Lack of DNA Damage Response at Low Radiation Doses in Adult Stem Cells Contributes to Organ Dysfunction.

PURPOSE: Radiotherapy for head and neck cancer may result in serious side effects, such as hyposalivation, impairing the patient's quality of life. Modern radiotherapy techniques attempt to reduce the dose to salivary glands, which, however, results in low-dose irradiation of the tissue stem cells. Here we assess the low-dose sensitivity of tissue stem cells and the consequences for tissue function. EXPERIMENTAL DESIGN: Postirradiation rat salivary gland secretory function was determined after pilocarpine induction. Murine and patient-derived salivary gland and thyroid gland organoids were irradiated and clonogenic survival was assessed. The DNA damage response (DDR) was analyzed in organoids and modulated using different radiation modalities, chemical inhibition, and genetic modification. RESULTS: Relative low-dose irradiation to the high-density stem cell region of rat salivary gland disproportionally impaired function. Hyper-radiosensitivity at doses <1 Gy, followed by relative radioresistance at doses ≥1 Gy, was observed in salivary gland and thyroid gland organoid cultures. DDR modulation resulted in diminished, or even abrogated, relative radioresistance. Furthermore, inhibition of the DDR protein ATM impaired DNA repair after 1 Gy, but not 0.25 Gy. Irradiation of patient-derived salivary gland organoid cells showed similar responses, whereas a single 1 Gy dose to salivary gland-derived stem cells resulted in greater survival than clinically relevant fractionated doses of 4 × 0.25 Gy. CONCLUSIONS: We show that murine and human glandular tissue stem cells exhibit a dose threshold in DDR activation, resulting in low-dose hyper-radiosensitivity, with clinical implications in radiotherapy treatment planning. Furthermore, our results from patient-derived organoids highlight the potential of organoids to study normal tissue responses to radiation.

The In Vitro Response of Tissue Stem Cells to Irradiation With Different Linear Energy Transfers.

PURPOSE: A reduction in the dose, irradiated volume, and sensitivity of, in particular, normal tissue stem cells is needed to advance radiation therapy. This could be obtained with the use of particles for radiation therapy. However, the radiation response of normal tissue stem cells is still an enigma. Therefore, in the present study, we developed a model to investigate the in vitro response of stem cells to particle irradiation. METHODS AND MATERIALS: We used the immortalized human salivary gland (HSG) cell line resembling salivary gland (SG) cells to translate the radiation response in 2-dimensional (2D) to 3-dimensional (3D) conditions. This response was subsequently translated to the response of SG stem cells (SGSCs). Dispersed single cells were irradiated with photons or carbon ions at different linear energy transfers (LETs; 48.76 ± 2.16, 149.9 ± 10.8, and 189 ± 15 keV/µm). Subsequently, 2D or 3D clonogenicity was determined by counting the colonies or secondary stem cell-derived spheres in Matrigel. γH2AX immunostaining was used to assess DNA double strand break repair. RESULTS: The 2D response of HSG cells showed a similar increase in dose response to increasing higher LET irradiation as other cell lines. The 3D response of HSG cells to increasing LET irradiation was reduced compared with the 2D response. Finally, the response of mouse SGSCs to photons was similar to the 3D response of HSG cells. The response to higher LET irradiation was reduced in the stem cells. CONCLUSIONS: Mouse SGSC radiosensitivity seems reduced at higher LET radiation compared with transformed HSG cells. The developed model to assess the radiation response of SGSCs offers novel possibilities to study the radiation response of normal tissue in vitro.

Human amniotic fluid-derived mesenchymal cells from fetuses with a neural tube defect do not deposit collagen type i protein after TGF-ß1 stimulation in vitro.

In spina bifida, the neural tube fails to close during the embryonic period. Exposure of the neural tube to the amniotic fluid during pregnancy causes additional neural damage. Intrauterine tissue engineering using a biomaterial seeded with stem cells might prevent this additional damage. For this purpose, autologous cells from the amniotic fluid are an attractive source. To close the defect, it is important that these cells deposit an extracellular matrix. However, it is not known if amniotic fluid mesenchymal cells (AFMCs) from a fetus with a neural tube defect (NTD) share the same characteristics as AFMCs from a healthy fetus. We found that cells derived from fetuses with a NTD, in contrast to healthy human amniotic fluid cells, did not deposit collagen type I. Furthermore, the NTD cells showed, compared with both healthy amniotic fluid cells and fetal fibroblasts, much lower mRNA expression levels of genes that are involved in collagen biosynthesis [procollagen C-endopeptidase enhancer proteins (PCOLCE), PCOLCE2, ADAM metallopeptidase with thrombospondin type 1 motif, 2 (ADAMTS2), ADAMTS14]. This indicates that NTD-AFMCs have different characteristics compared with healthy AFMCs and might not be suitable for fetal therapy to close the defect in spina bifida patients.

