Stem Cells and Irradiation.

The main difficulty of radiotherapy is to destroy cancer cells without depletion of healthy tissue [...].

Musashi expression in intestinal stem cells attenuates radiation-induced decline in intestinal permeability and survival in Drosophila.

Exposure to genotoxic stress by environmental agents or treatments, such as radiation therapy, can diminish healthspan and accelerate aging. We have developed a Drosophila melanogaster model to study the molecular effects of radiation-induced damage and repair. Utilizing a quantitative intestinal permeability assay, we performed an unbiased GWAS screen (using 156 strains from the Drosophila Genetic Reference Panel) to search for natural genetic variants that regulate radiation-induced gut permeability in adult D. melanogaster. From this screen, we identified an RNA binding protein, Musashi (msi), as one of the possible genes associated with changes in intestinal permeability upon radiation. The overexpression of msi promoted intestinal stem cell proliferation, which increased survival after irradiation and rescued radiation-induced intestinal permeability. In summary, we have established D. melanogaster as an expedient model system to study the effects of radiation-induced damage to the intestine in adults and have identified msi as a potential therapeutic target.

The Role of Autophagy in Skin Fibroblasts, Keratinocytes, Melanocytes, and Epidermal Stem Cells.

Human skin acts as a barrier to protect our bodies from UV rays and external pathogens and to prevent water loss. Phenotypes of aging, or natural aging due to chronic damage, include wrinkles and the reduction of skin thickness that occur because of a loss of skin cell function. The dysregulation of autophagy, a lysosome-related degradation pathway, can lead to cell senescence, cancer, and various human diseases due to abnormal cellular homeostasis. Here, we discuss the roles and molecular mechanisms of autophagy involved in the anti-aging effects of autophagy and the relationship between autophagy and aging in skin cells.

Krüppel-like Factor 5 Regulates Stemness, Lineage Specification, and Regeneration of Intestinal Epithelial Stem Cells.

BACKGROUND & AIMS: Self-renewal and multipotent differentiation are cardinal properties of intestinal stem cells (ISCs), mediated in part by WNT and NOTCH signaling. Although these pathways are well characterized, the molecular mechanisms that control the 'stemness' of ISCs are still not well defined. Here, we investigated the role of Krüppel-like factor 5 (KLF5) in regulating ISC functions. METHODS: We performed studies in adult Lgr5EGFP-IRES-creERT2;Rosa26LSLtdTomato (Lgr5Ctrl) and Lgr5EGFP-IRES-creERT2;Klf5fl/fl;Rosa26LSLtdTomato (Lgr5ΔKlf5) mice. Mice were injected with tamoxifen to activate Cre recombinase, which deletes Klf5 from the intestinal epithelium in Lgr5ΔKlf5 but not Lgr5Crtl mice. In experiments involving irradiation, mice were subjected to 12 Gy total body irradiation (TBI). Tissues were collected for immunofluorescence (IF) analysis and next generation sequencing. Oganoids were derived from fluoresecence activated cell sorted- (FACS-) single cells from tamoxifen-treated Lgr5ΔKlf5 or Lgr5Crtl mice and examined by immunofluorescence stain. RESULTS: Lgr5+ ISCs lacking KLF5 proliferate faster than control ISCs but fail to self-renew, resulting in a depleted ISC compartment. Transcriptome analysis revealed that Klf5-null Lgr5+ cells lose ISC identity and prematurely differentiate. Following irradiation injury, which depletes Lgr5+ ISCs, reserve Klf5-null progenitor cells fail to dedifferentiate and regenerate the epithelium. Absence of KLF5 inactivates numerous selected enhancer elements and direct transcriptional targets including canonical WNT- and NOTCH-responsive genes. Analysis of human intestinal tissues showed increased levels of KLF5 in the regenerating epithelium as compared to those of healthy controls. CONCLUSION: We conclude that ISC self-renewal, lineage specification, and precursor dedifferentiation require KLF5, by its ability to regulate epigenetic and transcriptional activities of ISC-specific gene sets. These findings have the potential for modulating ISC functions by targeting KLF5 in the intestinal epithelium.

