iPSC-derived megakaryocytes and platelets accelerate wound healing and angiogenesis

Abstract

Background: Platelet-rich plasma (PRP), which is prepared by concentrating platelets in autologous blood, shows efficacy in chronic skin wounds via multiple growth factors. However, it exhibits heterogeneity across patients, leading to unstable therapeutic efficacy. Human induced pluripotent stem cell (iPSC)-derived megakaryocytes and platelets (iMPs) are capable of providing a stable supply, holding promise as materials for novel platelet concentrate-based therapies. In this context, we evaluated the effect of iMPs on wound healing and validated lyophilization for clinical applications.

Methods: The growth factors released by activated iMPs were measured. The effect of the administration of iMPs on human fibroblasts and human umbilical vein endothelial cells (HUVECs) was investigated in vitro. iMPs were applied to dorsal skin defects of diabetic mice to assess the wound closure rate and quantify collagen deposition and angiogenesis. Following the storage of freeze-dried iMPs (FD-iMPs) for three months, the stability of growth factors and their efficacy in animal models were determined.

Result: Multiple growth factors that promote wound healing were detected in activated iMPs. iMPs specifically released FGF2 and exhibited a superior enhancement of HUVEC proliferation compared to PRP. Moreover, an RNA-seq analysis revealed that iMPs induce polarization to stalk cells and enhance ANGPTL4 gene expression in HUVECs. Animal studies demonstrated that iMPs promoted wound closure and angiogenesis in chronic wounds caused by diabetes. We also confirmed the long-term stability of growth factors in FD-iMPs and their comparable effects to those of original iMPs in the animal model.

Conclusion: Our study demonstrates that iMPs promote angiogenesis and wound healing through the activation of vascular endothelial cells. iMPs exhibited more effectiveness than PRP, an effect attributed to the exclusive presence of specific factors including FGF2. Lyophilization enabled the long-term maintenance of the composition of the growth factors and efficacy of the iMPs, therefore contributing to stable supply for clinical application. These findings suggest that iMPs provide a novel treatment for chronic wounds.

Keywords: Growth factors; Immortalized megakaryocyte cell lines; Lyophilization; Platelets; Wound healing.

Fig. 1

Preparation of iMPs and measurement of growth factors. A Schema of the preparation. Following 5 days of imMKCL differentiation with DOX removal, a mixture of mature megakaryocytes and platelets were collected as iMPs. B Concentration of growth factors in iMPs that have been previously reported to promote wound healing. Following the activation, PDGF-BB, TGF-β, EGF, VEGF, FGF2, and IGF1 in the supernatant were quantified using ELISA. n = 3. C Thrombin dependency of the growth factors released from iMPs. Their concentrations were determined with and without thrombin during the activation. n = 3. Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. NS: not significant. ND: not detected

Fig. 2

Effects of iMPs on fibroblasts in vitro. A Shema of the assay using the NICO-1 co-culture system. This system has a filter of 0.6 μm between two linked wells, enabling evaluation of the paracrine effect in a biological wound environment. Primary cells were seeded in one well, while culture medium containing 10% PBS, PRP, or iMPs filled the other well. B Fibroblast proliferation. Following 72 h of co-culture, the number of fibroblasts per well was counted. n = 6. C Cell cycle of fibroblasts. Representative plots show 7AAD staining after 72 h of co-culture. The positive rate of 7AAD is indicated in the bar graph. n = 6. D Gene expressions of ACTA2, FGF2, and VEGF in fibroblasts. ACTA2 is a differentiation marker of myofibroblasts. FGF2 and VEGF are secreted from fibroblasts and contribute to cell-cell interactions in wound healing. n = 6. E Concentrations of FGF2 and VEGF released into the culture medium from fibroblasts following the co-culture. n = 6. Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Fig. 3

Effect of iMPs on vascular endothelial cells in vitro. A HUVEC proliferation. Following 72 h of co-culture, the number of HUVECs per well were counted. n = 6. B Cell cycle of HUVECs. Representative plots show 7AAD and Ki67 staining after 72 h of co-culture. G₀, 7AAD⁻Ki67⁻; G₁, 7AAD⁻Ki67⁺; G₂/M, 7AAD⁺Ki67⁺. The percentage of cells in G₀, G₁, and G₂/M is indicated in the bar graphs. n = 6. C Evaluation of HUVEC migration by the transwell assay. Cells that penetrated into the lower side of the membrane, which was placed on the well containing iMPs or PRP, were stained with crystal violet. The calculated area of stained cells is shown in the bar graph. n = 6. D Effect of FGF2 on HUVEC proliferation. 1.5 or 10 ng/mL FGF2 was added to the culture medium, and the cell number was counted after 72 h of incubation. n = 6. E FGFR-inhibition assay. The FGFR inhibitors Pemigatinib and Futibatinib or the control DMSO were administered to HUVECs at the initiation of the co-culture with iMPs. The number of HUVECs was determined after 72 h of co-culture. n = 6. Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 4

