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Defining functionally distinct subpopulations of human adipose-derived stromal cells through single cell transcriptional analysis
S92
Surgical Forum Abstracts
CONCLUSIONS: Dermal collagen hydrogels viably sustain MSCs in vitro and augment early wound healing. Hydrogel delivery of MSCs improves cell survival following implantation compared to local injection. MSC-seeded hydrogels accelerate normal wound healing and are a promising cell-scaffold construct for skin regeneration.
Trogocytosis links the chimerism level to the selection of tolerant host NK cells following in utero hematopoietic cellular transplantation (IUHCT) Amir M Alhajjat, MD, Aimen F Shaaban MD University of Iowa Hospitals and Clinics, Iowa City, IA INTRODUCTION: Host NK cells are potent effectors of rejection in low-level chimeric mice (rejecter mice) following IUHCT and are rendered hyporesponsive in high-level chimeric mice (engrafter mice). Transfer of donor MHC (trogocytosis) during development via donor-specific inhibitory receptors is a mechanism in which the chimerism level might indirectly “select” tolerant NK cell phenotypes and “delete” hostile ones. To explore this mechanism of NK cell selection, we queried differences in the development of host NK cells with tolerant or hostile phenotypes in chimeric mice and directly measured trogocytosis in each group. METHODS: Balb/c (H2d) fetal liver hematopoietic cells at E14 were transplanted into age-matched B6 (H2b) recipients. Trogocytosis and co-expression of donor-specific activating (Ly49D) and inhibitory (Ly49AFG) receptors were serially measured on host NK cells from engrafter and rejecter mice. Lastly, Ly49D⫹ host NK cells were depleted from a separate group of rejecter mice to determine the impact on graft rejection. Results were compared using a two-tailed Student t test. RESULTS: Trogocytosis was significantly higher in engrafters when compared to rejecters and was confined to Ly49D⫹Ly49AFG⫹ NK cell subset amongst Ly49D⫹ cells. Furthermore, the frequency of the double-positive Ly49D⫹Ly49AFG⫹ cell subset was dramatically higher in engrafter mice when compared to controls (79.6% vs. 44.2% P⬍0.0005). This shift was not found on the irrelevant Ly49D-Ly49AFG⫹ cell subsets. Lastly, elimination of Ly49D⫹ host NK cells in rejecter mice universally abrogated graft rejection and preserved engraftment. CONCLUSIONS: Trogocytosis offers a plausible link between the chimerism level and the development of tolerant NK cell phenotypes following IUHCT.
Elimination of the maternally induced adaptive immune response allows uniform achievement of high level allogeneic chimerism by in utero hematopoietic cell transplantation and postnatal minimal conditioning BMT Matthew T Santore MD, Demetri J Merianos MD, Carlyn A Todorow BA, Alan W Flake MD, FACS Children’s Hospital of Philadelphia/University of Pennsylvania, Philadelphia, PA, INTRODUCTION: We have previously demonstrated that donor hematopoietic chimerism after in utero hematopoietic cell transplantation
J Am Coll Surg
(IUHCT) is limited by an adaptive alloimmune response induced by maternal antibodies transferred in breast milk. If this transfer is eliminated by foster nursing recipients with naı¨ve mothers, 100% of recipients remain chimeric. We hypothesized that fostered mice would be tolerant to the donor and that high levels of donor chimerism could be uniformly achieved using a strategy of postnatal enhancement of chimerism by a minimal conditioning same donor BMT. METHODS: E14 Balb/c fetuses received 20 million B6-GFP BM cells/fetus by intravascular injection. The pups were fostered at P1. At P28 a minimally ablative dose of busulfan (15mg/kg) was administered followed by transplantation of T-cell depleted B6-GFP BM cells. Chimerism was assessed by flow cytometry as the percentage of CD45⫹ cells that were GFP⫹. Skin grafts were placed from donor and third party mice to assess donor specific tolerance. RESULTS: At one, three and five months after the postnatal transplant an increasing level of chimerism was observed relative to fostered controls that did not receive the postnatal BMT (2M-66.7% vs. 31.7%, 3M-78.2% vs. 26.4%, 5M-90.1% vs. 24.4%, p⬍0.001). Enhancement of chimerism was associated with donor specific tolerance as demonstrated by acceptance of the B6-GFP skin graft and rejection of third party grafts. CONCLUSIONS: This study demonstrates for the first time that high level allogeneic chimerism can be achieved in 100% of recipients by the potential clinical strategy of IUHCT followed by a minimal conditioning same donor postnatal BMT.
