MacDermott 1,2

MacDermott 1,2

1 Department of Physiology and Cellular Biophysics, Columbia University, New York, NY.

2 Department of Neuroscience, Columbia University, New York, NY.

3 Project A.L.S./Jenifer Estess Laboratory for Stem Cell Research, New York, NY.

4 Departments of Pathology, Neurology and Neuroscience, Center for Motor Neuron Biology and Disease (MNC), and Columbia Stem Cell Initiative (CSCI), Columbia University, New York, NY. Motor neurons generated from patient-derived induced pluripotent stem cells

have the potential to be excellent cellular models for ALS and functional characterization of these neurons provides a useful approach to determine pathophysiological mechanisms. Live cell techniques such as Ca 2+ imaging and electrophysiology are particularly well-suited for this purpose as they provide a method to directly monitor neuronal function. In order to investigate the potential role of changes to Ca 2+ homeostasis in ALS, Ca 2+ imaging experiments were carried out in induced pluripotent stem cell-derived motor neurons (iPS- MNs) generated from an ALS patient with an SOD1 A4V mutation and control iPS-MNs generated from a healthy individual. Ca 2+ transients were evoked by a

2 s application of kainate and the time course of Ca 2+ clearance from the cytosol was determined. There was a small but significant decrease in time course of Ca 2+ recovery in the ALS iPS-MNs compared to the controls. The relationship of this change to ALS pathophysiology remains to be determined. Action potential firing characteristics of the control and ALS iPS-MNs were determined using current clamp recording. Significant changes in the firing properties of the cells indicative of a decrease in excitability were observed in the ALS iPS-MNs, but variability of the firing characteristics makes interpretation of the results uncertain. Future experiments using iPS-MNs expressing a motor neuron 2 s application of kainate and the time course of Ca 2+ clearance from the cytosol was determined. There was a small but significant decrease in time course of Ca 2+ recovery in the ALS iPS-MNs compared to the controls. The relationship of this change to ALS pathophysiology remains to be determined. Action potential firing characteristics of the control and ALS iPS-MNs were determined using current clamp recording. Significant changes in the firing properties of the cells indicative of a decrease in excitability were observed in the ALS iPS-MNs, but variability of the firing characteristics makes interpretation of the results uncertain. Future experiments using iPS-MNs expressing a motor neuron

Gremlin 1 labels a mesenchymal progenitor cell in the gastrointestinal tract, bone marrow and cancer microenvironment. Worthley DL, Si Y, Asfaha S, Westphalen CB, Tailor Y, Carpenter J,

Quante M, Churchill M, Pradere J-P, Troeger J, Mukherjee S, Schwabe R, Wang TC.

Introduction: The cellular origin and kinetics of mesenchymal stem cells (MSCs) in the adult gastrointestinal tract and bone marrow (BM) is uncertain. More specific markers of MSCs are needed to allow for lineage tracing and in vitro analysis of these cells in health and disease. We recently reported that the BMP antagonist Gremlin 1 (Grem1) was a potential marker of MSCs in the BM (CFU-F and differentiation) and that BM-derived cells, including Grem1 positive cells, were recruited from the BM to generate cancer-associated fibroblasts.1,2 We hypothesized that Grem1 marked the MSC origin of both physiological and tumor-associated mesenchyme. Methods: To address this we developed a Grem1-BAC-CreERT transgenic line. We also generated 3 other stromal- dire ted tra sge i li es α“MA-BAC-CreERT, Grem1-BAC-EGFP-peptide 2A-DTR-peptide 2A-CreERT lines and a Vimentin-BAC- CreERT to characterize the function and cellular kinetics of gastrointestinal and BM mesenchyme. These mice were crossed to either the R26-mT/mG or the R26- TdTo ato reporter. The ellular fate of the Gre

, α“MA a d Vi e ti

expressing cells, was analyzed by direct fluorescence microscopy and immunostaining at several time points (up to 6 months) following tamoxifen induction as well as in several cancer models. Results: Grem1 recombination was identified in a very rare population of CD45/Ter119 negative cells (i.e. in the non-hematopoietic stromal fraction) within the BM (0.0008% of all nucleated BM cells by FACS, Fig 1). Grem1(+) cells dramatically expanded within BM-derived MSC cultures (from 0.0008% to 0.5% after one week in culture) forming large colony forming units (CFU-F, Fig 2A). These Grem1-recombined cells could be differentiated in culture into adipo tes, osteo lasts a d α“MA + MFs Fig B-D). Chondrocyte cultures are ongoing. In the gastrointestinal tract, 24 hours after tamoxifen induction, there were single Grem1 recombined cells found immediately subjacent to the basement membrane (Fig 3). This location was confirmed in our Grem1-EGFP reporter line (Grem1-EGFP-peptide 2A-DTR-peptide 2A- CreE‘T . α“MA a d vimentin recombination, however, identified broader and partially overlapping populations of mesenchymal cells. In keeping with Grem1 labeling a mesenchymal progenitor within the gastrointestinal tract, these initially rare α“MA -) recombined cells expanded over the next 6 months giving rise to differe tiated α“MA + ells ithi the gastrointestinal subepithelial MF sheath. If Grem1 expression, however, truly marked an MSC in mesenchymal development one would expect that earlier induction of Grem1-recombination (before complete mesenchymal development) would lead to even greater tracing. Therefore, we compared recombination of the gastrointestinal tract 3 weeks after induction of either a P2 pup versus a 6-8 week old adult mouse. The earlier induction was associated with dramatically increased stromal recombination in all tissues examined. These findings are consistent with Grem1 being a marker of a multipotent mesenchymal progenitor cell in health. Importantly, Grem1 also marked a cellular origin of cancer-associated mesenchyme. We performed syngeneic tumor studies using subcutaneous MC38 cells as well as a hepatic metastatic model (splenic injection of MC38, C57Bl/6 colorectal cancer cell line) in our Grem1-BAC-CreERT x R26mT/mG mice two weeks after tamoxifen induction. Recombined (EGFP +) stromal cells were found invading the tumor in both models (Fig 4). This suggests that Grem1 cells can be recruited and generate peritumoral mesenchyme. Conclusions: Our data presents compelling evidence that Grem1-expression labels a mesenchymal progenitor in the BM and gut that can give rise to multiple mesenchymal lineages during development, adulthood and in the peritumoral stroma. We are currently examining the functional relevance of this cell in supporting the normal and pathological stem cell niche. References