Epithelial-to-mesenchymal transition in fibrosis: collagen type I expression is highly upregulated after EMT, but does not contribute to collagen deposition.

The hallmark of fibrosis is an accumulation of fibrillar collagens, especially of collagen type I. There is considerable debate whether in vivo type II epithelial-to-mesenchymal transition (EMT) is involved in organ fibrosis. Lineage tracing experiments by various groups show opposing data concerning the relative contribution of epithelial cells to the pool of myofibroblasts. We hypothesized that EMT-derived cells might directly contribute to collagen deposition. To study this, EMT was induced in human epithelial lung and renal cell lines in vitro by means of TGF-ß1 stimulation, and we compared the collagen type I (COL1A1) expression levels of transdifferentiated cells with that of myofibroblasts obtained by TGF-ß1 stimulation of human dermal and lung fibroblasts. COL1A1 expression levels of transdifferentiated epithelial cells appeared to be at least one to two orders of magnitude lower than that of myofibroblasts. This was confirmed at immunohistochemical level: in contrast to myofibroblasts, collagen type I deposition by EMT-derived cells was not or hardly detectable. We postulate that, even when type II EMT occurs in vivo, the direct contribution of EMT-derived cells to collagen accumulation is rather limited.

Cell plasticity in wound healing: paracrine factors of M1/ M2 polarized macrophages influence the phenotypical state of dermal fibroblasts.

BACKGROUND: Macrophages and fibroblasts are two major players in tissue repair and fibrosis. Despite the relevance of macrophages and fibroblasts in tissue homeostasis, remarkably little is known whether macrophages are able to influence the properties of fibroblasts. Here we investigated the role of paracrine factors secreted by classically activated (M1) and alternatively activated (M2) human macrophages on human dermal fibroblasts (HDFs). RESULTS: HDFs stimulated with paracrine factors from M1 macrophages showed a 10 to > 100-fold increase in the expression of the inflammatory cytokines IL6, CCL2 and CCL7 and the matrix metalloproteinases MMP1 and MMP3. This indicates that factors produced by M1 macrophages induce a fibroblast phenotype with pro-inflammatory and extracellular matrix (ECM) degrading properties. HDFs stimulated with paracrine factors secreted by M2 macrophages displayed an increased proliferation rate. Interestingly, the M1-activated pro-inflammatory fibroblasts downregulated, after exposure to paracrine factors produced by M2 macrophages or non-conditioned media, the inflammatory markers as well as MMPs and upregulated their collagen production. CONCLUSIONS: Paracrine factors of M1 or M2 polarized macrophages induced different phenotypes of HDFs and the HDF phenotypes can in turn be reversed, pointing to a high dynamic plasticity of fibroblasts in the different phases of tissue repair.

Intra-uterine tissue engineering of full-thickness skin defects in a fetal sheep model.

In spina bifida the neural tube fails to close during the embryonic period and it is thought that prolonged exposure of the unprotected spinal cord to the amniotic fluid during pregnancy causes additional neural damage. Intra-uterine repair might protect the neural tissue from exposure to amniotic fluid and might reduce additional neural damage. Biodegradable collagen scaffolds may be useful in case of fetal therapy for spina bifida, but biochemical properties need to be studied. The aim of this study was to investigate whether biodegradable collagen scaffolds can be used to treat full-thickness fetal skin defects. We hypothesized that the pro-angiogenic growth factors VEGF and FGF2 would enhance vascularization, epidermialization and lead to improved wound healing. To investigate the effect of these two growth factors, a fetal sheep model for skin defects was used. Compared to wounds treated with bare collagen scaffolds, wounds treated with growth factor-loaded scaffolds showed excessive formation of capillaries and less myofibroblasts were present in these wounds, leading to less contraction. This study has demonstrated that collagen scaffolds can be used to treat fetal skin defects and that the combination of collagen scaffolds with VEGF and FGF2 had a beneficial effect on wound healing.