Enhanced wound healing promotion by immune response-free monkey autologous iPSCs and exosomes vs. their allogeneic counterparts

Abstract

Background: Comparing non-inbred autologous and allogeneic induced pluripotent stem cells (iPSCs) and their secreted subcellular products among non-human primates is critical for choosing optimal iPSC products for human clinical trials.

Methods: iPSCs were induced from skin fibroblastic cells of adult male rhesus macaques belonging to four unrelated consanguineous families. Teratoma generativity, host immune response, and skin wound healing promotion were evaluated subsequently.

Findings: All autologous, but no allogeneic, iPSCs formed teratomas, whereas all allogeneic, but no autologous, iPSCs caused lymphocyte infiltration. Macrophages were not detectable in any wound. iPSCs expressed significantly more MAMU A and E of the major histocompatibility complex (MHC) class I but not more other MHC genetic alleles than parental fibroblastic cells. All topically disseminated autologous and allogeneic iPSCs, and their exosomes accelerated skin wound healing, as demonstrated by wound closure, epithelial coverage, collagen deposition, and angiogenesis. Allogeneic iPSCs and their exosomes were less effective and viable than their autologous counterparts. Some iPSCs differentiated into new endothelial cells and all iPSCs lost their pluripotency in 14 days. Exosomes increased cell viability of injured epidermal, endothelial, and fibroblastic cells in vitro. Although exosomes contained some mRNAs of pluripotent factors, they did not impart pluripotency to host cells.

Interpretation: Although all of the autologous and allogeneic iPSCs and exosomes accelerated wound healing, allogeneic iPSC exosomes were the preferred choice for "off-the shelf" iPSC products, owing to their mass-production, with no concern of teratoma formation. FUND: National Natural Science Foundation of China and National Key R&D Program of China.

Keywords: Allogeneic stem cells; Autologous stem cells; Exosomes; Immune response; Non-human primate; Stem cell therapy; Teratoma; Wound healing; Yamanaka factors.

Graphical abstract

Fig. 1

Teratoma formation and expression of genetic alleles of major histocompatibility complex I and II in autologous and allogeneic macaque iPSCs. (a) Allogeneic iPSCs failed to generate teratomas. (b-f) Autologous iPSCs formed mature teratomas with three germ layers. (g) Teratoma generation rates of autologous and allogeneic iPSCs. (h) Expression of genetic alleles of the major histocompatibility complex I and II in monkey iPSCs and skin fibroblastic cells.

Fig. 2

Macaque iPSCs accelerate wound healing. Wound closure was measured for 14 days. (a) Representative images of wounds treated with PBS, autologous and allogeneic iPSCs on 0, 3, 7, 10, and 14 days after wound punching. (b) Percentages of wound closure. Data represent the means ± SD. n = 4 for autologous and PBS treatments, respectively. n = 12 for allogeneic treatment.

Fig. 3

Transplanted iPSC survival in wounds. (a) Transplanted iPSCs (red) in 3D re-constructed skin wounds by laser confocal microscopy. The layer with the red fluorescent signal was between 7 and 16 μM. (b) Percentage of accumulated area of the red fluorescent signal in the wounds. Data represent the means ± SD. n = 4 for PBS or autologous iPSC treatment. n = 12 for allogeneic iPSC treatment. (c) Presence of administered cells (red), newly produced endothelial cells (green), and co-localization of the two (yellow). (d) Absence of pluripotent reprograming markers (OCT4 and SSEA4) in the skin lesions as demonstrated by immunohistochemistry. Macaque iPSCs smeared on glass slides were used as positive controls. (e) Skin lesions were negative for pluripotency markers (OCT4 and SSEA4), as demonstrated by western blotting; iPSC homogenates served as positive controls. n = 4 for autologous iPSC treatment. n = 12 for allogeneic iPSC treatment. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Macaque iPSCs promote epithelial proliferation and collagen deposition. (a) Representative images of wounds treated with PBS, and autologous and allogeneic iPSCs. Scale bars, 1000 μm. (b) Percentage of collagen area. (c) Epidermal thickness. Data represent the means ± SD. n = 4 for PBS or autologous iPSC treatment. n = 12 for allogeneic iPSC treatment.

Fig. 5

Macaque iPSCs accelerate angiogenesis in wounds. (a) Representative images of wound sections stained for CD34, a marker of new endothelial cells, on day 7 and 14 after wound punching and PBS, autologous, or allogeneic iPSC treatment. Scale bars = 200 μm. (b) Statistical analyses were performed on numbers of CD34-positive cells per field. Data represent the means ± SD. n = 4 for PBS or autologous iPSC treatment. n = 12 for allogeneic iPSC treatment.

Fig. 6

Macaque iPSC-derived exosomes promote wound healing. (a) iPSC exosomes were labelled with red fluorescent membrane dye (PKH 26) before being topically applied to new punched skin wounds. Representative images of the red fluorescent signal in the wounds at 7 days after exosome application. (b) Percentage of accumulated area of red fluorescent signal in the wounds. (c) Percentages of wound closure. (d) Cross-comparison of wound closure among lesions treated with autologous and allogeneic iPSCs and their exosome counterparts. (e-f) Macaque iPSC-derived exosome therapy promoted epithelial proliferation and collagen deposition as demonstrated by epidermal thickness and the percentage of collagen area out of the whole area observed. (g) Angiogenesis in the wounds. (h) Exosomes promoted cell viability of cisplatin-injured epidermal, endothelial, and skin fibroblastic cells. Data represent the means ± SD. In panels b-g, n = 4 for PBS or autologous iPSC treatment, and n = 12 for allogeneic iPSC treatment. In panel h, n = 4. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Macaque iPSC-derived exosomes did not induce pluripotency of host cells in skin lesions. (a) The exosomes were negative for pluripotent reprogramming markers (OCT4, SOX2, KLF4, and c-Myc), and positive for exosome markers (TSG101 and ALIX) as shown by western blotting. (b) Host cells in skin lesions were negative for pluripotency markers (OCT4 and SSEA4), as demonstrated by immunohistochemistry. Macaque iPSCs, smeared on glass slides, were used as positive controls. (c) Skin lesions were negative for pluripotency markers (OCT4 and SSEA4), as demonstrated by western blotting. Homogenate of iPSCs served as positive controls; n = 4 for autologous iPSC treatment; n = 12 for allogeneic iPSC treatment. (d) OCT4, SOX2, KLF4, and Nanog mRNA levels in fibroblastic cells, iPSCs, and iPSC-derived exosomes. (e) OCT4, SOX2, KLF4, and Nanog mRNA levels in fibroblastic cells treated with iPSC exosome-containing medium or exosome-free medium. mRNAs from iPSCs served as positive controls. Immunofluorescence staining (f) and Western blotting (g) of fibroblastic cells treated with iPSC exosome-containing medium and iPSCs. Exo 4 h and Exo 16 h represent cells incubated with exosome-containing medium for 4 h and 16 h, respectively. Media represent cells incubated in iPSC exosome-free medium. n = 3.

Fig. 8

Effects of autologous and allogeneic iPSCs and their exosomes on infiltration of immune cells. (a) Presence of CD3, CD20, and CD68 proteins in iPSC- or exosome-containing skin. Western blot images and the detectable rates of corresponding markers are presented. Protein of autologous peripheral blood mononuclear cells served as positive control. (b-c) Presence of CD3, CD20, and CD68 proteins in skin lesions three days after iPSC or exosome treatment. The membranes were over-exposed to avoid false negative result.