- Email: [email protected]
Transplantation of neural crest progenitor cells for Hirschsprung’s disease
Vol. 205, No. 3S, September 2007
Transcriptional profiling of human amniotic fluid derived stem cells and their multilineage differentiation Jennifer Hipp, Bryan Tillman, MD, PhD, Jason Hipp, PhD, Steve Hodges, MD, Anthony Atala, MD, Shay Soker, PhD Wake Forest University School of Medicine, Winston-Salem, NC INTRODUCTION: We have recently isolated a subpopulation of pluripotent stem cells from human amniotic fluid (Amniotic Fluid Stem Cells, AFSC). These cells have a high self renewal capacity, with a short doubling time, do not require a feeder layer, and can be grown in basic medium supplemented. AFSC can be differentiated in vitro into osteogenic, adipogenic, myogenic, neurogenic, vasculogenic, and hepatic phenotypes. The goal of this study is to understand the genetic processes involved in stem cell differentiation that would identify targets to increase the efficiency of differentiation. METHODS: Microarrays were performed at day 20 and 30 on myogenic and osteogenic differentiation, day 14 and 30 on hepatogenic differentiation, and day 30 on endothelial differentiation using the Affymetrix HU-133A GeneChip. Data files were analyzed for differential gene expression and for gene ontologic analyses. RESULTS: By combining data sets from the myogenic, osteogenic, endothelia and hepatogenic lineages, we identified a genetic signature of 1017 genes. By subtracting the ’universally’ up-regulated genes from those identified as being up-regulated from each lineage, we were able to identify a ’lineage specific’ genetic signature of differentiation. By clustering ’lineage specific’ targets, we were able to identify tissue specific processes such as sterol, lipid, and cholesterol metabolism for hepatogenic differentiation, extracellular matrix production genes (which have a structural function) for osteogenic and endothelia, and muscle processes for the myogenic lineage. CONCLUSIONS: These studies highlight that genomics may give us insight into the processes that are activated during stem cell differentiation. These results offer new avenues for applying stem cells to tissue engineering.
In vitro expansion of embryonic stem cells for progenitor isolation as a source of cells for tissue regeneration Paul J Kokorowski, MD, Laura Perin, PhD, Federico Tozzi, MD, Casey Brewer, BS, Sargis Sedrakyan, BS, Stefano Da Sacco, BS, Roger DeFilippo, MD Children’s Hospital Los Angeles, Keck School of Medicine, USC, Los Angeles, CA INTRODUCTION: Our laboratory focuses on alternative cell sources to embryonic stem cells (ESC) for kidney regeneration. One of the major concerns with the use of embryonic stem cells is their potential to form teratomas in vivo. A heterogeneous population of partially differentiated cells typically surround the periphery of ESC colonies. These cells are discarded to preserve undifferentiated cells centrally. By allowing these peripheral cells to undergo early steps of differentiation we believe a more clinically useful population can be obtained that avoid tumor formation in vivo.
Surgical Forum Abstracts
S65
METHODS: Mouse ESC were cultured without a feeder layer. Floating cells in early passages were separately cultured as well. Cells were cultured for more than 20 passages and exposed to differentiation media in vitro. Differentiation was confirmed with histology, immunocytochemistry and PCR. Cells were also injected into NuNu mice to asses for teratoma formation. RESULTS: Cells assumed a flattened fibroblastic morphology with sheet like growth without colonies or embryoid bodies. Embryonic markers brachury, nanog, Sox2 and Oct-4 persisted during early passages and were lost with passages above 20. Cells from late passages differentiated into adipocytes, osteocytes, myocytes, and endothelial cells with associated markers. Injected cells from early passages formed palpable tumors in all mice by three weeks in vivo. Cells derived from late passages (⬎20) demonstrated no tumor formation thus far. CONCLUSIONS: A more clinically useful multipotent cell population may exist within ESC cultures. These may prove useful for future regenerative applications by avoiding teratoma formation.
Transplantation of neural crest progenitor cells for Hirschsprung’s disease Hong Qin, MD, James Dunn, MD, PhD UCLA School of Medicine, Los Angeles, CA INTRODUCTION: Neural crest progenitor cells can potentially generate the enteric nervous system in Hirschsprung’s disease. We hypothesize that isolated neural crest progenitor cells can survive and differentiate into mature neural cells when they are transplanted into the rectum. METHODS: Neural crest progenitor cells were isolated from fetal mice. These cells expressed p75, a marker for neural progenitor cells. Cells were labeled with DiI, a red-fluorescent membrane marker, for the purpose of tracking these cells. One million cells were injected transanally into the rectum of 10 mice. The animals were sacrificed right after injection and at 14 days. The retrieved specimens were immunostained for peripherin, a mature neuronal marker. RESULTS: The transplanted cells were delivered into the submucosal or the subserosal space by transanal injections. Red-fluorescent cells and non-fluorescent cells were observed in the injected cell clusters at 14 days. Red-fluorescent cells expressed peripherin but not p75, indicating the differentiation of injected cells. Non-fluorescent cells in the cluster expressed p75 but not peripherin, indicating the proliferation of immature neural progenitor cells. CONCLUSIONS: Neural progenitor cells can proliferate and differentiate when transplanted into rectum. Whether these transplanted cells can form a functional enteric nervous system remains to be determined.
