Susannah Kassmer

Abstract 2615 Since 1998, there have been many discoveries that bone marrow derived cells (BMDC) possess the ability to cross lineage barriers and differentiate into mature, non-hematopoietic cells of multiple tissues. In recent years, several studies demonstrated engraftment of BMDC as epithelial cells of the lung. One of the main unanswered questions is which population(s) of BMDC are responsible for this engraftment. Previous investigations reported a lack of marrow derived lung epithelial cells (MDLE) after transplantation of hematopoietic stem cells (Thy1loLSK), while others found MDLE in the lungs of mice undergoing whole bone marrow transplantation. We therefore hypothesized that primitive, non-hematopoietic stem cells residing in the BM are the source of MDLE, and tested directly whether nonhematopoietic bone marrow derived cells have the capacity to engraft as epithelial cells of the lung. To test this hypothesis, we used Vav-cre transgenic mice, in which Cre recombinase is expressed uniquely within cells of the hematopoietic lineage, to determine which BM cell populations can engraft as surfactant protein C (SPC) expressing type 2 pneumocytes in surfactant-protein-C-null (SPC-KO) mice. Live hematopoietic and non-hematopoietic BM cells were separated by FACS-sorting based on expression of YFP in cells from ROSA-YFP/Vav-Cre mice that express YFP only in cells committed to the hematopoiesis. YFP positive (hematopoietic) or YFP negative (non-hematopoietic) cells were transplanted into irradiated SPC-KO mice. For transplant recipients of YFP negative cells, 1 million whole bone marrow cells from a recipient type SPC-KO mouse were cotransplanted to provide hematopoietic rescue following irradiation. MDLE were assessed by detection of alveolar type 2 cells expressing wild type SPC, which could only be derived from the donor cells in SPC-KO recipients. Methods of detection included confocal microscopic analysis of sorted CD45 negative lung cells and tissue sections stained for SPC and epithelial specific cytokeratin or TTF1 by immunofluorescence. In addition, isolated lung cells were stained in suspension and analyzed for donor derived type 2 cells by Imagestream technology for expression of SPC and cytokeratin. SPC positive type 2 cells were found in the lungs of 4 out of 6 mice receiving YFPnegative, non-hematopoietic BM cells, with 1 out of 16,000 total nucleated cells expressing SPC on lung tissue sections. In contrast, donor derived SPC positive type 2 pneumocytes were exceedingly rare in mice receiving YFPpositive, hematopoietic cells and were found in just 1 out of 9 mice, with only 1 out of 144,000 total nucleated cells being positive for SPC on tissue sections. These findings were confirmed by highly sensitive RT-PCR for wild type SPC mRNA. We conclude that the non-hematopoietic fraction of murine BM contains cells capable of engrafting as epithelial cells of the lung. Our data support the hypothesis of a primitive, pluripotent stem cell population residing in adult bone marrow. Disclosures: No relevant conflicts of interest to declare.

The colonial ascidian Botryllus schlosseri regenerates the germline during repeated cycles of asexual reproduction. Germline stem cells (GSCs) circulate in the blood and migrate to new germline niches as they develop and this homing process is directed by a Sphigosine-1-Phosphate (S1P) gradient. Here, we find that inhibition of ABC transporter activity reduces migration of GSCs towards low concentrations of S1P in vitro. In addition, inhibiting phospholipase A2 (PLA2) or lipoxygenase (Lox) blocks chemotaxis towards low concentrations of S1P. These effects can be rescued by addition of the 12-Lox product 12-S-HETE. Blocking ABC transporter, PLA2 or 12-Lox activity also inhibits homing of germ cells in vivo. Using a live-imaging chemotaxis assay in a 3D matrix, we show that a shallow gradient of 12-S-HETE enhances chemotaxis towards low concentrations of S1P and stimulates motility. A potential homolog of the human receptor for 12-S-HETE, gpr31, is expressed on GSCs and differentiatin...