Defining functionally distinct subpopulations of human adipose-derived stromal cells through single cell transcriptional analysis Jason P Glotzbach MD, Benjamin Levi MD, Victor W Wong MD, Michael Januszyk MD, I Nick Vial BA, Aaron W James MD, Michael Sorkin MD, Michael T Longaker MD, MBA, FACS, Geoffrey C Gurtner MD, FACS Stanford University, Stanford, CA INTRODUCTION: Human adipose-derived stromal cells (hASCs) are heterogeneous multipotent cells that can be readily isolated from lipoaspirate specimens. It is unknown whether the heterogeneity of hASCs is functionally relevant. To address this question, we employed a single cell transcriptional analysis to characterize hASC gene expression and define functionally distinct subpopulations. METHODS: Human ASCs were isolated from clinical lipoaspirate specimens from 4 female patients and single cell-sorted by FACS. After lysis and reverse transcription, a microfluidic chip-based system was used to perform real-time quantitative PCR on individual cell isolates across 48 genes. A clustering algorithm based on the mathematical principles of fuzzy set theory was used to group cells based on transcriptional patterns. In vitro osteogenesis, adipogenesis, and vasculogenesis were analyzed using Alizarin red staining, Oil Red O staining, and Matrigel tubulization assays. RESULTS: We analyzed the transcriptional profile of 500 individual hASCs and identified three unique subpopulations of hASCs with distinct patterns of expression across osteogenic, adipogenic, and vasculo-
Vol. 211, No. 3S, September 2010
genic genes. By associating these transcriptional profiles with surface antigen expression and applying these patterns to subsequent sorts, we prospectively isolated multiple subpopulations of hASCs that demonstrated differential in vitro osteo-, adipo- and vasculogenic capacity. CONCLUSIONS: While hASCs hold great promise for tissue engineering and cell-based therapies, the heterogeneous nature of these cells must be characterized before they can be used clinically. By using single cell transcriptional analysis to characterize hASC gene expression patterns, we demonstrate that selection of hASC subpopulations targeted for specific needs, such as for bone, adipose, or cardiovascular tissue regeneration, is feasible.
Human mesenchymal stem cells express adrenergic receptors David Yonick MD, Kuzhali Muthu PhD, Richard L Gamelli MD, FACS Loyola University Medical Center, Maywood, IL INTRODUCTION: Mesenchymal stem cells (MSC) are multipotent cells that provide a reservoir of progenitor cells necessary for normal tissue repair. Adrenergic receptors are expressed on a variety of tissues and play an important role in wound healing. We sought to characterize the expression of adrenergic receptors on human MSCs and the potential of adrenergic receptor engagement to modulate osteogenic differentiation of MSCs. METHODS: Human bone marrow derived MSCs were indentified by the cell surface markers SSEA-4, CD73, CD90 and CD105 by fluorescence activated cell sorting (FACS). SSEA-4⫹ MSCs were labeled with specific antibodies against alpha-1, alpha-2 and beta-2 adrenergic receptors and analyzed by FACS and confocal microscopy. MSCs were differentiated into osteoblasts in a standard differentiation medium with and without the beta agonist, Isoproterenol (1 micro-molar). RESULTS: Cultured human MSCs demonstrated 87% expression of SSEA-4. The SSEA-4⫹ cells had 99% expression of CD73, CD90 and CD105. The MSCs had similar levels of expression of alpha-1, alpha-2 and beta-2 receptors as compared to control monocytes. Confocal microscopy further demonstrated alpha-1, alpha-2 and beta-2 adrenergic receptor expression on MSCs. The MSCs exposed to Isoproterenol showed increased mineralization of the in-vitro matrix compared to untreated differentiation controls. CONCLUSIONS: Human MSC have adrenergic receptors present on their cell surface. The beta-2 adrenergic receptors appear to cause epigenetic changes in the differentiating MSC that result in increased in-vitro mineralization.
