acase@tulane.edu / The ability of humans to regenerate complex tissue structures after amputation is not completely absent. Clinical reports have described random spontaneous cases of digit tip regeneration in young adults. Regeneration of a structure such as a limb or a digit requires tight orchestration of environmental cues and cells that come together and coordinate the regeneration of the missing body part. Studies on animal models have been crucial to have a better understanding on relevant components and mechanisms that are involved in epimorphic regeneration. Mechanistic studies however, are difficult to perform due to the lack of spatial and temporal control of microenvironmental factors. The overall approach of this proposal is to develop a blastema-like in vitro model to conduct comparative studies between connective tissue cells from regeneration-competent (P3) and incompetent (P2) regions of the mouse digit tip, and to control the cellular microenvironment to modulate P2 cells regeneration-incompetent behavior. A 3D spheroid culture model was identified to serve as a 3D biomimetic blastema model that preserves the inherent regenerative properties of regeneration-incompetent and regeneration-competent phenotypes. Relevant factors associated with either wound healing or a regenerative response, were evaluated in both P2 and P3 cells cultured as spheroids. It was found that the expression of wound healing markers, particularly known to be involved in scar tissue formation, were significantly higher in P2 spheroids. Conversely, the expression of markers indicative of a regeneration-permissive microenvironment was significantly higher in P3 spheroids. We evaluated the effects of oxygen modulation in P2 during 2D expansion and/or during 3D spheroid culture and found that preconditioning of P2 cells in 2D increases P2 cell number and promotes spontaneous aggregation. We also found that modulation of oxygen concentration during 3D culture significantly decreases expression of both, wound healing and regenerative markers. This physiologically relevant in vitro model provides a platform to characterize cellular processes involved in the wound healing and regenerative responses. Additionally, it allows for the incorporation of environmental cues, such as oxygen concentration, to better understand the key target mechanisms to shift the default wound response from scar formation to epimorphic regeneration. / 1 / Lina M. Quijano

Research using the adult mammalian model shows that regeneration in the limb is limited to the distal most portion of the terminal phalanx. Recent studies suggest that the cellular contributions made to the regenerating system are lineage restricted and that the niche bone marrow hematopoietic stem cell population’s contributions are minimal. These studies however, do not address other residing populations within the bone marrow, specifically the mesenchymal and endothelial stem cell populations. One of the residing populations, the reputed pericyte or perivascular cells, possesses the ability to differentiate into multiple other cell types. To assess the potential contribution of perivascular cells to the regeneration competency of the terminal phalanges, we began by identifying perivascular cells within the terminal phalanx by using two accepted pericyte markers: nerve-glial antigen 2 (NG2) and endosialin (TEM1). Using NG2 and TEM1 in conjunction with vascular marker Tie2 in the Tie2-EGFP murine reporter line, we confirm a large number of perivascular cells in the bone marrow’s unusually well-developed and organized vasculature and a lower density within the connective tissue microvasculature; implicating a great potential contribution from the bone marrow. Post-amputation, we observe a large population of NG2+ and TEM1+ cells within the regenerating blastema region. Co-immunohistochemical studies reveal the blastema have cells that co-express osteogenic and pericyte markers; strongly suggestive of a transdifferentiation event. We attempt to confirm our hypotheses made in our initial assessment by utilizing two independent cell tracing studies: a DiI labeling of the bone marrow of the terminal phalanx to identify a marrow derived cellular contribution to the regenerate and a genetic fate tracing study using transgenic NG2CreERTamR26REYFP mice to confirm a transdifferentiation event. Using a novel in vivo method , we DiI-label the bone marrow content before amputation and trace DiI labeled bone marrow derived cellular contributions to the regenerate. DiI labeled cells were observed within the blastema expressing either endothelial, perivascular, or osteogenic markers, confirming the bone marrow contributes multiple cell types during the regeneration process. Using a similar experimental design, we genetically label the terminal phalanx NG2 expressing cells using systemic tamoxifen induction of NG2CreERTamR26REYFP mice. We fate trace the initially labeled population during blastema formation and re-differentiation and observed transdifferentiation events of the perivascular cells into two distinctive lineages, endothelial and osteoprogenitor cells. Establishing a direct correlation between peri-vasculature and re-differentiation, we address NG2/perivascular necessity with a series of temporal loss of function studies using a blocking antibody (iNG2). We implant iNG2 soaked microcarrier beads into various regions of the terminal phalanx and during different stages of the regeneration process. The experiments confirm the necessity of NG2 expression for distal bone elongation, as well as ascertain the temporal nature of the NG2 expression in different microenvironments. These results establish the importance of NG2+ cells in the bone marrow during early stages of regeneration, with early iNG2 bone marrow implantation resulting in a complete failure of the regeneration process. In an attempt to rescue this iNG2 failed regeneration we employ an established position-specific fibroblast cell line that displays a surprising plasticity as a cell-based therapeutic. Through a series of RNAi lentiviral transfection of inhibitors of the TGFβ-BMP pathway we induce osteogenic plasticity in the line. These results reveal regeneration competency associated with the mammalian terminal phalanx is in part due to the ability to recruit local perivascular multipotent populations, which has great translational relevancy. / acase@tulane.edu

