelerated wound healing
We next used Matrigel to simulate ECM, with agarose as a nonprotein matrix control, to quantify the effect of the ECM on healing. We created a full-thickness excisional cutaneous wound on the dorsal skin of mice and transplanted iPSCs embedded in Matrigel or agarose topically, followed by macroscopic evaluation 2 days later to quantify wound healing. Healing with Matrigelembedded WT-iPSCs was significantly decreased relative to that with agarose-embedded WT-iPSCs on days 2 (22.14 ± 3.69% vs. 29.59 ± 2.54%) and 4 (37.78 ± 4.18% vs. 43.51 ± 4.41%) post-treatment, whereas we observed no significant difference between Matrigel-and agaroseembedded Itgb1 −/− -iPSC treatment groups on any day (Fig. 5a).

Absence of ITGß1 rescues Matrigel-mediated inhibition of accelerated angiogenesis and blood perfusion
We next performed IHC staining for CD31 to assess the effect of the ECM on angiogenesis at days 6 and 12 posttreatment. CD31 expression with Matrigel-embedded WT-iPSC treatment was significantly lower than that with agarose-embedded WT-iPSC treatment on day 12 (6.36 ± 1.09% vs. 9.30 ± 2.14%), whereas there was no significant difference in CD31 expression between Matrigelembedded Itgb1 −/− -iPSC and agarose-embedded WT-iPSC treatments on days 6 and 12 post-treatment (Fig. 5b). Quantitative RT-PCR revealed significantly lower Cd34 expression with Matrigel-embedded WT-iPSC treatment relative to that with agarose-embedded WT-iPSC treatment on day 6 (0.83 ± 0.12 vs. 1.00 ± 0.09) post-treatment, whereas there was no significant difference observed between the Matrigel-embedded Itgb1 −/− -iPSCs and agarose-embedded WT-iPSC treatments on either day (Fig. 5c, left panel). Moreover, relative Cd31 expression with Matrigel-embedded WT-iPSC treatment was significantly lower than that with agaroseembedded WT-iPSC treatment on day 12 (0.88 ± 0.10 vs. 1.00 ± 0.09) post-treatment, whereas no significant difference was observed between the Matrigel-embedded and agarose-embedded Itgb1 −/− -iPSC treatments on days 6 and 12 post-treatment (Fig. 5c, right panel). We then performed serial laser Doppler imaging of the dorsal skin of mice to measure iPSC-mediated acceleration of blood perfusion during wound healing. Perfusion with Matrigel-embedded WT-iPSC treatment was significantly lower than that with agarose-embedded WT-iPSC treatment on days 2 (385.20 ± 60.75 PUs vs. 483.20 ± 41.80 PUs) and 4 (354.10 ± 45.25 PUs vs. 465.40 ± 103.32 PUs) post-treatment, whereas no significant difference in perfusion was observed between the Matrigel-embedded and agarose-embedded Itgb1 −/− -iPSC treatments on any day post-treatment (Fig. 5d).

Absence of ITGß1 rescues Matrigel-mediated inhibition of iPSC survival in skin wounds
We then measured Tet-on expression to evaluate iPSC survival on days 6 and 12 post-treatment. We found significantly lower expression with Matrigel-embedded WT-iPSC treatment was significantly lower than that with agarose-embedded WT-iPSC treatment on days 6 (0.62 ± 0.19 vs. 0.84 ± 0.17) and 12 (0.66 ± 0.34 vs. 1.08 ± 0.68), whereas no significant difference was observed between the Matrigel-embedded and agarose-embedded Itgb1 −/− -iPSC treatments on either day (Fig. 5e).

Tracking of fluorescently labeled cells in skin wounds
We used iPSCs labeled using PKH26-and GFP-overexpressing iPSCs to detect their presence in the skin wounds at days 6 and 12. Although fluorescent cells were identified, they were difficult to quantify because of their uneven distribution; moreover, they were difficult to differentiate from autofluorescence, regardless of Itgb1 genotype, Matrigel embedding, or agarose embedding (Additional file 1: Figs. S1 and S2).

Absence of the integrin alpha-4 subunit alters neither iPSC behavior nor therapeutic efficacy
To test for a role for integrin alpha subunits, we knocked out Itga4 in iPSCs using CRISPR-Cas9; the targeting was confirmed by DNA sequencing (Additional file 1: Fig.  S3). The absence of ITGα4 had no effect on iPSC pluripotency, function, or survival in culture (Additional file 1: Figs. S3, S4 and S5); nor did it affect wound healing, angiogenesis, blood perfusion, or survival in skin wounds in vivo (Additional file 1: Figs. S1, S2 and S6), independent of Matrigel or agarose-embedding.