Surgical Forum Abstracts
S93
factor-1 (SCF-1) is involved in cellular recruitment. We have shown that SCF-1 overexpression can induce mesenchymal stem cell migration in vitro, however its role in epidermal stem cell migration is unknown. We hypothesized that SCF-1 overexpression would increase recruitment of epidermal stem cells in vivo. METHODS: The skin from 8-week-old animals injected in utero with lenti-RFP demonstrated a mosaic of RFP⫹ cells, consistent with stem cell transfection. Following wounding, these RFP⫹ cells migrated towards the wound. Overexpression of SCF-1 resulted in even greater recruitment of RFP⫹ stem cells compared to lenti-GFP controls (42.8 cells ⫾ 17.2 versus 24.7 ⫾ 11.2, p ⫽ 0.032). RESULTS: At 8 weeks of age the skin from animals injected in utero with lenti-RFP demonstrated a mosaic of RFP⫹ cells, consistent with stem cell transfection. Following wounding, these RFP⫹ cells migrated towards the wound. Overexpression of SCF-1 resulted in even greater recruitment of RFP⫹ stem cells compared to lenti-GFP controls (42.8 cells ⫾ 17.2 versus 24.7 ⫾ 11.2, p ⫽ 0.032). CONCLUSIONS: This is the first report of SCF-1 mediating increased migration of epidermal stem cells in vivo. Overexpression of SCF-1 may have a potential therapeutic role in the treatment of chronic wounds. We also report the development of a novel model to examine recruitment of epidermal stem cells in vivo. Further studies are needed to better define the role SCF-1 plays in normal and impaired wound healing.
Burn injury shifts the commitment patterns of hematopoietic stem and progenitor cells Joseph A Posluszny Jr MD, Joseph A Posluszny Jr MD, Kuzhali Muthumalaiappan PhD, Li-Ke He MD, Andrea L Szilagyi BS, Richard L Gamelli MD, FACS, Ravi Shankar PhD Loyola University Medical Center, Maywood, IL INTRODUCTION: Burn injury (BI) results in a significant shift in hematopoietic commitment, leading to anemia. Although all blood cells originate from hematopoietic stem cells (HSC), burn-induced temporal changes in HSC proliferation and commitment patterns are not clear. We determined the burn-induced temporal changes in HSC and progenitor cell (PC) populations.
Dustin M Bermudez MD, Benjamin J Herdrich MD, David Stitelman MD, Antoneta Radu MS, Haiying Li MS, Philip W Zoltick MD, Marc E Mitchell MD University of Pennsylvania School of Medicine, Philadelphia, PA
METHODS: B6D2F1 mice (n⫽6) were administered a 15% TBSA scald burn. Sham mice were not burned. On post burn days (PBD) 2, 5, 7 and 14, bone marrow cells (5x106cells) were separated into HSCs including long-term (LT) HSCs, short-term (ST) HSCs, multi-potential progenitors (MPPs) and PCs including common lymphoid (CLP), common myeloid (CMP), granulocytemacrophage (GMP) and megakaryocyte-erythroid progenitors (MEP) by FACS, and absolute cell numbers were determined. Cell proliferation in HSC-PC compartments was determined by bromodeoxyuridine (BrdU) incorporation.