Changes in HUVEC gene expressions induced by PRP, FGF2, and iMPs. Total RNA of HUVECs in each group was extracted for RNA-seq following the co-culture described in Fig. 3. n = 3. A The number of differentially expressed genes (DEGs) compared with the control group. B The top 20 most significantly upregulated pathways in the GSEA. The respective normalized enrichment score (NES) is indicated in the bar graphs. The overall data is available in supplementary table S1. C Enrichment of the cell cycle pathway in the iMPs and FGF2 groups. D Enrichment of the collagen formation pathway in the PRP group. In C and D, the fragments per kilobase of exon per million reads mapped (FPKM) of individual genes included in the gene sets is indicated. Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus the control group. † p < 0.05, †† p < 0.01, ††† p < 0.001, †††† p < 0.0001

Fig. 5

Pairwise comparison of HUVEC gene expressions between the FGF2 and iMPs groups. A The number of differentially expressed genes (DEGs) in the iMPs group in comparison with the FGF2 group. B Pathways detected using g: profiler. Both upregulated and downregulated genes in the iMPs group compared to the FGF2 group were analyzed with g: profiler. The top 10 most significantly enriched pathways included in the GO: biological process are shown. All enriched pathways are available in supplementary table S2. C A volcano plot of individual genes in both groups. Significantly upregulated or downregulated genes that are included in the vascular development pathway are indicated as red dots. D Expressions of genes related to the polarity of endothelial cells. Marker genes of stalk cells and tip cells that showed significantly different expression between the two groups are represented in the heatmaps. The fragments per kilobase of exon per million reads mapped (FPKM) values were used to generate the heatmaps. ** p < 0.01, *** p < 0.001, **** p < 0.0001. E Expression of ANGPTL4 gene in the two groups. n = 3. F Knockdown of ANGPTL4. siANGPTL4 or non-targeting control siRNA was transfected into HUVECs. Afterwards, the cells were co-cultured with iMPs or administered FGF2. The number of HUVECs in each group was counted after 72 h. n = 6. G Knockdown efficiency of siRNA. The expression of ANGPTL4 in HUVECs was evaluated by RT-qPCR 72 h after the transfection. Three independent experiments were performed in technical duplicate. Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. NS: not significant

Fig. 6

Effectiveness of iMPs in wound healing of mouse skin. A Schematic illustration representing the in vivo assay using a diabetic mouse model. Full thickness skin defects of 20 mm in diameter were introduced on the back. On days 0 and 3, PBS, PRP, or iMPs was applied onto the wound surface. Until day 20, the wound was photographed, and the non-epithelialized area was calculated. Histological analysis was performed on day 10. B Chronological analysis of the wound closure rate. Representative images of each group are presented. Statistical differences on days 10 to 20 are indicated in the bar graph. n = 9. C Evaluation of collagen formation with Masson’s trichrome staining. The area where the loss of hair follicles was observed was identified as the original wound, and the total area of the blue-stained area within the wound was measured using analysis software. n = 9. D Evaluation of angiogenesis with immunohistochemical staining for CD31. High magnification images were taken at 10 random fields of view within the wound. The average number of stained blood vessels was calculated using analysis software. n = 9. Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Fig. 7

Validation of FD-iMPs. A Photograph of FD-iMPs in a test tube. iMPs were lyophilized and stored at 4 °C for 90 days. B Stability of growth factors in FD-iMPs. Following suspension in PBS to the same volume as before lyophilization, the concentration of growth factors in FD-iMPs was measured and compared to that in the original iMPs. n = 3. C Efficacy of FD-iMPs in the animal model. The in vivo wound healing assay was performed similarly as with the original iMPs described in Fig. 6. n = 9. Data are presented as the mean ± SD. ** p < 0.01, *** p < 0.001, **** p < 0.0001