Distinct response of adult neural stem cells to low versus high dose ionising radiation.

Radiosusceptibility is the sensitivity of a biological organism to ionising radiation (IR)-induced carcinogenesis, an outcome of IR exposure relevant following low doses. The tissue response is strongly influenced by the DNA damage response (DDR) activated in stem and progenitor cells. We previously reported that in vivo exposure to 2 Gy X-rays activates apoptosis, proliferation arrest and premature differentiation in neural progenitor cells (transit amplifying cells and neuroblasts) but not in neural stem cells (NSCs) of the largest neurogenic region of the adult brain, the subventricular zone (SVZ). These responses promote adult quiescent NSC (qNSC) activation after 2 Gy. In contrast, neonatal (P5) SVZ neural progenitors continue proliferating and do not activate qNSCs. Significantly, the human and mouse neonatal brain is radiosusceptible. Here, we examine the response of stem and progenitor cells in the SVZ to low IR doses (50-500 mGy). We observe a linear dose-response for apoptosis but, in contrast, proliferation arrest and neuroblast differentiation require a threshold dose of 200 or 500 mGy, respectively. Importantly, qNSCs were not activated at doses below 500 mGy. Thus, full DDR activation in the neural stem cell compartment in vivo necessitates a threshold dose, which can be considered of significance when evaluating IR-induced cancer risk and dose extrapolation.

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.

Radiation Sensitivity of Adipose-Derived Stem Cells Isolated from Breast Tissue.

Within their niche, adipose-derived stem cells (ADSCs) are essential for homeostasis as well as for regeneration. Therefore, the interest of physicians is to use ADSCs as a tool for radiation oncology and regenerative medicine. To investigate related risks, this study analyses the radiation response of adult stem cells isolated from the adipose tissue of the female breast. To avoid donor-specific effects, ADSCs isolated from breast reduction mammoplasties of 10 donors were pooled and used for the radiobiological analysis. The clonogenic survival fraction assay was used to classify the radiation sensitivity in comparison to a more radiation-sensitive (ZR-75-1), moderately sensitive (MCF-7), and resistant (MCF10A) cell lines. Afterwards, cytotoxicity and genotoxicity of irradiation on ADSCs were investigated. On the basis of clonogenic cell survival rates of ADSCs after irradiation, we assign ADSCs an intermediate radiation sensitivity. Furthermore, a high repair capacity of double-strand breaks is related to an altered cell cycle arrest and increased expression of cyclin-dependent kinase (CDK) inhibitor p21. ADSCs isolated from breast tissue exhibit intermediate radiation sensitivity, caused by functional repair mechanisms. Therefore, we propose ADSCs to be a promising tool in radiation oncology.

Effects of pulsing of light on the dentinogenesis of dental pulp stem cells in vitro.

Low power light (LPL) treatment has been widely used in various clinical trials, which has been known to reduce pain and inflammation and to promote wound healing. LPL was also shown to enhance differentiation of stem cells into specific lineages. However, most studies have used high power light in mW order, and there was lack of studies about the effects of very low power light in µW. In this study, we applied 810 nm LPL of 128 µW/cm2 energy density in vitro. Upon this value, continuous wave (CW) irradiation did not induce any significant changes for differentiation of human dental pulp stem cells (hDPSCs). However, the membrane hyperpolarization, alkaline phosphatase activity, and intracellular oxidative stress were largely enhanced in the pulsed wave (PW) with 30% of duty cycle and 300-3000 Hz frequencies-LPL in which LED driver work in the form of square wave. After 21 days of daily LPL treatment, Western blot revealed the dentinogenesis in this condition in vitro. This study demonstrates that the very low power light at 810 nm enhanced significant differentiation of hDPSCs in the PW mode and there were duty cycle dependency as well as pulsing frequency dependency in the efficiency.

The GS-nitroxide JP4-039 improves intestinal barrier and stem cell recovery in irradiated mice.