Novel etiology of infantile hemangioma Chia J Chung, MD, Susan Fisher, PhD, Matthew Gormley, BS, William Hoffman, MD, David M Young, MD, FACS, Scott Hansen, MD University of California-San Francisco, San Francisco, CA INTRODUCTION: Infantile hemangiomas, the most prevalent benign tumor of infancy, have a predictable life cycle beginning with
Transcriptional profiling of human amniotic fluid derived stem cells and their multilineage differentiation Jennifer Hipp, Bryan Tillman, MD, PhD, Jason Hipp, PhD, Steve Hodges, MD, Anthony Atala, MD, Shay Soker, PhD Wake Forest University School of Medicine, Winston-Salem, NC INTRODUCTION: We have recently isolated a subpopulation of pluripotent stem cells from human amniotic fluid (Amniotic Fluid Stem Cells, AFSC). These cells have a high self renewal capacity, with a short doubling time, do not require a feeder layer, and can be grown in basic medium supplemented. AFSC can be differentiated in vitro into osteogenic, adipogenic, myogenic, neurogenic, vasculogenic, and hepatic phenotypes. The goal of this study is to understand the genetic processes involved in stem cell differentiation that would identify targets to increase the efficiency of differentiation. METHODS: Microarrays were performed at day 20 and 30 on myogenic and osteogenic differentiation, day 14 and 30 on hepatogenic differentiation, and day 30 on endothelial differentiation using the Affymetrix HU-133A GeneChip. Data files were analyzed for differential gene expression and for gene ontologic analyses. RESULTS: By combining data sets from the myogenic, osteogenic, endothelia and hepatogenic lineages, we identified a genetic signature of 1017 genes. By subtracting the ’universally’ up-regulated genes from those identified as being up-regulated from each lineage, we were able to identify a ’lineage specific’ genetic signature of differentiation. By clustering ’lineage specific’ targets, we were able to identify tissue specific processes such as sterol, lipid, and cholesterol metabolism for hepatogenic differentiation, extracellular matrix production genes (which have a structural function) for osteogenic and endothelia, and muscle processes for the myogenic lineage. CONCLUSIONS: These studies highlight that genomics may give us insight into the processes that are activated during stem cell differentiation. These results offer new avenues for applying stem cells to tissue engineering.
In vitro expansion of embryonic stem cells for progenitor isolation as a source of cells for tissue regeneration Paul J Kokorowski, MD, Laura Perin, PhD, Federico Tozzi, MD, Casey Brewer, BS, Sargis Sedrakyan, BS, Stefano Da Sacco, BS, Roger DeFilippo, MD Children’s Hospital Los Angeles, Keck School of Medicine, USC, Los Angeles, CA INTRODUCTION: Our laboratory focuses on alternative cell sources to embryonic stem cells (ESC) for kidney regeneration. One of the major concerns with the use of embryonic stem cells is their potential to form teratomas in vivo. A heterogeneous population of partially differentiated cells typically surround the periphery of ESC colonies. These cells are discarded to preserve undifferentiated cells centrally. By allowing these peripheral cells to undergo early steps of differentiation we believe a more clinically useful population can be obtained that avoid tumor formation in vivo.
Surgical Forum Abstracts
S65
METHODS: Mouse ESC were cultured without a feeder layer. Floating cells in early passages were separately cultured as well. Cells were cultured for more than 20 passages and exposed to differentiation media in vitro. Differentiation was confirmed with histology, immunocytochemistry and PCR. Cells were also injected into NuNu mice to asses for teratoma formation. RESULTS: Cells assumed a flattened fibroblastic morphology with sheet like growth without colonies or embryoid bodies. Embryonic markers brachury, nanog, Sox2 and Oct-4 persisted during early passages and were lost with passages above 20. Cells from late passages differentiated into adipocytes, osteocytes, myocytes, and endothelial cells with associated markers. Injected cells from early passages formed palpable tumors in all mice by three weeks in vivo. Cells derived from late passages (⬎20) demonstrated no tumor formation thus far. CONCLUSIONS: A more clinically useful multipotent cell population may exist within ESC cultures. These may prove useful for future regenerative applications by avoiding teratoma formation.
Transplantation of neural crest progenitor cells for Hirschsprung’s disease Hong Qin, MD, James Dunn, MD, PhD UCLA School of Medicine, Los Angeles, CA INTRODUCTION: Neural crest progenitor cells can potentially generate the enteric nervous system in Hirschsprung’s disease. We hypothesize that isolated neural crest progenitor cells can survive and differentiate into mature neural cells when they are transplanted into the rectum. METHODS: Neural crest progenitor cells were isolated from fetal mice. These cells expressed p75, a marker for neural progenitor cells. Cells were labeled with DiI, a red-fluorescent membrane marker, for the purpose of tracking these cells. One million cells were injected transanally into the rectum of 10 mice. The animals were sacrificed right after injection and at 14 days. The retrieved specimens were immunostained for peripherin, a mature neuronal marker. RESULTS: The transplanted cells were delivered into the submucosal or the subserosal space by transanal injections. Red-fluorescent cells and non-fluorescent cells were observed in the injected cell clusters at 14 days. Red-fluorescent cells expressed peripherin but not p75, indicating the differentiation of injected cells. Non-fluorescent cells in the cluster expressed p75 but not peripherin, indicating the proliferation of immature neural progenitor cells. CONCLUSIONS: Neural progenitor cells can proliferate and differentiate when transplanted into rectum. Whether these transplanted cells can form a functional enteric nervous system remains to be determined.
Novel etiology of infantile hemangioma Chia J Chung, MD, Susan Fisher, PhD, Matthew Gormley, BS, William Hoffman, MD, David M Young, MD, FACS, Scott Hansen, MD University of California-San Francisco, San Francisco, CA INTRODUCTION: Infantile hemangiomas, the most prevalent benign tumor of infancy, have a predictable life cycle beginning with