INTRODUCTION: Epidermal stem cells play a central role in reepithelialization in the normal wound-healing process. Stem cell
RESULTS: BI increases the absolute number of ST-HSCs and MPPs by 215% by PBD2 and 236% by PBD5 compared to sham
Increased epithelial stem cell migration in vivo in response to stem cell factor-1
Surgical Forum Abstracts
CONCLUSIONS: Dermal collagen hydrogels viably sustain MSCs in vitro and augment early wound healing. Hydrogel delivery of MSCs improves cell survival following implantation compared to local injection. MSC-seeded hydrogels accelerate normal wound healing and are a promising cell-scaffold construct for skin regeneration.
Trogocytosis links the chimerism level to the selection of tolerant host NK cells following in utero hematopoietic cellular transplantation (IUHCT) Amir M Alhajjat, MD, Aimen F Shaaban MD University of Iowa Hospitals and Clinics, Iowa City, IA INTRODUCTION: Host NK cells are potent effectors of rejection in low-level chimeric mice (rejecter mice) following IUHCT and are rendered hyporesponsive in high-level chimeric mice (engrafter mice). Transfer of donor MHC (trogocytosis) during development via donor-specific inhibitory receptors is a mechanism in which the chimerism level might indirectly “select” tolerant NK cell phenotypes and “delete” hostile ones. To explore this mechanism of NK cell selection, we queried differences in the development of host NK cells with tolerant or hostile phenotypes in chimeric mice and directly measured trogocytosis in each group. METHODS: Balb/c (H2d) fetal liver hematopoietic cells at E14 were transplanted into age-matched B6 (H2b) recipients. Trogocytosis and co-expression of donor-specific activating (Ly49D) and inhibitory (Ly49AFG) receptors were serially measured on host NK cells from engrafter and rejecter mice. Lastly, Ly49D⫹ host NK cells were depleted from a separate group of rejecter mice to determine the impact on graft rejection. Results were compared using a two-tailed Student t test. RESULTS: Trogocytosis was significantly higher in engrafters when compared to rejecters and was confined to Ly49D⫹Ly49AFG⫹ NK cell subset amongst Ly49D⫹ cells. Furthermore, the frequency of the double-positive Ly49D⫹Ly49AFG⫹ cell subset was dramatically higher in engrafter mice when compared to controls (79.6% vs. 44.2% P⬍0.0005). This shift was not found on the irrelevant Ly49D-Ly49AFG⫹ cell subsets. Lastly, elimination of Ly49D⫹ host NK cells in rejecter mice universally abrogated graft rejection and preserved engraftment. CONCLUSIONS: Trogocytosis offers a plausible link between the chimerism level and the development of tolerant NK cell phenotypes following IUHCT.
Elimination of the maternally induced adaptive immune response allows uniform achievement of high level allogeneic chimerism by in utero hematopoietic cell transplantation and postnatal minimal conditioning BMT Matthew T Santore MD, Demetri J Merianos MD, Carlyn A Todorow BA, Alan W Flake MD, FACS Children’s Hospital of Philadelphia/University of Pennsylvania, Philadelphia, PA, INTRODUCTION: We have previously demonstrated that donor hematopoietic chimerism after in utero hematopoietic cell transplantation
J Am Coll Surg
(IUHCT) is limited by an adaptive alloimmune response induced by maternal antibodies transferred in breast milk. If this transfer is eliminated by foster nursing recipients with naı¨ve mothers, 100% of recipients remain chimeric. We hypothesized that fostered mice would be tolerant to the donor and that high levels of donor chimerism could be uniformly achieved using a strategy of postnatal enhancement of chimerism by a minimal conditioning same donor BMT. METHODS: E14 Balb/c fetuses received 20 million B6-GFP BM cells/fetus by intravascular injection. The pups were fostered at P1. At P28 a minimally ablative dose of busulfan (15mg/kg) was administered followed by transplantation of T-cell depleted B6-GFP BM cells. Chimerism was assessed by flow cytometry as the percentage of CD45⫹ cells that were GFP⫹. Skin grafts were placed from donor and third party mice to assess donor specific tolerance. RESULTS: At one, three and five months after the postnatal transplant an increasing level of chimerism was observed relative to fostered controls that did not receive the postnatal BMT (2M-66.7% vs. 31.7%, 3M-78.2% vs. 26.4%, 5M-90.1% vs. 24.4%, p⬍0.001). Enhancement of chimerism was associated with donor specific tolerance as demonstrated by acceptance of the B6-GFP skin graft and rejection of third party grafts. CONCLUSIONS: This study demonstrates for the first time that high level allogeneic chimerism can be achieved in 100% of recipients by the potential clinical strategy of IUHCT followed by a minimal conditioning same donor postnatal BMT.
