Kelly Hiatt

Characterization of Definitive Lymphohematopoietic Stem Cells in the Day 9 Murine Yolk Sac

The site of origin of lymphohematopoietic stem cells (HSC) that initiate definitive blood cell production in the murine fetal liver is controversial. Contrary to reports that the preliver yolk sac does not contain definitive HSC, we observed that CD34+ day 9 yolk sac cells repopulated multiple blood cell lineages in newborn hosts for at least 1 year. Furthermore, 100 CD34+c-Kit+ day 9 yolk sac or para-aortic splanchnopleura (P-Sp) cells, known to give rise to embryonic HSC, similarly repopulated hematopoiesis in recipient hosts. Surprisingly, 37-fold more CD34+c-Kit+ cells reside in the day 9 yolk sac than in the P-Sp. In sum, definitive HSC are coexistent, but not equal in number, in the murine yolk sac and P-Sp prior to fetal liver colonization.

Neurofibromin GTPase-activating Protein-related Domains Restore Normal Growth in Nf1−/− Cells

Members of the Ras superfamily of signaling proteins modulate fundamental cellular processes by cycling between an active GTP-bound conformation and an inactive GDP-bound form. Neurofibromin, the protein product of the NF1 tumor suppressor gene, and p120GAP are GTPase-activating proteins (GAPs) for p21Ras (Ras) and negatively regulate output by accelerating GTP hydrolysis on Ras. Neurofibromin and p120GAP differ markedly outside of their conserved GAP-related domains (GRDs), and it is therefore unknown if the respective GRDs contribute functional specificity. To address this question, we expressed the GRDs of neurofibromin and p120GAP in primary cells from Nf1 mutant mice in vitro and in vivo. Here we show that expression of neurofibromin GRD, but not the p120GAP GRD, restores normal growth and cytokine signaling in three lineages of primaryNf1-deficient cells that have been implicated in the pathogenesis of neurofibromatosis type 1 (NF1). Furthermore, utilizing a GAP-inactive mutant NF1 GRD identified in a family with NF1, we demonstrate that growth restoration is a function ofNF1 GRD GAP activity on p21Ras. Thus, the GRDs of neurofibromin and p120GAP specify nonoverlapping functions in multiple primary cell types.

Loss of the Nf1 Tumor Suppressor Gene Decreases Fas Antigen Expression in Myeloid Cells

Genetic loss of surface Fas antigen expression leads to reduced apoptosis of myeloid and lymphoid progenitor cells, and a propensity to develop autoimmunity and myeloid leukemia in mouse models. Oncogenic p21(ras) decreases surface Fas antigen expression and renders fibroblasts resistant to Fas mediated apoptosis. Neurofibromin, which is encoded by NF1, is a GTPase activating protein that negatively regulates p21(ras) activity. NF1 loss leads to deregulation of p21(ras)-effector pathways, which control myeloid cell survival. Heterozygous inactivation of Nf1 increases mast cell numbers in Nf1 +/- mice, and enhances mast cell survival in response to c-kit ligand (kit-L). Here, we show that Nf1-deficient mast cells have reduced surface Fas antigen expression in response to kit-L and are resistant to Fas ligand-mediated apoptosis. Using genetic intercrosses between Nf1 +/- and class I (A)-PI-3K-deficient mice, we demonstrate that hyperactivation of the p21(ras)-class I(A) PI-3K pathway is the mechanism for this phenotype. Finally, we demonstrate that mast cells from both Fas antigen-deficient mice and Nf1 +/- mice are resistant to apoptosis following kit-L withdrawal in vivo. Thus, therapies designed to decrease p21(ras) activity and up-regulate Fas antigen expression may limit the pathological accumulation of myeloid cells in disease states where p21(ras) is hyperactivated.

Neurotrophic factor-secreting autologous muscle stem cell therapy for the treatment of laryngeal denervation injury†‡§

To determine if the spontaneous reinnervation that characteristically ensues after recurrent laryngeal nerve (RLN) injury could be selectively promoted and directed to certain laryngeal muscles with the use of neurotrophic factor (NF)‐secreting muscle stem cell (MSC) vectors while antagonistic reinnervation is inhibited with vincristine (VNC).

Murine yolk sac and bone marrow hematopoietic cells with high proliferative potential display different capacities for producing colony-forming cells ex vivo.