Background Botryllid ascidians are a group of marine invertebrate chordates that are colonial and grow by repeated rounds of asexual reproduction to form a colony of individual bodies, called zooids, linked by a common vascular network. Two distinct processes are responsible for zooid regeneration. In the first, called blastogenesis, new zooids arise from a region of multipotent epithelium from a pre-existing zooid. In the second, called whole body regeneration (WBR), mobile cells in the vasculature coalesce and are the source of the new zooid. In some botryllid species, blastogenesis and WBR occur concurrently, while in others, blastogenesis is used exclusively for growth, while WBR only occurs following injury or exiting periods of dormancy. In species such as Botrylloides diegensis, injury induced WBR is triggered by the surgical isolation of a small piece of vasculature. However, Botryllus schlosseri has unique requirements that must be met for successful injury induced WBR. Our...

Vascular diseases affect over 1 billion people worldwide and are highly prevalent among the elderly, due to a progressive deterioration of the structure of vascular cells. Most of our understanding of these age-related cellular changes comes from in vitro studies on human cell lines. Further studies of the mechanisms underlying vascular aging in vivo are needed to provide insight into the pathobiology of age-associated vascular diseases, but are difficult to carry out on vertebrate model organisms. We are studying the effects of aging on the vasculature of the invertebrate chordate, Botryllus schlosseri. This extracorporeal vascular network of Botryllus is transparent and particularly amenable to imaging and manipulation. Here we use a combination of transcriptomics, immunostaining and live-imaging, as well as in vivo pharmacological treatments and regeneration assays to show that morphological, transcriptional, and functional age-associated changes within vascular cells are key hal...

Colonial ascidians are the only chordates able to undergo whole body regeneration (WBR), during which entire new bodies can be regenerated from small fragments of blood vessels. Here, we show that during the early stages of WBR in Botrylloides diegensis, proliferation occurs only in small, blood-borne cells that express integrin-alpha-6 (IA6), pou3 and vasa. WBR cannot proceed when proliferating IA6+ cells are ablated with Mitomycin C, and injection of a single IA6+ Candidate stem cell can rescue WBR after ablation. Lineage tracing using EdU-labeling demonstrates that donor-derived IA6+ Candidate stem cells directly give rise to regenerating tissues. Inhibitors of either Notch or canonical Wnt signaling block WBR and reduce proliferation of IA6+ Candidate stem cells, indicating that these two pathways regulate their activation. In conclusion, we show that IA6+ Candidate stem cells are responsible for whole body regeneration and give rise to regenerating tissues.

2615 Since 1998, there have been many discoveries that bone marrow derived cells (BMDC) possess the ability to cross lineage barriers and differentiate into mature, non-hematopoietic cells of multiple tissues. In recent years, several studies demonstrated engraftment of BMDC as epithelial cells of the lung. One of the main unanswered questions is which population(s) of BMDC are responsible for this engraftment. Previous investigations reported a lack of marrow derived lung epithelial cells (MDLE) after transplantation of hematopoietic stem cells (Thy1loLSK), while others found MDLE in the lungs of mice undergoing whole bone marrow transplantation. We therefore hypothesized that primitive, non-hematopoietic stem cells residing in the BM are the source of MDLE, and tested directly whether nonhematopoietic bone marrow derived cells have the capacity to engraft as epithelial cells of the lung. To test this hypothesis, we used Vav-cre transgenic mice, in which Cre recombinase is expresse...

In the colonial ascidianBotryllus schlosseri, long-lived germline stem cells (GSCs) migrate to new germline niches as they develop during repetitive cycles of asexual reproduction. ABC-transporters are involved in the export of lipid-signaling molecules, but their roles in germ cell migration are poorly understood. Here, we show that inBotryllus, abcc1andabcb1are highly expressed in germ cells, and inhibition of ABC-transporter activity leads to failure of germ cell migration. Phospholipase A2 (PLA2) produces arachidonic acid, which is further metabolized to eicosanoid signaling molecules. In humans, 12-lipoxygenase (LOX) metabolizes arachidonic acid to12-Hydroxyeicosatetraenoic acid (12-S-HETE), which stimulates migration of mammalian cancer cells and smooth muscle cells. We show that PLA2 and LOX activity are required for germ cell migration. A potential homolog to the human receptor for 12-S-HETE,BSgpr31, is expressed in germ cells. 12-S-HETE rescues migration towards S1P in the ...