Defining functionally distinct subpopulations of human adipose-derived stromal cells through single cell transcriptional analysis Jason P Glotzbach MD, Benjamin Levi MD, Victor W Wong MD, Michael Januszyk MD, I Nick Vial BA, Aaron W James MD, Michael Sorkin MD, Michael T Longaker MD, MBA, FACS, Geoffrey C Gurtner MD, FACS Stanford University, Stanford, CA INTRODUCTION: Human adipose-derived stromal cells (hASCs) are heterogeneous multipotent cells that can be readily isolated from lipoaspirate specimens. It is unknown whether the heterogeneity of hASCs is functionally relevant. To address this question, we employed a single cell transcriptional analysis to characterize hASC gene expression and define functionally distinct subpopulations. METHODS: Human ASCs were isolated from clinical lipoaspirate specimens from 4 female patients and single cell-sorted by FACS. After lysis and reverse transcription, a microfluidic chip-based system was used to perform real-time quantitative PCR on individual cell isolates across 48 genes. A clustering algorithm based on the mathematical principles of fuzzy set theory was used to group cells based on transcriptional patterns. In vitro osteogenesis, adipogenesis, and vasculogenesis were analyzed using Alizarin red staining, Oil Red O staining, and Matrigel tubulization assays. RESULTS: We analyzed the transcriptional profile of 500 individual hASCs and identified three unique subpopulations of hASCs with distinct patterns of expression across osteogenic, adipogenic, and vasculo-
Vol. 211, No. 3S, September 2010
genic genes. By associating these transcriptional profiles with surface antigen expression and applying these patterns to subsequent sorts, we prospectively isolated multiple subpopulations of hASCs that demonstrated differential in vitro osteo-, adipo- and vasculogenic capacity. CONCLUSIONS: While hASCs hold great promise for tissue engineering and cell-based therapies, the heterogeneous nature of these cells must be characterized before they can be used clinically. By using single cell transcriptional analysis to characterize hASC gene expression patterns, we demonstrate that selection of hASC subpopulations targeted for specific needs, such as for bone, adipose, or cardiovascular tissue regeneration, is feasible.
Human mesenchymal stem cells express adrenergic receptors David Yonick MD, Kuzhali Muthu PhD, Richard L Gamelli MD, FACS Loyola University Medical Center, Maywood, IL INTRODUCTION: Mesenchymal stem cells (MSC) are multipotent cells that provide a reservoir of progenitor cells necessary for normal tissue repair. Adrenergic receptors are expressed on a variety of tissues and play an important role in wound healing. We sought to characterize the expression of adrenergic receptors on human MSCs and the potential of adrenergic receptor engagement to modulate osteogenic differentiation of MSCs. METHODS: Human bone marrow derived MSCs were indentified by the cell surface markers SSEA-4, CD73, CD90 and CD105 by fluorescence activated cell sorting (FACS). SSEA-4⫹ MSCs were labeled with specific antibodies against alpha-1, alpha-2 and beta-2 adrenergic receptors and analyzed by FACS and confocal microscopy. MSCs were differentiated into osteoblasts in a standard differentiation medium with and without the beta agonist, Isoproterenol (1 micro-molar). RESULTS: Cultured human MSCs demonstrated 87% expression of SSEA-4. The SSEA-4⫹ cells had 99% expression of CD73, CD90 and CD105. The MSCs had similar levels of expression of alpha-1, alpha-2 and beta-2 receptors as compared to control monocytes. Confocal microscopy further demonstrated alpha-1, alpha-2 and beta-2 adrenergic receptor expression on MSCs. The MSCs exposed to Isoproterenol showed increased mineralization of the in-vitro matrix compared to untreated differentiation controls. CONCLUSIONS: Human MSC have adrenergic receptors present on their cell surface. The beta-2 adrenergic receptors appear to cause epigenetic changes in the differentiating MSC that result in increased in-vitro mineralization.
