Preparation and culture of human WJ-MSCs

This study was approved by the Institutional Review Board of Asan Medical Center (No. 20150303), and the WJ-MSCs were provided by the Stem Cell Center, Asan Institute for Life Sciences, Seoul, Korea. All experiments were performed in accordance with relevant guidelines and regulations. Informed consent from the mothers was obtained for the use of umbilical cords. Umbilical cords were cut into 0.31.0cm pieces without blood vessels. The matrix was minced and transferred to culture dishes in minimal essential medium supplemented with 10% fetal bovine serum and an antibioticantimycotic mixture at 37C in a 5% CO2 incubator in vitro as described previously45. When the cells reached 80% confluency, they were replated at a 1:3 split ratio.

This study complied with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of Asan Medical Center and Ulsan University College of Medicine (No. 2017-12-127), and all the following methods were performed in accordance with the relevant guidelines and regulations. After isoflurane induction, rats were euthanized by CO2 inhalation. Sciatic nerve segments (10mm in length) were harvested from male SpragueDawley (SD) rats (78weeks old, weight 250350g) (Orient Bio Inc., Seongnam, Korea). To prepare ANGs, sciatic nerve pieces were decellularized using a series of detergents as described by Shin et al.2. Briefly, the nerves were treated with detergents, including aprotinin, CHAPS, and DNase and RNase solutions. Then, the decellularized segments were washed several times with phosphate-buffered saline (PBS) to remove residual reagents and stored in PBS at 4C until use. All solutions were autoclaved or filter-sterilized before use.

Seventy-four adult male SD rats were randomly assigned to two groups: WJ-MSCs group (which was implanted with WJ-MSCs-laden ANG; n=37) and control group (which was implanted with ANG only; n=37). After anesthetization, the left sciatic nerve of rats was exposed and transected, and 10mm of the nerve was removed. The 10-mm piece of WJ-MSCs-laden ANG or ANG was sutured using 90 nylon (Ethicon, Somerville, NY) under a microscope.

Seven rats were selected from each group, and their ankle angles at the toeoff phase were measured at 4, 8, 12, and 16weeks postoperatively to evaluate serial functional recovery. A walking track (length 1m, width 10cm, and height 10cm) was built for this test. During the test, video was acquired with a digital camera (Canon SX730HS, Canon, Tokyo, Japan) at a distance of 1m and calibrated to prevent optical distortion. Records were repeated until three satisfactory trials were obtained per rat. The ankle angle at the toeoff phase was measured at maximal plantar flexion in the experimental lateral ankle joint. After the foot and leg segments were manually identified in the video frames, the ankle angles at the toeoff phase were displayed in degrees.

All rats were anesthetized at 16weeks postoperatively, and the maximum isometric tetanic force was measured. The sciatic nerve was fully exposed through previous operation incision, and another skin incision was made anterior to the ankle to expose and transect the tibialis anterior (TA) tendon distally. The TA tendon was connected to a force transducer using a custom clamp with the knee and ankle joints immobilized to a platform. A bipolar stimulator (Grass S88, Grass Instrument Corp, Quincy, MA) was used to generate stimulus and processed on a computer using LabVIEW software (National Instruments, Austin, TX). All contractions were performed at supramaximal voltage to ensure maximal activation of all TA motor units. The strength of muscles was standardized as a percentage of the value from the contralateral side.

Sciatic nerve axonal regeneration in each group was directly examined using toluidine blue staining at 16weeks postoperatively. The implanted sciatic nerves were harvested by including the distal sites, and 2.5% glutaraldehyde solution was used for fixation. The harvested nerves were further fixed in 1% osmium tetroxide, dehydrated in ethanol, and embedded in EPON resin (Miller-Stephenson Chemical Co., Sylmar, CA, USA). Cross-sections (1m thick) were stained with toluidine blue to visualize myelin with light microscopy. Digital images of nonoverlapping fields were taken at 400magnification using unbiased random sampling. The total number of myelinated axons was calculated using ImageJ software (National Institutes of Health, Bethesda, MD).