Total body irradiation (TBI) leads to dose- and tissue-specific lethality. In the current study, we demonstrate that a mitochondrion-targeted nitroxide JP4-039 given once 24 hours after 9-10 Gy TBI significantly improves mouse survival, and the recovery of intestinal barrier, differentiation and stem cell functions. The GI-protective effects are associated with rapid and selective induction of tight junction proteins and cytokines including TGF-ß, IL-10, IL-17a, IL-22 and Notch signaling long before bone marrow depletion. However, no change was observed in crypt death or the expression of prototypic pro-inflammatory cytokines such as TNF-α, IL-6 or IL-1ß. Surprisingly, bone marrow transplantation (BMT) performed 24 hours after TBI improves intestinal barrier and stem cell recovery with induction of IL-10, IL-17a, IL-22, and Notch signaling. Further, BMT-rescued TBI survivors display increased intestinal permeability, impaired ISC function and proliferation, but not obvious intestinal inflammation or increased epithelial death. These findings identify intestinal epithelium as a novel target of radiation mitigation, and potential strategies to enhance ISC recovery and regeneration after accidental or medical exposures.

Total body irradiation tremendously impair the proliferation, differentiation and chromosomal integrity of bone marrow-derived mesenchymal stromal stem cells.

Total body irradiation (TBI) is frequently used in hematopoietic stem cell transplantation (HSCT) and is associated with many complications due to radiation injury to the normal cells, including normal stem cells. Nevertheless, the effects of TBI on the mesenchymal stromal stem cell (MSC) are not fully understood. Bone marrow-derived MSCs (BM-MSCs) isolated from normal adults were irradiated with 200 cGy twice daily for consecutive 3 days, a regimen identical to that used in TBI-conditioning HSCT. The characteristics, differentiation potential, cytogenetics, hematopoiesis-supporting function, and carcinogenicity of the irradiated BM-MSCs were then compared to the non-irradiated control. The irradiated and non-irradiated MSCs shared similar morphology, phenotype, and hematopoiesis-supporting function. However, irradiated MSCs showed much lower proliferative and differentiative potential. Irradiation also induced clonal cytogenetic abnormalities of MSCs. Nevertheless, the carcinogenicity of irradiated MSCs is low in vitro and in vivo. In parallel with the ex vivo irradiation experiments, decreased proliferative and differentiative abilities and clonal cytogenetic abnormalities can also be found in MSCs isolated from transplant recipients who had received TBI-based conditioning previously. Thus, TBI used in HSCT drastically injury MSCs and may contribute to the development of some long-term complications associated with clonal cytogenetic abnormality and poor adipogenesis and osteogenesis after TBI.

Epithelial-mesenchymal transition transcription factors control pluripotent adult stem cell migration in vivo in planarians.

Migration of stem cells underpins the physiology of metazoan animals. For tissues to be maintained, stem cells and their progeny must migrate and differentiate in the correct positions. This need is even more acute after tissue damage by wounding or pathogenic infection. Inappropriate migration also underpins metastasis. Despite this, few mechanistic studies address stem cell migration during repair or homeostasis in adult tissues. Here, we present a shielded X-ray irradiation assay that allows us to follow stem cell migration in planarians. We demonstrate the use of this system to study the molecular control of stem cell migration and show that snail-1, snail-2 and zeb-1 EMT transcription factor homologs are necessary for cell migration to wound sites and for the establishment of migratory cell morphology. We also observed that stem cells undergo homeostatic migration to anterior regions that lack local stem cells, in the absence of injury, maintaining tissue homeostasis. This requires the polarity determinant notum Our work establishes planarians as a suitable model for further in-depth study of the processes controlling stem cell migration in vivo.

Accelerated senescence in skin in a murine model of radiation-induced multi-organ injury.

