Embryonic stem cells as a potential treatment modality for pulmonary hypoplasia

Vol. 207, No. 3S, September 2008

Functional differences between adult and neonatal stem cells are abolished by VEGF siRNA Troy A Markel MD, Paul R Crisostomo MD, Nathan M Novotny MD, Yue Wang PhD, Rinki Ray MD, Jiangning Tan PhD, Daniel R Meldrum MD, FACS Indiana University School of Medicine, Indianapolis, IN INTRODUCTION: Adult bone marrow mesenchymal stem cells (aMSCs), unlike their neonatal counterparts (nMSCs), provide protection to ischemic myocardium. nMSCs produce lower levels of VEGF, which may prove to be a distinguishing factor in their lack of myocardial protection. We hypothesized that: 1) VEGF is an essential paracrine mediator of aMSC cardioprotection following I/R; and, 2) ablating VEGF in aMSCs will abolish functional differences between adult and neonatal stem cells. METHODS: MSCs were harvested from adult and 2.5 week old mice. VEGF was ablated in aMSCs by transfecting 50nM VEGF siRNA with Lipofectamine transfection reagent. Rat hearts were isolated via Langendorff and subjected to the same I/R protocol. Prior to ischemia, 1 million MSCs were infused into the coronary circulation based on the following groups: 1) Acellular vehicle, 2) aMSC, 3) nMSC, 4) aMSC Negative Control siRNA, 5) aMSC VEGF siRNA. Cardiac functional parameters were continuously recorded. p⬍0.05 significant via ANOVA. RESULTS: aMSCs provided enhanced post-ischemic myocardial recovery compared to vehicle, while nMSCs provided no postischemic protection. Hearts infused with VEGF ablated aMSCs had significantly worse recovery of developed pressure, ⫹dp/dt, and ⫺dp/dt compared to that seen with standard aMSCs (Adult DP: 54.8⫾3.7%, ⫹dp/dt: 60.6⫾2.6%, ⫺dp/dt 58.3⫾4.0% vs. VEGF siRNA DP: 35.0⫾0.7%, ⫹dp/dt: 32.4⫾1.6%, ⫺dp/dt: 30.9⫾ 1.6%). Hearts infused with VEGF ablated aMSCs also had similar functional parameters as those infused with nMSCs. CONCLUSIONS: VEGF is an essential paracrine factor for stem cell mediated post-ischemic myocardial protection. Decreased VEGF production in nMSCs may be responsible for their lack of cardioprotection following I/R.

Embryonic stem cells as a potential treatment modality for pulmonary hypoplasia Blair Roszell MS, Mark Mondrinos PhD, Ariel Seaton, Guo Hua Fong PhD, Peter Lelkes PhD, Christine Finck MD, FACS UConn Health Center, Farmington, CT INTRODUCTION: Pulmonary Hypoplasia (PH) is characterized by poorly differentiated alveolar epithelium and reduced surfactant production. Clinically, embryonic stem cells (ESCs) could be useful in cell-based therapy for augmentation of diseased tissue. Recently, efficient induction of endoderm from murine ESCs using defined growth factors that recapitulate developmental cues during embryogenesis has been established. We hypothesize that an enriched population of ESC-derived endoderm can be differentiated toward lung epithelial cells, specifically alveolar epithelial type II cells (AEII) with high doses of FGF2, a potent regulator of lung development.

Surgical Forum Abstracts

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METHODS: Murine ESCs were cultured in growth factor defined conditions or in conditioned medium of an AEII-like cell line, A549, for 6 days. Cells were analyzed using RT-PCR and immunostaining for definitive endoderm markers. This enriched population was then cultured in high doses of FGF2 and screened for AEII markers. SP-C expression was quantified and compared across conditions. Receptor blocking studies were performed to identify the specific effects of FGF2. RESULTS: Induction of definitive endoderm markers was reliable in conditioned Medium (55%) or Activin A (46%). Subsequent exposure to FGF2 induced SP-C expression in 12% of cell populations. SP-C expression was inhibited by the addition of soluble receptor FGFR2IIIc. CONCLUSIONS: We demonstrate effective methods for deriving AEII-like cells from pluripotent ESCs and provide evidence that FGF2 plays a role in AEII cell differentiation. Once purified, these ESC-derived AEII cells could be used as a potential cell source for augmenting developmentally impaired lungs in premature newborns with pulmonary hypoplasia.

In vitro microdistraction of human adipose-derived stem cells: Osteogenic and cytoskeletal gene expression Navanjun S Grewal MD, Kristy L Wasson BA, Rebekah Ashley BA, Luciana Yacomotti MD, Vahe Melkonyan MD, Scott Mitchell MD, Joubin S Gabbay MD, James P Bradley MD, Patricia A Zuk PhD UCLA, Los Angeles, CA INTRODUCTION: Distraction osteogenesis has been used to correct hypoplastic and asymmetric bony deformities in patients; yet its underlying cellular mechanisms are poorly understood. Using a novel system capable of applying linear stresses to cells in vitro (i.e. the microdistractor), the effect of stress on osteogenic and cytoskeletal gene expression in an adult stem cell population was studied. METHODS: Adipose-derived stem cells (ASCs) were obtained from raw human lipoaspirates. The microdistractor system was used to apply linear stresses to the ASCs. The effect of three types of stresses on ASCs was studied: (1) as outward linear distraction force, (2) an alternating series of outward distraction and inward compression oscillation, and (3) a static control in which inward retraction forces are resisted (no force). Real-time PCR was used to quantitate the expression of established osteogenic genes (CBFA-1, alkaline phosphatase, osteonectin, osteopontin, and osteocalcin) and cytoskeletal markers (rac2, rhoD, and rhoA, alpha-actinin, beta-integrin, and vimentin). RESULTS: The application of linear stresses to ASCs produced variable results with respect to osteogenic and cytoskeletal gene expression. CBFA-1, the most essential transcription factor for bone formation, showed substantial increase with distraction. Further targets, osteonectin and osteopontin similarly showed positive responses to distraction and oscillation. In contrast, no significant changes were seen in most of the cytoskeletal genes with distraction or oscillation.