The colonial ascidian Botryllus schlosseri is an ideal model organism for studying gonad development and hermaphroditism. B. schlosseri has been reared in laboratories for over half a century, and its unique biology allows investigators to probe the processes of germ cell migration and gonad formation, resorption, and regeneration. Following metamorphosis, colonies of B. schlosseri show a synchronized and sequential fertility program that, under standard laboratory conditions, begins with a juvenile stage with no visible gonads and subsequently develops testes at 9 weeks followed later by the development of oocytes - thus resulting in hermaphroditic individuals. The timing of oocyte production varies according to the season, and adult B. schlosseri colonies can cycle among infertile and both male and hermaphrodite fertile states in response to changing environmental conditions. Thus, these acidians are amenable to studying the molecular mechanisms controlling fertility, and recent g...

Colonial ascidians are marine invertebrate chordates that are the closest invertebrate relative to the vertebrates. Colonies of Botryllus schlosseri undergo a continuous asexual reproduction process that involves the regeneration of entire new bodies, which include all somatic and germline tissues. This adult regenerative process depends on signaling pathways known to regulate the formation of tissues and organs during embryonic development. The formation of gonads within regenerated bodies depends on migration and homing of germ cell precursors to niches within the developing bodies, and Botryllus colonies can cycle between fertile and infertile states. The vasculature that connects all individuals within the colony is highly regenerative, and is a valuable tool for the study of angiogenesis in adult blood vessels. The tremendous regenerative capacity of the vasculature even results in regeneration of entire new bodies solely from fragments of blood vessels upon surgical removal of...

The invertebrate chordate Botrylloides leachii regenerates entire new bodies from small fragments of blood vessels in a process called whole body regeneration (WBR). During the early stages of WBR, proliferation occurs only in small, blood borne cells expressing integrin-alpha-6, pou3 and vasa. Proliferating blood cells can be ablated using Mitomycin C (MMC), and MMC treatment blocks regeneration. However, regeneration could be rescued by injection of blood borne cells from healthy individuals, and using limiting dilution approaches we found that injection of a single Integrin-alpha-6 (IA6)-positive cell is capable of rescuing whole body regeneration. The contribution of IA6+ cells to WBR was confirmed via lineage tracing: following transplantation of EdU-labeled IA6+ cells into MMC treated recipients, EdU label is detected in regenerating tissues. Our data suggest that ia6+pou3+ cells are proliferating blood borne stem cells that are responsible for whole body regeneration, and we ...

One of the challenges in stem cell research is to avoid transformation during cultivation. We studied high passage subventricular zone derived neural stem cells (NSCs) cultures of adult rats in the absence of growth factors epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). We termed this culture exogenous growth factor independent neural stem cells (GiNSCs). GiNSCs expressed stemness markers, displayed a high constitutive NF-kappaB activity and an increased, aberrant, polyploid DNA content. GiNSCs showed a tumorigenic phenotype and formed colonies in a soft agar assay. Microarray analysis showed the up-regulation of the NF-kappaB target gene vascular endothelial growth factor (VEGF). In contrast, proneuronal genes were down-regulated. Under neuronal differentiation conditions GiNSCs adopted a glioma-like phenotype, with nuclear p53, preserving high amounts of Nestin positive cells and prolonged proliferation. Neutralization of VEGF strongly inhibited proliferation and induced differentiation. In a gain of function approach, the transfection of NSCs with constitutively active upstream kinase IKK-2 led to constitutively activated NF-kappaB, proliferation in absence of growth factors and augmented VEGF secretion. In a rescue experiment a reduction of NF-kappaB activity by overexpression of IkappaB-AA1 was able to shift the morphology toward an elongated cell form, increased cell death, and decreased proliferation. Thus GiNSCs may provide a potent tool in cancer research, as their exogenous cytokine independent proliferation and their constitutively high NF-kappaB expression presumes cancerous properties observed in gliomas. In addition, this study might add a novel mechanism for detecting oncogenic transformation in therapeutic stem cell cultures.

