Institute for Stem Cell Biology and Regenerative Medicine

We report here a systematic, quantitative population analysis of transcription factor expression within developmental progenitors, made possible by a microfluidic chip-based "digital RT-PCR" assay that can count template molecules in cDNA samples prepared from single cells. In a survey encompassing five classes of early hematopoietic precursor, we found markedly heterogeneous expression of the transcription factor PU.1 in hematopoietic stem cells and divergent patterns of PU.1 expression within flk2- and flk2+ common myeloid progenitors. The survey also revealed significant differences in the level of the housekeeping transcript GAPDH across the surveyed populations, which demonstrates caveats of normalizing expression data to endogenous controls and underscores the need to put gene measurement on an absolute, copy-per-cell basis.

Ongoing thymopoiesis requires continual seeding from progenitors that reside within the bone marrow (BM), but the identity of the most proximate prethymocytes has remained controversial. Here we take a comprehensive approach to prospectively identify the major source of thymocyte progenitors that reside within the BM and blood, and find that all thymocyte progenitor activity resides within a rare Flk2(+)CD27(+) population. The BM Flk2(+)CD27(+) subset is predominantly composed of common lymphoid progenitors (CLPs) and multipotent progenitors. Of these 2 populations, only CLPs reconstitute thymopoiesis rapidly after intravenous injection. In contrast, multipotent progenitor-derived cells reconstitute the thymus with delayed kinetics only after they have reseeded the BM, self-renewed, and generated CLPs. These results identify CLPs as the major source of thymocyte progenitors within the BM.

Pulmonary artery intimal sarcoma is an uncommon tumor with a poor prognosis. We report a case of a 75-year-old man with a pulmonary artery sarcoma, recurrent following surgical resection. To palliate symptoms of this recurrence, he underwent CyberKnife stereotactic radiosurgery with a clinical and radiographic response of his treated disease. No acute or sub-acute toxicity was seen until the patient's death due to metastatic disease 10 weeks following treatment. The feasibility and short-term safety of this technique are reviewed, with emphasis on the stereotactic planning considerations, such as mediastinal organ movement and radiation tolerance.

BACKGROUND: Side effects of current pediatric brain tumor therapeutics, usually resulting from neural cell toxicity, call for testing of novel therapeutic modalities on appropriate preclinical models, which recapitulates normal human neural environment with malignant brain tumors. Here, we present a new model to study toxicity of humanized antibodies against xenografted human normal neural cells sequentially co-transplanted with primary medulloblastoma (MB) cells. METHODS: Human neural progenitor cells (HuNPCs) derived from human fetal neural tissue, were infected with td-Tomato-luciferase expressing virus and injected into newborn pups (p1-p3 days old). HuNPC engraftment was confirmed with bioluminescence imaging and previously characterized for multi-potent differentiation into neurons, astrocytes and oligodendrocytes. HuNPC engrafted mice were then injected with unlabeled human MB cells at 1.5 months of age. MB cells used were previously confirmed to have 100% penetrance in multiple cohorts. After 2 weeks of tumor cell transplantation, mice were randomized and treated with either humanized anti-CD47 monoclonal antibody (HuCD47 mAb) or control. Human neural cells viability and proliferation in all mice were measured through subsequent bioluminescence imaging. RESULTS: Follow-up bioluminescence imaging revealed minimal change in flux values of treated mice with HuCD47 mAb, representing viability of engrafted normal neural cell, compared to control. Moreover, survival rates as well as subsequent H & E staining show HuCD47 mAb dramatically decreased tumor size and subsequently increased the survival of mice treated with HuCD47 mAb compared to control similar to experiments carried out in mice transplanted with human MB cells only. CONCLUSION: The presented in vivo model provides a new platform to test any novel potential therapeutic (here, HuCD47 mAb) in the context of xenografted human normal neural environment. This way we not only measure effect of the proposed drug on tumor regression but impact of developed agent on human normal neural tissue in the vicinity of the tumor.