Discussion
The therapeutic potential of iPSCs is enormous [17]. We have previously demonstrated that topical administration of iPSCs to treat skin wounds promotes angiogenesis, accelerates wound healing, and avoids the side effect of teratoma formation [18]. However, the extent of the therapeutic efficacy of these administered cells remains limited to the site on which the cells are delivered, which also limits the distance through which they can act [19,20]. Previous reports indicate that integrins mediate adhesion between stem cells and ECM [21][22][23]; we previously reported that ITGβ1 is expressed at high levels on the surface of iPSCs. The expression level of β1 was more than 1000-fold higher than that of β2 [8]. We hypothesized that although stem cell-ECM adhesion mediates stem-cell anchoring, adhesion also decreases the migratory potential of stem cells, thereby reducing their area of action. Furthermore, ITGβ1 inhibition or gene knockout reduces iPSC adhesion to the ECM, extends iPSCs distribution in skin tissues, and promotes wound healing. The mechanisms by which iPSCs function in cutaneous wound healing include accelerating re-epithelialization and promoting angiogenesis and blood perfusion [8].
In the present study, the Itgb1 knockout had no influence on pluripotency or in vitro function in the absence of Matrigel. Using a full-thickness excisional cutaneous wound model, we found that topical administration of iPSCs promoted early-stage (days 2-6 post-treatment) wound healing, angiogenesis, blood perfusion, and iPSCs survival with WT-iPSC treatment relative to controls and that these activities were significantly enhanced by Itgb1 −/− -iPSCs relative to the WT-iPSCs. During wound healing, blood flow changes over time. High perfusion occurs in the early stage of wound repair. With the granulation tissue remodeled into a scar in the advanced stage of wound repair, blood flow decreases to normal levels gradually, because in normal tissue only a portion of capillaries open under the control of capillary sphincters. Thus, at the time that the treated lesions had finished repairing while untreated lesions had not, blood perfusion in untreated lesions was expected to be higher.
The clinical efficacy of stem-cell transplantation depends on the quantity of stem cells needed to treat damaged sites; therefore, the development of more efficient methods for their delivery is essential for enhancing the survival of transplanted cells and maximizing their therapeutic efficacy [24,25]. A previous study reported that ITGβ1 plays an essential role in adhesion to the ECM and adjacent cells, serving as the link between extracellular molecules and the intracellular cytoskeleton [26]. In the present study, we showed that knocking out Itgb1 improved the efficacy of iPSCs in accelerating cutaneous wound healing, suggesting that ITGβ1 mediates interactions between transplanted iPSCs and the ECM, limiting the extent of iPSC activity to the site of their delivery.
To investigate the effect of ITGβ1-mediated cell-ECM interaction on cutaneous wound healing, we used Matrigel to simulate the ECM, with agarose as a nonprotein matrix control, and compared their effects on WT and Itgb1-knockout iPSCs. Agarose gel has a similar pore size and stiffness as Matrigel, but does not contain ITGβ1-binding sites. We observed significantly lower levels of wound healing, angiogenesis, blood perfusion, and iPSC survival with Matrigel embedding than with agarose embedding during early wound healing (days 2-6) whereas we observed no significant differences between Matrigel and agarose embedding of Itgb1 −/− -iPSCs throughout healing. Our results suggest that the absence of ITGβ1 is beneficial in wound healing when cells are applied topically. When embedded in agarose gel, the effect of iPSCs was not altered by knocking out Itgb1. Since collagen is a primary ligand of integrin β1 in the ECM, we used it as a known ligand control, in addition to agarose. The effects of collagen and Matrigel embedding were similar, further confirming the importance of the presence of ITGβ1-binding sites.
Our result showed knockout of integrin α4 did not change the interaction between iPSCs and ECM; nor did it influence the therapeutic efficacy in our model.
The high apoptosis rate of transplanted stem cells in the niche of recipient tissue is a major problem. Integrinmediated adhesion between cells and the ECM promotes cell migration [27,28]. Our in vitro data showed that Matrigel promotes migration and increases apoptosis in Itgb1 −/− -iPSCs compared to WT-iPSCs, which may lead to a decreased presence of Itgb1 −/− -iPSCs in wounds. However, our in vivo experiments showed that Itgb1 −/− -iPSCs are more abundant than WT-iPSCs in lesions. Cell survival depends on both cell-intrinsic factors and microenvironment. Because the Itgb1 knockout promotes cell migration, the topically applied iPSCs leave the wound surface earlier and migrated more deeply. Therefore, such iPSCs may inhabit a microenvironment that better promotes their survival.
We tried to track the fate of fluorescently labeled iPSCs in the wound lesions, but were unsuccessful. It is possible that the fluorescence faded and/or cells expressing GFP were killed by antibodies against GFP. The possibility of being killed by antibodies against GFP is supported by the existence of many commercially available mouse antibodies against GFP. It was also one of the reasons that we did not use GFP-expressing iPSCs for the main experiment.
Stem cells have been widely used in wound healing. The stromal vascular fraction (SVF) containing adipose stem cells (ASCs) has been routinely used for regenerative medicine and surgical applications. ASC-based therapies could be based on the use of decellularized ECM as a scaffold re-cellularized by ASCs of the recipient, as the safety and efficacy of allogenic ASCs and ECM transplants have been demonstrated without major side effects [29,30]. Tissue bioengineering is an ideal therapeutic approach to wound healing and soft-tissue defects; platelet-rich plasma (PRP) plays an important role, as the