Noninvasive application of mesenchymal stem cell spheres derived from hESC accelerates wound healing in a CXCL12-CXCR4 axis-dependent manner

Mesenchymal stem cells (MSC) derived from adult tissues effectively promote wound healing. However, MSC quality varies, and the quantity of MSC is limited, as MSC are acquired through donations. Moreover, the survival and functioning of dissociated MSC delivered to an inflammatory lesion are subject to challenges. Methods: Here, spheres (EMSCSp) generated from human embryonic stem cell-derived MSC (EMSC) were directly dropped onto excised wounds in mice; the effects of EMSCSp were compared to those of dissociated EMSC (EMSCDiss). Following transplantation, we measured the extent of wound closure, dissected the histological features of the wounds, determined transcriptomic changes in cells isolated from the treated and control wounds, and evaluated the molecular mechanism of the effects of EMSC. Results: The application of EMSCSp onto murine dermal wounds substantially increased survival and efficacy of EMSC compared to the topical application of EMSCDiss. RNA sequencing (RNA-Seq) of cells isolated from the wounds highlighted the involvement of CXCL12-CXCR4 signaling in the effects of EMSCSp, which was verified in EMSC via CXCL12 knockdown and in target cells (vascular endothelial cells, epithelial keratinocytes, and macrophages) via CXCR4 inhibition. Finally, we enhanced the biosafety of EMSCSp by engineering cells with an inducible suicide gene. Conclusions: Together, these data suggest the topical application of EMSCSp as an unlimited, quality-assured, safe, and noninvasive therapy for wound healing and the CXCL12-CXCR4 axis as a key player in this treatment.

iC9-P2A-Neo cassette was then cloned into EcoRI/SalI sites of pCDH-CAG-MCS-T2A-Puro as described above to obtain the pCDH-CAG-iC9-P2A-Neo. The resultant lentiviral vector was packaged as described above. Lentiviruses were collected to transduce H9 hESC, followed by G418 selection for 2 weeks. Single cell-derived colonies were picked via serial dilution to establish the iC9 stable hESC line.
For the construction of the shCXCL12 lentiviral vector, oligonucleotides for shCXCL12 were synthesized and annealed, and the resultant DNA fragment was cloned into the KpnI/EcoRI sites of pLKO_TRC005. The shLacZ lentiviral vector TRCN0000072225 (a gift from William Hahn via Addgene #78160) was used as a control. Both vectors were packaged as described above, and the resultant lentiviruses were collected and used to transduce EMSC followed by puromycin selection for 2 weeks. Primers and oligonucleotides used for the vector constructions are listed in Table   S2.

Histologic examinations and immunostaining
Tissue specimens were fixed in 4% paraformaldehyde, dehydrated in a graded ethanol series and embedded in paraffin. Sections in 5-µm thickness were stained with hematoxylin and eosin for microscopy. Each slide was given a histological score, according to parameters including re-epithelialization, cell infiltration, granulation formation, and angiogenesis as reported [3] with slight modification as detailed in Table   1. For Masson trichrome collagen staining, the whole procedure was performed according to the manual of Heart Biological Technology.
Immunofluorescence staining was performed using a standard procedure. In brief, after antigen retrieval, tissue sections were treated with 0.3% Triton X-100 for 10 min. and washed extensively in PBS, blocked with 5% BSA for 1 h, then the sections were stained with specific primary antibodies: EMSC (Envy), endothelial cells, smooth muscle cells, keratinocytes, and macrophage were detected by antibodies against GFP, CD31, SMA, epidermal keratin subunits, and MAC2, respectively with the nucleus counterstained with DAPI. Cell proliferation was detected with a primary antibody against Ki67. All the staining was visualized with fluorescent-conjugated secondary antibodies. Images were captured with Carl Zeiss Axio Observer microscope.