Sublinhagens de camundongos AIRmax e AIRmin homozigotas para os alelos R ou S do gene Slc11a1 apresentam distinta capacidade regenerativa à perfuração de suas orelhas. Animais AIRmaxSS exibiram regeneração tecidual precoce em comparação aos animais AIRmaxRR, sugerindo que o alelo S favorece a regeneração nestes animais. Camundongos das sublinhagens AIRmin não apresentaram regeneração após perfuração de suas orelhas. Em resposta ao estímulo, animais AIRmaxSS exibiram inflamação local mais intensa e tardia do que animais AIRmaxRR, demonstrando elevados níveis de MPO e edema, e influxo celular predominantemente de neutrófilos. Ensaios de expressão gênica global demonstraram genes diferencialmente expressos entre as sublinhagens, evidenciando genes sobre-representados no tema biológico proliferação celular em ambas sublinhagens, enquanto somente nos animais AIRmaxSS ocorreu sobre-representação para resposta inflamatória nos genes ativados e para contração muscular nos genes reprimidos. Os resultados de microarray foram validados por qPCR. / Homozygous AIRmax and AIRmin sublines for Slc11a1 R and S alleles present distinct regenerative capacity to the ear hole. AIRmaxSS mice exhibited early tissue regeneration compared to AIRmaxRR animals, suggesting that the Slc11a1 S allele promotes regeneration in these animals. AIRmin sublines didnt show regeneration after ear punch. In response to the stimulus, AIRmaxSS animals exhibited more intense and later local inflammation than AIRmaxRR animals, presenting elevated levels of MPO, edema and cellular influx predominantly of neutrophils. Global gene expression analysis showed differentially-expressed genes between the sublines, in which over-represented biological theme is cell proliferation in both sublines. AIRmaxSS animals displayed over-representation of inflammatory response in up-regulated genes and of muscle contraction in down-regulated genes. Microarray results were validated by using quantitative PCR.

Sublinhagens de camundongos AIRmax e AIRmin homozigotas para os alelos R ou S do gene Slc11a1 apresentam distinta capacidade regenerativa à perfuração de suas orelhas. Animais AIRmaxSS exibiram regeneração tecidual precoce em comparação aos animais AIRmaxRR, sugerindo que o alelo S favorece a regeneração nestes animais. Camundongos das sublinhagens AIRmin não apresentaram regeneração após perfuração de suas orelhas. Em resposta ao estímulo, animais AIRmaxSS exibiram inflamação local mais intensa e tardia do que animais AIRmaxRR, demonstrando elevados níveis de MPO e edema, e influxo celular predominantemente de neutrófilos. Ensaios de expressão gênica global demonstraram genes diferencialmente expressos entre as sublinhagens, evidenciando genes sobre-representados no tema biológico proliferação celular em ambas sublinhagens, enquanto somente nos animais AIRmaxSS ocorreu sobre-representação para resposta inflamatória nos genes ativados e para contração muscular nos genes reprimidos. Os resultados de microarray foram validados por qPCR. / Homozygous AIRmax and AIRmin sublines for Slc11a1 R and S alleles present distinct regenerative capacity to the ear hole. AIRmaxSS mice exhibited early tissue regeneration compared to AIRmaxRR animals, suggesting that the Slc11a1 S allele promotes regeneration in these animals. AIRmin sublines didnt show regeneration after ear punch. In response to the stimulus, AIRmaxSS animals exhibited more intense and later local inflammation than AIRmaxRR animals, presenting elevated levels of MPO, edema and cellular influx predominantly of neutrophils. Global gene expression analysis showed differentially-expressed genes between the sublines, in which over-represented biological theme is cell proliferation in both sublines. AIRmaxSS animals displayed over-representation of inflammatory response in up-regulated genes and of muscle contraction in down-regulated genes. Microarray results were validated by using quantitative PCR.