1. Quante, M. et al. Bone Marrow-Derived Myofibroblasts Contribute to the Mesenchymal Stem Cell Niche and Promote Tumor Growth. Cancer Cell 19,

2. Worthley, D. L. et al. Human gastrointestinal neoplasia-associated myofibroblasts can develop from bone marrow-derived cells following allogeneic stem cell transplantation. Stem Cells 27, 1463 –1468 (2009).

A novel extracellular matrix-derived hydrogel for stem cell-based meniscus tissue engineering

1 X. Yuan , D. E. Arkonac 1 , A. B. Taubman 1 , and G. Vunjak-

Novakovic 1

1 Columbia University, New York, NY

Introduction: The knee meniscus is a fibrocartilaginous tissue with high load- bearing properties due to its water content and unique extracellular matrix

(ECM), composed of collagenous proteins and glycosaminoglycans (GAG) 1 . However, the limited extent of innate meniscal repair after injury has led to efforts towards tissue-engineered replacements. Recently, promising new approaches have incorporated natural scaffolds made from native ECM, which retain tissue-specific molecules that guide the behavior of new cells

repopulating the material and facilitate tissue development 2 . This study sought to create a novel scaffold from meniscus ECM, which contains biological cues that support the growth of viable replacement tissue. Materials and Methods: The menisci of juvenile bovine calves were dissected

and minced to 1-2 mm 3 pieces, and incubated in 0.1% peracetic acid for 2 h at 25°C with agitation, followed by three cycles of washes in sterile water and PBS.

The pieces were then lyophilized for 24 h, before digestion at 10 mg/mL in 0.1% pepsin in 0.01 M HCl for 16 h at 25°C. The resulting meniscus ECM (mECM)

digestion solution was allowed to repolymerize, using a previously established protocol 2 . Human mesenchymal stem cells (hMSCs; passage 7; 30×10 6 cells/mL)

were encapsulated into disks (4 mm Ø × 2 mm thickness) composed of either mECM (3 mg/mL) or type I collagen (3 mg/mL) hydrogel, and cultured for 28 days in chondrogenic media supplemented with 10 ng/mL TGF- β . “a ples were collected at days 0, 14, and 28 for biochemical (DNA, sulfated GAG

o te t a d histologi al [he ato li a d eosi H&E , Masso s tri hro e, Alcian blue] analyses. All data are presented as mean ± SEM. Statistics were

performed by unpaired t-test. Results and Discussion: Encapsulation in meniscus ECM hydrogel enhanced chondrogenesis of hMSCs compared to cells in type I collagen alone. No differences in cell proliferation were observed between the two types of hydrogel constructs, but sulfated GAG content was significantly greater in mECM constructs relative to type I collagen only, as early as at day 14 (Fig. 1A). Histological staining of mECM constructs at day 28 demonstrates the production of a matrix rich in proteoglycans (Fig. 1B; Alcian blue), but only faint stai i g for ollage s Fig. B; Masso s tri hro e . Conclusions: Meniscus ECM hydrogel retains native, tissue-specific molecules that cue stem cells to form new meniscus tissue, as demonstrated by enhanced sulfated GAG content and staining for proteoglycans. mECM hydrogel and digestion solution are therefore promising new starting materials for the future design of scaffolds that recapitulate the complex architecture of the meniscus. Acknowledgments: Funding by the NIH is gratefully acknowledged.

References: 1) Sweigart MA, et al. Tissue Eng, 2001, 7, 111-129. 2) Freytes DO, et al. Biomaterials, 2008, 29, 1630-1637.

Lineage analysis of basal epithelial cells reveals their unexpected plasticity and supports a cell of origin model for prostate cancer heterogeneity. Zhu A. Wang, Antonina Mitrofanova, Sarah K. Bergren, Cory

Abate-Shen, Andrea Califano, and Michael M. Shen.

Departments of Medicine and Genetics & Development, Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, NY 10032.

A key issue in cancer biology is whether oncogenic transformation of different cell types of origin within an adult tissue gives rise to distinct tumor subtypes that differ in their prognosis and/or treatment response. In the case of prostate A key issue in cancer biology is whether oncogenic transformation of different cell types of origin within an adult tissue gives rise to distinct tumor subtypes that differ in their prognosis and/or treatment response. In the case of prostate

Retinal Ganglion

Cell

Generation

and

Differentiation in the Albino Mouse Visual System

Punita Bhansali 1,3 , Ilana Rayport and Carol Mason 1,2