Surgical Forum Abstracts
S93
factor-1 (SCF-1) is involved in cellular recruitment. We have shown that SCF-1 overexpression can induce mesenchymal stem cell migration in vitro, however its role in epidermal stem cell migration is unknown. We hypothesized that SCF-1 overexpression would increase recruitment of epidermal stem cells in vivo. METHODS: The skin from 8-week-old animals injected in utero with lenti-RFP demonstrated a mosaic of RFP⫹ cells, consistent with stem cell transfection. Following wounding, these RFP⫹ cells migrated towards the wound. Overexpression of SCF-1 resulted in even greater recruitment of RFP⫹ stem cells compared to lenti-GFP controls (42.8 cells ⫾ 17.2 versus 24.7 ⫾ 11.2, p ⫽ 0.032). RESULTS: At 8 weeks of age the skin from animals injected in utero with lenti-RFP demonstrated a mosaic of RFP⫹ cells, consistent with stem cell transfection. Following wounding, these RFP⫹ cells migrated towards the wound. Overexpression of SCF-1 resulted in even greater recruitment of RFP⫹ stem cells compared to lenti-GFP controls (42.8 cells ⫾ 17.2 versus 24.7 ⫾ 11.2, p ⫽ 0.032). CONCLUSIONS: This is the first report of SCF-1 mediating increased migration of epidermal stem cells in vivo. Overexpression of SCF-1 may have a potential therapeutic role in the treatment of chronic wounds. We also report the development of a novel model to examine recruitment of epidermal stem cells in vivo. Further studies are needed to better define the role SCF-1 plays in normal and impaired wound healing.
Burn injury shifts the commitment patterns of hematopoietic stem and progenitor cells Joseph A Posluszny Jr MD, Joseph A Posluszny Jr MD, Kuzhali Muthumalaiappan PhD, Li-Ke He MD, Andrea L Szilagyi BS, Richard L Gamelli MD, FACS, Ravi Shankar PhD Loyola University Medical Center, Maywood, IL INTRODUCTION: Burn injury (BI) results in a significant shift in hematopoietic commitment, leading to anemia. Although all blood cells originate from hematopoietic stem cells (HSC), burn-induced temporal changes in HSC proliferation and commitment patterns are not clear. We determined the burn-induced temporal changes in HSC and progenitor cell (PC) populations.
Dustin M Bermudez MD, Benjamin J Herdrich MD, David Stitelman MD, Antoneta Radu MS, Haiying Li MS, Philip W Zoltick MD, Marc E Mitchell MD University of Pennsylvania School of Medicine, Philadelphia, PA
METHODS: B6D2F1 mice (n⫽6) were administered a 15% TBSA scald burn. Sham mice were not burned. On post burn days (PBD) 2, 5, 7 and 14, bone marrow cells (5x106cells) were separated into HSCs including long-term (LT) HSCs, short-term (ST) HSCs, multi-potential progenitors (MPPs) and PCs including common lymphoid (CLP), common myeloid (CMP), granulocytemacrophage (GMP) and megakaryocyte-erythroid progenitors (MEP) by FACS, and absolute cell numbers were determined. Cell proliferation in HSC-PC compartments was determined by bromodeoxyuridine (BrdU) incorporation.
INTRODUCTION: Epidermal stem cells play a central role in reepithelialization in the normal wound-healing process. Stem cell
RESULTS: BI increases the absolute number of ST-HSCs and MPPs by 215% by PBD2 and 236% by PBD5 compared to sham
Increased epithelial stem cell migration in vivo in response to stem cell factor-1