Increasing evidence suggests that hematopoietic stem and progenitor cells from embryonic and fetal tissues demonstrate proliferative capacities greater than cells isolated from adult hematopoietic tissues. Few studies have explored the organization of the high proliferative potential hematopoietic progenitor hierarchy in the murine yolk sac. We have demonstrated the appearance of high proliferative potential colony-forming cells (HPP-CFC) in the yolk sac as early as embryonic day 8 (E8). Yolk sac HPP-CFC colony size and differentiated cellular composition were similar to adult marrow HPP-CFC. The frequency of yolk sac HPP-CFC at E11 was greater than HPP-CFC frequency in the adult marrow. Replating of primary yolk sac HPP-CFC resulted in significantly greater HPP-CFC and multipotent progenitors than replated adult marrow primary HPP-CFC. Similar results were obtained when AA4.1-expressing yolk sac and adult marrow cells that bind wheat germ agglutinin (WGA) were isolated via flow cytometry. These results support growing evidence that fetal, and perhaps embryonic, hematopoietic tissues may be excellent alternative sources of highly proliferative hematopoietic cells as targets for somatic gene therapy.

Local neurotoxins for prevention of laryngeal synkinesis after recurrent laryngeal nerve injury.

Objectives: Persistent vocal fold motion impairment after recurrent laryngeal nerve (RLN) injury is not characteristically due to absent reinnervation, but often results from spontaneous aberrant reinnervation (synkinesis). We administered local neurotoxins to selected laryngeal muscles after RLN injury to determine whether aberrant reinnervation could be selectively inhibited. Methods: Unilateral RLN transection was performed in 24 male rats. Three weeks later, the denervated laryngeal adductor complex was injected with phenol, high- or low-dose vincristine sulfate (VNC), or saline solution. One month later, rat larynges were evaluated via videolaryngoscopy and laryngeal electromyography (LEMG). Larynges from euthanized animals were analyzed via immunofluorescent staining for the presence of reinnervation. Results: One animal that received phenol and 3 animals that received high-dose VNC died of toxicity-related complications. In the surviving neurotoxin-treated animals, videolaryngoscopy showed increased lateralization of the immobile vocal fold. Only 1 phenol-injected rat had adductor complex motor recruitment (score of 3+) with LEMG. The other neurotoxin-treated animals demonstrated an absence of adductor complex reinnervation, with only insertional activity and fibrillations (no motor units/recruitment). Spontaneous ipsilateral abductor reinnervation was not affected by the adductor injections. Conclusions: Low-dose VNC injections appear to be relatively safe and effective in selectively inhibiting spontaneous aberrant reinnervation after RLN injury in an animal model.

Optimization of autologous muscle stem cell survival in the denervated hemilarynx.

Objective: Current treatments for vocal fold paralysis are suboptimal in that they fail to restore dynamic function. Autologous muscle stem cell (MSC) therapy is a promising potential therapy for vocal fold paralysis in that it can attenuate denervation‐induced muscle atrophy and provide a vehicle for delivery of neurotrophic factors, thereby potentially selectively guiding reinnervation. The goal of this project was to characterize optimal conditions for injected autologous MSC survival in the thyroarytenoid (TA) muscle following recurrent laryngeal nerve (RLN) injury by local administration of adjuvant factors.

Ciliary neurotrophic factor (CNTF) promotes skeletal muscle progenitor cell (MPC) viability via the phosphatidylinositol 3-kinase–Akt pathway

Muscle progenitor cells (MPCs) are currently being investigated as cellular vectors to deliver neurotrophic factor (NF) for the promotion of re‐innervation after axonal injury. Ideally NF delivery in such a model would enhance axonal regeneration while simultaneously promoting MPC viability. To date, insulin‐like growth factor 1 (IGF‐1) is one of the few NFs known to promote both re‐innervation and MPC viability. We herein identify ciliary neurotrophic factor (CNTF) as a factor that promotes MPC viability in culture, and demonstrate CNTF to impart greater viability effects on MPCs than IGF‐1. We demonstrate that pharmacological inhibition via LY294002 results in abrogation of CNTF‐mediated viability, suggesting that the CNTF‐mediated MPC viability benefit occurs via the PI3–Akt pathway. Finally, we employ a genetic model, establishing MPC cultures from mice deficient in class IA PI‐3 K (p85α−/−) mice, and demonstrate that the viability benefit imparted by CNTF is completely abrogated in PI‐3 K‐deficient MPCs compared to wild‐type controls. In summary, our investigations define CNTF as a promoter of MPC viability beyond IGF‐1, and reveal that the CNTF‐mediated MPC viability effects occur via the PI3–Akt pathway. Copyright

Characterization of laryngeal muscle stem cell survival and proliferation.

Objectives/Hypothesis: Laryngeal muscle and skeletal muscle stem cells (MSC) have been shown to differ in physiological basal activity and responsiveness to stimuli. Given these differences, it is the purpose of this investigation to characterize the in vitro proliferation and survival of laryngeal and skeletal MSC to determine whether intrinsic differences exist that may account for differences noted in vivo.

The impact of obesity on adult tracheostomy complication rate

Prospectively compare tracheostomy‐related complications in obese patients with complications in nonobese patients.