Accidental high-dose radiation exposures can lead to multi-organ injuries, including radiation dermatitis. The types of cellular damage leading to radiation dermatitis are not completely understood. To identify the cellular mechanisms that underlie radiation-induced skin injury in vivo, we evaluated the time-course of cellular effects of radiation (14, 16 or 17 Gy X-rays; 0.5 Gy/min) in the skin of C57BL/6 mice. Irradiation of 14 Gy induced mild inflammation, observed histologically, but no visible hair loss or erythema. However, 16 or 17 Gy radiation induced dry desquamation, erythema and mild ulceration, detectable within 14 days post-irradiation. Histological evaluation revealed inflammation with mast cell infiltration within 14 days. Fibrosis occurred 80 days following 17 Gy irradiation, with collagen deposition, admixed with neutrophilic dermatitis, and necrotic debris. We found that in cultures of normal human keratinocytes, exposure to 17.9 Gy irradiation caused the upregulation of p21/waf1, a marker of senescence. Using western blot analysis of 17.9 Gy-irradiated mice skin samples, we also detected a marker of accelerated senescence (p21/waf1) 7 days post-irradiation, and a marker of cellular apoptosis (activated caspase-3) at 30 days, both preceding histological evidence of inflammatory infiltrates. Immunohistochemistry revealed reduced epithelial stem cells from hair follicles 14-30 days post-irradiation. Furthermore, p21/waf1 expression was increased in the region of the hair follicle stem cells at 14 days post 17 Gy irradiation. These data indicate that radiation induces accelerated cellular senescence in the region of the stem cell population of the skin.

In vivo sensitivity of the embryonic and adult neural stem cell compartments to low-dose radiation.

The embryonic brain is radiation-sensitive, with cognitive deficits being observed after exposure to low radiation doses. Exposure of neonates to radiation can cause intracranial carcinogenesis. To gain insight into the basis underlying these outcomes, we examined the response of the embryonic, neonatal and adult brain to low-dose radiation, focusing on the neural stem cell compartments. This review summarizes our recent findings. At E13.5-14.5 the embryonic neocortex encompasses rapidly proliferating stem and progenitor cells. Exploiting mice with a hypomorphic mutation in DNA ligase IV (Lig4(Y288C) ), we found a high level of DNA double-strand breaks (DSBs) at E14.5, which we attribute to the rapid proliferation. We observed endogenous apoptosis in Lig4(Y288C) embryos and in WT embryos following exposure to low radiation doses. An examination of DSB levels and apoptosis in adult neural stem cell compartments, the subventricular zone (SVZ) and the subgranular zone (SGZ) revealed low DSB levels in Lig4(Y288C) mice, comparable with the levels in differentiated neuronal tissues. We conclude that the adult SVZ does not incur high levels of DNA breakage, but sensitively activates apoptosis; apoptosis was less sensitively activated in the SGZ, and differentiated neuronal tissues did not activate apoptosis. P5/P15 mice showed intermediate DSB levels, suggesting that DSBs generated in the embryo can be transmitted to neonates and undergo slow repair. Interestingly, this analysis revealed a stage of high endogenous apoptosis in the neonatal SVZ. Collectively, these studies reveal that the adult neural stem cell compartment, like the embryonic counterpart, can sensitively activate apoptosis.

How electromagnetic fields can influence adult stem cells: positive and negative impacts.

The electromagnetic field (EMF) has a great impact on our body. It has been successfully used in physiotherapy for the treatment of bone disorders and osteoarthritis, as well as for cartilage regeneration or pain reduction. Recently, EMFs have also been applied in in vitro experiments on cell/stem cell cultures. Stem cells reside in almost all tissues within the human body, where they exhibit various potential. These cells are of great importance because they control homeostasis, regeneration, and healing. Nevertheless, stem cells when become cancer stem cells, may influence the pathological condition. In this article we review the current knowledge on the effects of EMFs on human adult stem cell biology, such as proliferation, the cell cycle, or differentiation. We present the characteristics of the EMFs used in miscellaneous assays. Most research has so far been performed during osteogenic and chondrogenic differentiation of mesenchymal stem cells. It has been demonstrated that the effects of EMF stimulation depend on the intensity and frequency of the EMF and the time of exposure to it. However, other factors may affect these processes, such as growth factors, reactive oxygen species, and so forth. Exploration of this research area may enhance the development of EMF-based technologies used in medical applications and thereby improve stem cell-based therapy and tissue engineering.