Cell migration via Transwell assays
The chemotactic motility of cells was performed using a Transwell plate with an upper insert containing a polycarbonate membrane at 3.0-μm pore size (Corning). For migration assay, 2 x 10 5 HUVEC or macrophage per well were suspended in fresh medium and seeded into the upper insert, and spheres containing a total of 1x10 6 EMSC were seeded in the lower compartment. After 24-h incubation, cells migrated across the membrane of the insert were stained with 0.5% crystal violet and counted in seven random fields using a light microscope at 20X magnification. For inhibition of CXCR4, HUVEC or HaCaT cells were pretreated with AMD3100 for 30 min, followed by thorough washing and seeding the cells to the insert of Transwell as above.

Detection of EMSC retained in various organs of the host following transplantation
At various days after transplantation of EMSCSp (Envy) onto mouse wounds, genomic DNA (gDNA) was isolated from the wound skin and some major organs of the mice, using a gDNA extraction kit (Tiangen), according to manufacturer's instructions. The DNA concentration and purity were estimated by measuring the optical density. Two hundred ng of purified DNA from each sample were amplified using the TaqMan Universal PCR Master Mix. Testing of target DNA fragments was performed via realtime quantitative PCR (qPCR) with primers and probes for the transgene and GAPDH listed in Table S2. gDNA isolated from 1 x 10 6 Envy hESC was used as a positive control, and a standard curve was generated using qPCR to calculate the amount of the transgene (ng) per µg of gDNA, which reflects the relative number of EMSC (Envy) retained in each sample.
For long-term observation, four months after transplantation with EMSCSp or EMSCDiss onto wounds, gDNA from the wound-site skin and several other organs were isolated. qPCR was performed to measure the amount of gDNA for human thymidine kinase-1 (hTK1) [4]. Vehicle-treated control was tested as the negative control, and EMSC-transplanted day-1 wound as positive controls. The hTK1 level was normalized by the amount of total gDNA.

Teratoma formation assay
1×10 6 hESC or EMSC were injected subcutaneously into a hind leg of NOD/SCID mice.
After 8 weeks, resulting teratomas were surgically dissected out of the mice and fixed with 4% paraformaldehyde. The samples were embedded in paraffin, sectioned into 5µm slices, and stained with hematoxylin and eosin.

Assays of apoptotic, live, and dead cells
For EMSC stably transduced with iC9, the AP20187 was added at 20 nM to the cell culture. The cells were harvested at various times, stained with Annexin V and propidium iodide (PI) to determine the percentage of apoptotic cells via flow cytometry.
For isolated skin wound, the tissues were digested as described above, and resultant single cells stained with acridine orange (AO)/PI. The stained cells were photographed and quantified on Cellometer.

Enzyme-linked immunosorbent assay (ELISA)
Condition medium was collected as above, CXCL12 protein was measured by using an ELISA kit (Thermo Scientific) according to the standard procedures provided by the manufacturer. A microplate reader was used to read the optical density at 450 nm.

EMSC and macrophage interaction assay
Co-culture of EMSC with RAW264.7 macrophage was performed in a Transwell plate with an inset per well which contained a polycarbonate membrane with an 8.0-μm pore size (Corning). Two x 10 5 EMSC per well were suspended in fresh medium and seeded in the upper insert, and 2 x 10 5 RAW macrophage seeded in the lower compartment.
After 48-h incubation, the insert with EMSC was removed, and the macrophages in the bottom were subjected to the following experiments, either immunostaining for the M2 macrophage marker CD206 or qRT-PCR with primers whose sequences are listed in Table S2.

Macrophage phagocytosis assay
EMSC or nothing seeded in the upper insert of a Transwell and RAW264.7 macrophage seeded in the lower compartment were co-cultured as above. Forty-eight hours later, the macrophage were incubated with GFP-labeled E. coli bioparticles (200 μg/ml).
After 1-h incubation, the cells were fixed, counterstained with DAPI for the nuclei, and photographed. The number of macrophage (DAPI + ) with phagocytosis (GFP + ) over the total number of macrophage per view was calculated as % of phagocytosis.           The blue peaks represent isotype controls and red peaks depict the expression of the cell surface markers. Since Envy hESC-derived EMSC were GFP + , they were not suitable for detection of CD90 could not be tested as the antibody used the green fluorescent channel FITC.