There are still no effective therapies for hyposalivation caused by irradiation. In our previous study, bone marrow stem cells can be transdifferentiated into acinar-like cells in vitro. Therefore, we hypothesized that transplantation with bone marrow stem cells or acinar-like cells may help functional regeneration of salivary glands. Bone marrow stem cells were labeled with nanoparticles and directly co-cultured with acinar cells to obtain labeled acinar-like cells. In total, 140 severely combined immune-deficiency mice were divided into 4 groups for cell therapy experiments: (1) normal mice, (2) mice receiving irradiation around their head-and-neck areas; (3) mice receiving irradiation and intra-gland transplantation with labeled stem cells; and (4) mice receiving irradiation and intra-gland transplantation with labeled acinar-like cells. Our results showed that salivary glands damaged due to irradiation can be rescued by cell therapy with either bone marrow stem cells or acinar-like cells for recovery of saliva production, body weight, and gland weight. Transdifferentiation of bone marrow stem cells into acinar-like cells in vivo was also noted. This study demonstrated that cell therapy with bone marrow stem cells or acinar-like cells can help functional regeneration of salivary glands, and that acinar-like cells showed better therapeutic potentials than those of bone marrow stem cells.
We have immortalized human normal proximal bronchial epithelial cells (HBECs) for the study of lung tumors arising in the central compartment of the lung, and now have over 30 of these strains (growing in defined KSFM media) including many isogenic derivatives with various oncogenic changes (Can Res 2004, Can Res 2006). We have extended this by immortalizing normal distal (small airway) epithelial cells that serve as models for the study of tumors arising in the peripheral compartment of the lung. Using Cdk4 and hTERT exogenous expression vectors (as used for HBECs) and serum free defined media, we have immortalized (N= 19) human small airway epithelial cells (HSAECs) from peripheral lung. We have characterized the HBECs and HSAECs and found they have the property of lung basal /stem cells. In the current study, we compared the genome-wide RNA expression profile of HBECs/ HSAECs with 108 lung tumor cell lines and identified a panel of genes that are differentially expressed in these two cell types. We found ∼ 1200 genes that are 2 fold over expressed in tumor cells lines compared with HBECs/HSAECs (p ≤ 0.005). Many known lung tumor oncogenes such as members in cancer/testis (CT) antigen families (XAGE/GAGE/MAGE), proliferation-promoting E2F2 transcription factors, and Kinesin family are over-expressed in tumor lines validating the comparison strategies. In addition, we found genes that had been shown to be involved in other types of tumors but had not been reported in lung tumors. Importantly, we found several genes that had not been implicated as oncogenes previously, thus these are potential novel oncogenes for lung tumor transformation. We also found ∼1300 genes that are 2 fold over expressed in HBECs/HSAECs compared with tumor cell lines (p≤ 0.006). Many of the genes are lung basal cell markers (Keratin and Laminin family members) or known tumor suppressor genes (p53 binding genes and metallothionein genes) as expected. Like in the case of tumor specific genes, we found several genes that had not been reported functioning in the inhibition of tumor transformation. Thus, these are candidate tumor suppressor genes. The current study showed that the immortalized HBEC and HSAECs are ideal models for the discovery of novel oncogenes and tumor suppressor genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2342. doi:1538-7445.AM2012-2342
Normal human lung epithelial cells that can be genetically manipulated to study the various steps in lung cancer pathogenesis and stem cell biology would be of great utility and also provide isogenic strains to develop and test new diagnostic (biomarkers), preventative and therapeutic strategies for lung cancer. Previously we immortalized normal proximal human lung bronchial epithelial cells (HBECs) for studying lung tumors arising in the central compartment of the lung and now have over 40 of these strains (growing in defined KSFM media) including many isogenic derivatives with various oncogenic changes (Can Res 2004, Can Res 2006). We have extended this by immortalizing normal distal (small airway) epithelial cells that serve as models for studying tumors arising in the peripheral compartment of the lung. Using Cdk4 and hTERT exogenous expression vectors (as used for HBECs) and SAGM defined media routinely immortalized (N= 15) human small airway epithelial cells (HSAECs) from peripheral lung. These HSAEC strains are highly enriched (>50%) for cells with stem like properties (e.g. Aldehyde dehydrogenase positivity), express basal cell markers but not Clara cell, type 1 or type 2 differentiation markers., However, HSAECs can be induced to express Clara cell, type 1 or type 2 cell markers when they are grown in matrigel (where they form cyst like structures which express such differentiation markers) and under other conditions favoring differentiation (such as insulin depletion, Ca2+ addition). HSAECs show anchorage dependent growth, do not form colonies in soft agar, and are non-tumorigenic in NOD/SCID mice. However, HSAECs can be genetically manipulated by introducing oncogenes (such as KRASV12, missense p53 and other oncogene combinations) which now allow HSAECs to progress towards a transformed phenotype (e.g. formation of soft agar colonies), and selection of a subset of HSAEC(KRAS, p53) large soft agar colonies identifies sublines that can now form poorly differentiated carcinomas. Thus, we have developed conditions to routinely immortalize lung epithelial cells derived from distal small airways (HSAECs) which grow in defined media, exhibit stem cell like characteristics, and can be genetically manipulated to give rise to poorly differentiated cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3230.
Most of our current understanding of stem cells in the adult lung is based on mouse models while information in the human lung remains poorly defined. A major advance would be the ability to reproducibly grow human lung stem or progenitor cells to allow their systematic study and genetic manipulation. Toward this goal, we have worked out conditions to routinely immortalized (NB = 9) human small airway epithelial cells (HSAECs) from peripheral lung using Cdk4 and hTERT expression vectors in these cells. As an example strain HSAEC1 grows in serum free SAGM medium (Clonetics), forms colonies in liquid media efficiently, but does not form colonies in soft agar or tumors in SCID/NOD mice. HSAEC1 expresses lung basal/stem cell markers (aldehyde dehydrogenase, p63, Cytokeratin14 and 17, and is resistant to 1 mM naphthalene) but very low or undetectable Clara cell, Type1 and Type2 cell markers in SAGM, which are then expressed when grown in Matrigel culture or in serum. Finally, the cells can be genetically manipulated toward oncogenesis (with KRASV12, and or missense p53). We conclude, that we have prepared multiple, immortalized strains of human peripheral lung epithelial cells with many characteristics of lung stem or progenitor cells that are experimentally tractable for studying lung differentiation and tumorigenesis.This work is supported by NCI SPORE P50CA70907, and NASA/DOE NSCOR NNJ05HD36G, DE‐AI02‐05ER64068
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