The Trp53-Trp53inp1-Tnfrsf10b pathway regulates the radiation response of mouse spermatogonial stem cells.

Germ cells are thought to exhibit a unique DNA damage response that differs from that of somatic stem cells, and previous studies suggested that Trp53 is not involved in the survival of spermatogonial stem cells (SSCs) after irradiation. Here, we report a critical role for the Trp53-Trp53inp1-Tnfrsf10b pathway during radiation-induced SSC apoptosis. Spermatogonial transplantation revealed that Trp53 deficiency increased the survival of SSCs after irradiation. Although Bbc3, a member of the intrinsic apoptotic pathway, was implicated in apoptosis of germ and somatic stem cells, Bbc3 depletion inhibited apoptosis in committed spermatogonia, but not in SSCs. In contrast, inhibition of Tnfrsf10b, an extrinsic apoptosis regulator, rescued SSCs. Tnfrsf10b, whose deficiency protected SSCs, was upregulated by Trp53inp1 upon irradiation. These results suggest that the Trp53-Trp53inp1-Tnfrsf10b pathway responds to genotoxic damage in SSCs and that stem and progenitor cells exhibit distinct DNA damage responses in self-renewing tissue.

Label-retaining cells in the adult murine salivary glands possess characteristics of adult progenitor cells.

Radiotherapy is the primary treatment for patients with head and neck cancer, which account for roughly 500,000 annual cases worldwide. Dysfunction of the salivary glands and associated conditions like xerostomia and dysphagia are often developed by these patients, greatly diminishing their life quality. Current preventative and palliative care fail to deliver an improvement in the quality of life, thus accentuating the need for regenerative therapies. In this study, a model of label retaining cells (LRCs) in murine salivary glands was developed, in which LRCs demonstrated proliferative potential and possessed markers of putative salivary progenitors. Mice were labeled with 5-Ethynyl-2'-deoxyuridine (EdU) at postnatal day 10 and chased for 8 weeks. Tissue sections from salivary glands obtained at the end of chase demonstrated co-localization between LRCs and the salivary progenitor markers keratin 5 and keratin 14, as well as kit mRNA, indicating that LRCs encompass a heterogeneous population of salivary progenitors. Proliferative potential of LRCs was demonstrated by a sphere assay, in which LRCs were found in primary and secondary spheres and they co-localized with the proliferation marker Ki67 throughout sphere formation. Surprisingly, LRCs were shown to be radio-resistant and evade apoptosis following radiation treatment. The clinical significance of these findings lie in the potential of this model to study the mechanisms that prevent salivary progenitors from maintaining homeostasis upon exposure to radiation, which will in turn facilitate the development of regenerative therapies for salivary gland dysfunction.

Accelerated differentiation of melanocyte stem cells contributes to the formation of hyperpigmented maculae.

It has been reported that the abnormal regulation of melanocyte stem cells (McSCs) causes hair greying; however, little is known about the role of McSCs in skin hyperpigmentation such as solar lentigines (SLs). To investigate the involvement of McSCs in SLs, the canonical Wnt signalling pathway that triggers the differentiation of McSCs was analysed in UVB-induced delayed hyperpigmented maculae in mice and human SL lesions. After inducing hyperpigmented maculae on dorsal skin of F1 mice of HR-1× HR/De, which was formed long after repeated UVB irradiation, the epidermal Wnt1 expression and the number of nuclear ß-catenin-positive McSCs were increased as compared to non-irradiated control mice. Furthermore, the expression of dopachrome tautomerase (Dct), a downstream target of ß-catenin, was significantly upregulated in McSCs of UVB-irradiated mice. The Wnt1 expression and the number of nuclear ß-catenin-positive McSCs were also higher in human SL lesions than in normal skin. Recombinant Wnt1 protein induced melanocyte-related genes including Dct in early-passage normal human melanocytes (NHEMs), an in vitro McSC model. These results demonstrate that the canonical Wnt signalling pathway is activated in SL lesions and strongly suggest that the accelerated differentiation of McSCs is involved in SL pathogenesis.

Dynamic Hedgehog signalling pathway activity in germline stem cells.