RT-qPCR was performed to evaluate the mRNA expression levels of factors related to peripheral nerve regeneration. First, macrophage markers (CD206 and interleukin 10 [IL10]) were assessed to investigate the immunomodulatory effects of WJ-MSCs. Second, NGF, BDNF, and VEGF were assessed to analyze the paracrine effects of WJ-MSCs on ANGs. Third, SC markers (S100 and MBP) were assessed to confirm the recruitment of SCs by WJ-MSCs in ANGs. All experiments were performed using nerve grafts harvested from five rats in each group at 3, 7, and 14days postoperatively. Gene expressions were analyzed as described previously5. Total RNA was isolated from ANGs or WJ-MSCs-laden ANGs using TRIzol (Thermo Fisher Scientific). Approximately 1g of total RNA was used for cDNA synthesis using a first-strand cDNA synthesis kit. Quantitative estimation of mRNA expression was conducted using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems/Thermo Fisher Scientific). All experiments were performed in triplicates and independently repeated more than three times. The following primers were used: CD206 (forward, TTA CTT TAA GGG GGC GTG TG; reverse, AGT TGG TTG GGG AGT GTC AG), IL10 (forward, CTC CAC CTG GCA AAC AAA AT; reverse, CTG CCT AGC CCA CAA AGA AG), NGF (forward, ACT CGG CTC CTT TGA GTT GA; reverse, CCC GTC CTA CAG AAG CAG AG), BDNF (forward, GAA GGT GAG GAA AGC AGC AC; reverse, TGC ACA GTC ATC TGG AAA GC), VEGF (forward, TGC TTC CTA GTG GGC TCT GT; reverse, CAC ACA TAC ACT CCG GCA TC), S100 (forward, GAA TTG GGG CAG AGA AAT GA; reverse, GGC TTG AGC TTC TTG GAA TG), MBP (forward, AAT GTT TCA GGG CAC CGT AG; reverse, AAA AAC CAG CCA GCT GAG AA), and GAPDH (forward, ATG GTG AAG GTC CCT GTG AAC G; reverse, CTT GCC GTG GGT AGA GTC AT). The comparative Ct method (2Ct)46 was used to analyze the relative amount of gene expression.

The protein levels of CD206, CD68, NGF, BDNF, VEGF, and S100 were evaluated using immunofluorescence staining at 3, 7, and 14days postoperatively. CD68 (a total marker of all macrophages) was analyzed to identify and quantify macrophages. In addition, double-staining for human nuclei and S100 was performed only in the WJ-MSCs group at 3 and 7days postoperatively to investigate the differentiation potential of WJ-MSCs in ANGs. All experiments were performed using nerve grafts harvested from five rats in each group.

The grafts were snap-frozen in liquid nitrogen with frozen section compound (Leica Biosystems, Wetzlar, Germany) as described previously47. Samples were cut into 6-m-thick cross-sections using a Cryo-Star HM560 freezing microtome (Thermo Fisher Scientific). After fixation, sections were permeabilized and then blocked with 10% normal goat serum. Thereafter, they were incubated overnight with the primary antibody at 4C. The following antibodies were used: anti-CD206 (ab64693; Abcam, Cambridge, UK), anti-CD68 (ab201340; Abcam), anti-NGF (ab6199; Abcam), anti-BDNF (ab108319; Abcam), anti-VEGF (ab1316), anti-S100b (ab52642; Abcam), and anti-hNuclei (MAB1281; Millipore, Burlington, MA, USA). Secondary antibodies, anti-rabbit Alexa 555 (A32732, Invitrogen), and anti-mouse Alexa 546 (Invitrogen A11003), were applied for 1h at room temperature in the dark. Finally, 4,6-diamidino-2-phenylindole was used to counterstain nuclei. Analysis of staining was performed using an LSM-810 confocal microscope (Zeiss, Oberkochen, Germany) at 100magnification.

All experiments were repeated three times or more. Values are presented as meanstandard error of the mean (SEM). Statistical significance was considered at p<0.05. All statistical analyses were performed using Students t-test in IBM SPSS Statistics for Windows v. 27.0 (IBM, USA).

Go here to read the rest:
Mechanisms of Wharton's Jelly-derived MSCs in enhancing ... - Nature.com