In Utero Amniotic Fluid Stem Cell Therapy Protects Against Myelomeningocele via Spinal Cord Coverage and Hepatocyte Growth Factor Secretion

Abstract

Despite the poor prognosis associated with myelomeningocele (MMC), the options for prenatal treatments are still limited. Recently, fetal cellular therapy has become a new option for treating birth defects, although the therapeutic effects and mechanisms associated with such treatments remain unclear. The use of human amniotic fluid stem cells (hAFSCs) is ideal with respect to immunoreactivity and cell propagation. The prenatal diagnosis of MMC during early stages of pregnancy could allow for the ex vivo proliferation and modulation of autologous hAFSCs for use in utero stem cell therapy. Therefore, we investigated the therapeutic effects and mechanisms of hAFSCs-based treatment for fetal MMC. hAFSCs were isolated as CD117-positive cells from the amniotic fluid of 15- to 17-week pregnant women who underwent amniocentesis for prenatal diagnosis and consented to this study. Rat dams were exposed to retinoic acid to induce fetal MMC and were subsequently injected with hAFSCs in each amniotic cavity. We measured the exposed area of the spinal cord and hepatocyte growth factor (HGF) levels at the lesion. The exposed spinal area of the hAFSC-treated group was significantly smaller than that of the control group. Immunohistochemical analysis demonstrated a reduction in neuronal damage such as neurodegeneration and astrogliosis in the hAFSC-treated group. Additionally, in lesions of the hAFSC-treated group, HGF expression was upregulated and HGF-positive hAFSCs were identified, suggesting that these cells migrated to the lesion and secreted HGF to suppress neuronal damage and induce neurogenesis. Therefore, in utero hAFSC therapy could become a novel strategy for fetal MMC. Stem Cells Translational Medicine 2019;8:1170-1179.

Keywords: Amniotic fluid stem cells; Fetal cellular therapy; Hepatocyte growth factor; Myelomeningocele; Spinal cord.

Figure 1

In utero human amniotic fluid stem cell (hAFSC) therapy reduces skin defect size and protects the exposed spinal cord. (A): Representative images of isolated myelomeningocele in retinoic acid (RA) group (left) and RA + hAFSC group (right). Scale bars: 500 μm. (B): Analysis of skin defect area (n = 5). (C): Representative images of H&E staining of spinal cross‐sections in RA group (left) and RA + hAFSC group (right). (D): Analysis of spinal cross‐section area (n = 5). (E): Representative images of Tubulin‐βIII and GFAP immunostaining of spinal cross‐sections in RA and RA + hAFSC group. (F): Analysis of Tubulin‐βIII‐positive area (n = 5), GFAP‐positive area (n = 5), and (G) GFAP/Tubulin‐βIII positive area ratio (n = 5). Images are representative of at least three independent experiments. Results are presented as mean ± SD; *, p < .05 compared with control.

Figure 2

Human amniotic fluid stem cells (hAFSCs)‐treatment increases hepatocyte growth factor production and reduces inflammatory reactions in exposed spinal cord. Retinoic acid was used to induce myelomeningocele in rats and hAFSCs‐treatment was performed. RT‐qPCR analysis of pro‐ or anti‐inflammatory cytokine levels in the exposed spinal cord. Results are presented as mean ± SD; *, p < .05 compared with control.

Figure 3

Human amniotic fluid stem cells (hAFSCs) engraft onto the surface of the defective spinal cord via CXCL12 signaling and differentiate into cytokeratin‐expressing cells. Retinoic acid (RA) was used to induce myelomeningocele in rats and hAFSCs‐treatment was performed. (A): Representative image of the response to hAFSCs‐treatment (scale bars: 500 μm). (B): Representative images of spinal cross‐sections stained with CXCL12 (×40; scale bars: 500 μm) in the RA and RA + hAFSC group. (C): Representative images of spinal cross‐sections stained with STEM121 and broad‐spectrum cytokeratin antibodies (×40; scale bars: 500 μm).

Figure 4

Engrafted human amniotic fluid stem cells (hAFSCs) produce hepatocyte growth factor (HGF). Retinoic acid (RA) was used to induce myelomeningocele in rats and hAFSCs‐treatment was performed. (A): RT‐qPCR analysis of growth factor levels in the exposed spinal cord. Results are presented as mean ± SD; *, p < .05 compared with control. (B): Representative images of STEM121 and HGF double staining (×40; scale bars: 500 μm). (C): Representative images of c‐Met and phosphorylated c‐Met staining of spinal cross‐sections in RA group and RA + hAFSC group (×40; scale bars: 500 μm). Images are representative of at least three independent experiments.

Figure 5

Human amniotic fluid stem cells (hAFSCs) integrate into myelomeningocele–rat amniotic fluid and produce hepatocyte growth factor. (A, B): Representative images of E21‐rat amniotic fluid cells (A) under phase contrast microscopy and STEM121 staining (×40; scale bars: 200 μm). (B): RT‐qPCR analysis of paracrine mediators in CD117 positive (CD117⁺) amniotic fluid cells obtained from E19 (48 hours after hAFSCs injection) and E21 (96 hours after hAFSCs injection) rat amniotic fluid cells compared with those from hAFSCs before injection into rat amniotic fluid. Results are presented as mean ± SD; *, p < .05 compared with control.