... Basel, Karger, 2010, vol 2, pp 7–27 Stem Cell Migration in Health and Disease Thomas DittmaraSusannah H. Kassmerd Benjamin Kasendab Jeanette Seidelc Bernd Niggemanna Kurt S. Zänkera aInstitute of Immunology, Witten/Herdecke University, Witten; bDepartment of ...

One of the challenges in stem cell research is to avoid transformation during cultivation. We studied high passage subventricular zone derived neural stem cells (NSCs) cultures of adult rats in the absence of growth factors epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). We termed this culture exogenous growth factor independent neural stem cells (GiNSCs). GiNSCs expressed stemness markers, displayed a high constitutive NF-kappaB activity and an increased, aberrant, polyploid DNA content. GiNSCs showed a tumorigenic phenotype and formed colonies in a soft agar assay. Microarray analysis showed the up-regulation of the NF-kappaB target gene vascular endothelial growth factor (VEGF). In contrast, proneuronal genes were down-regulated. Under neuronal differentiation conditions GiNSCs adopted a glioma-like phenotype, with nuclear p53, preserving high amounts of Nestin positive cells and prolonged proliferation. Neutralization of VEGF strongly inhibited proliferation and induced differentiation. In a gain of function approach, the transfection of NSCs with constitutively active upstream kinase IKK-2 led to constitutively activated NF-kappaB, proliferation in absence of growth factors and augmented VEGF secretion. In a rescue experiment a reduction of NF-kappaB activity by overexpression of IkappaB-AA1 was able to shift the morphology toward an elongated cell form, increased cell death, and decreased proliferation. Thus GiNSCs may provide a potent tool in cancer research, as their exogenous cytokine independent proliferation and their constitutively high NF-kappaB expression presumes cancerous properties observed in gliomas. In addition, this study might add a novel mechanism for detecting oncogenic transformation in therapeutic stem cell cultures.

The source of tissue turnover during homeostasis or following injury is usually due to proliferation of a small number of resident, lineage-restricted stem cells that have the ability to amplify and differentiate into mature cell types. We are studying vascular regeneration in a chordate model organism, Botryllus schlosseri, and have previously found that following surgical ablation of the extracorporeal vasculature, new tissue will regenerate in a VEGF-dependent process within 48 hrs. Here we use a novel vascular cell lineage tracing methodology to assess regeneration in parabiosed individuals and demonstrate that the source of regenerated vasculature is due to the proliferation of pre-existing vascular resident cells and not a mobile progenitor. We also show that these cells are bi-potential, and can reversibly adopt two fates, that of the newly forming vessels or the differentiated vascular tissue at the terminus of the vasculature, known as ampullae. In addition, we show that pre-existing vascular resident cells differentially express progenitor and differentiated cell markers including the Botryllus homologs of CD133, VEGFR-2, and Cadherin during the regenerative process.

Stromal cell-derived factor-1alpha (SDF-1alpha) is a strong migratory stimulant for hematopoietic stem and progenitor cells (HSPCs). The hematopoietic cytokines thrombopoietin (TPO), Flt3-ligand (FL), stem cell factor (SCF) and interleukin 11 (IL-11) are able to stimulate amplification of primitive murine hematopoietic stem cells (HSCs) in vitro. The effects of these cytokines on SDF-1alpha-induced migratory activity of murine Lin(-)c-kit+ HSPC were analyzed by cultivation of these cells in the presence of 12 combinations of FL, TPO, SCF and IL-11. Migratory activity was measured in a three-dimensional collagen matrix using time-lapse video microscopy. Each cytokine combination had a distinct effect on SDF-1alpha-stimulated migratory activity. For instance, FL- and SCF-cultivated cells showed a high migratory SDF-1alpha response, while cells cultivated with SCF, TPO and IL-11 did not react to SDF-1alpha stimulation with an elevated migration rate. Our data indicate that the differences in the migratory SDF-1alpha response are not related to different CXCR4 expression levels, but rather to the differential engagement of the CXCR4-dependent MAPK p42/44 and PI3K signal transduction pathways. This indicates that hematopoietic cytokines can have a significant impact on SDF-1alpha-stimulated migratory activity and the underlying intracellular signaling processes in cultivated HSPCs.