Although the contribution of Hedgehog (Hh) signalling to stem cell development and oncogenesis is well recognised, its importance for spermatogonial stem cells (SSCs) has not been established. Here we interrogate adult rat SSCs using an established model in which only undifferentiated spermatogonial cells remain in the testis at 15 weeks following irradiation, and spermatogonial differentiation is induced within 4 weeks by gonadotrophin-releasing hormone antagonist (GnRH-ant) administration. Synthesis of Hh pathway components in untreated adult rat testes was compared with that in irradiated testes prior to and after GnRH-ant exposure using in situ hybridization. In adult testes with complete spermatogenesis, the Desert Hedgehog ligand transcript, Dhh, was detected in Sertoli cells, some spermatogonia and in spermatocytes by in situ hybridization. Spermatogenic cells were identified as sites of Hh signalling through detection of transcripts encoding the Hh receptor, Ptc2 transcripts and proteins for the key downstream target of Hh signalling, Gli1 and the Hh transcriptional activator, Gli2. Remarkably, the undifferentiated spermatogonia present in irradiated adult rat testes contained Dhh in addition to Ptc2, Gli1 and Gli2, revealing the potential for an autocrine Hh signalling loop to sustain undifferentiated spermatogonial cells. These transcripts became undetectable by in situ hybridization following GnRH-ant induction of spermatogonial differentiation, however, detection of Gli1 protein in spermatogonia in all groups indicates that Hh signalling is sustained. This is the first evidence of active Hh signalling in mammalian male germline stem cells, as has been documented for some cancer stem cells.

P53-participated cellular and molecular responses to irradiation are cell differentiation-determined in murine intestinal epithelium.

AIM: Cells respond differently to DNA damaging agents, which may related to cell context and differentiation status. The aim of present study was to observe the cellular and molecular responses of cells in different differentiation status to ionizing irradiation (IR). METHODS: Crypt-villus unit of murine small intestine was adopted as a cell differentiation model. DNA damage responses (DDRs) of crypt and villus were observed 1-24 h after 12 Gy IR using gene expression microarray analysis, immunohistochemical staining, Western blotting and Electrophoretic Mobility Shift Assay. RESULTS: Microarray analysis revealed that most differentially expressed genes were related to p53 signaling pathway in crypt 4h after IR and in both crypt and villus 24h after IR. In crypt stem cells/progenitor cells, H2AX was phosphorylated and dephosphorylated quickly, Ki67 attenuated, cell apoptosis enhanced, phosphorylated P53 increased and translocated into nuclear with the ability to bind p53-specific sequence. In upper crypt (transit amplifying cells) and crypt-villus junction, cells kept survive and proliferate as indicated by retained Ki67 expression, suppressed p53 activation, and rare apoptosis. CONCLUSIONS: DDRs varied with cell differentiation status and cell function in small intestinal epithelium. P53 signaling pathway could be an important regulatory mechanism of DDRs.

Photo-mediated internalization of nanocomplex for effective gene delivery to adipose tissue-derived stem cells.

To deliver efficiently osteogenic, chondrogenic or adipogenic induction genes, such as Runx2, SOX9 and C/EBP-α, to adipose tissue-derived stem cells (ADSCs), a photo-mediated nanocomplex internalization gene delivery system was designed using chlorin e6 as a photosensitizer (PS) and polyethyleneimine (PEI) as a gene delivery carrier. In this system, gene delivery efficacy was significantly increased in ADSCs by photo irradiation. The gene transfection efficiency of Runx2, SOX9 and C/EBP-α was increased by 8.6-, 6.7- and 9.3-fold, respectively, by applying 0.7J/cm(2) of irradiation. Osteogenic, chondrogenic and adipogenic differentiation was confirmed by differentiation-related markers and histological analysis. ADSCs transfected with Runx2, SOX9 and C/EBP-α genes via photo irradiation indicated enhanced differentiation in comparison to the non-irradiated cells. These findings demonstrate that photo-mediated internalization is a promising system for efficient gene delivery and differentiation in ADSCs.