- Email: [email protected]
Chondrogenesis of human adipo-derived mesodermal stem cells
Vol. 191, No. 4S, October 2000
Results: Peak Tensile Wound Strength (Newtons) Treatment Group (n ⴝ 6) Control Normal ⫹ EGF Normal ⫹ EGF & eIF 4E
S47
Surgical Forum Abstracts
7 days
14 days
4.5 ⫾ 1.3 6.5 ⫾ 2.9 8.4 ⫾ 3.6
9.8 ⫾ 3.9 11.1 ⫾ 3.4 18.8 ⫾ 3.5*
* p ⬍ 0.01 compared to control or EGF by ANOVA
Conclusions: Simultaneous biolistic delivery of EGF mRNA with eIF4E mRNA significantly increases wound breaking strength compared to control animals or treatment with EGF mRNA alone without risk of cellular transformation. Further studies of translational activation to augment wound healing are warranted.
Composite tissue allograft (CTA): tolerance induction without graft vs host disease (GvHD) Vijay Gorantla MD, Gustavo Perez-Abadia MD, Kaustubha Prabhune MD, Haldun Orhun MD, Thanos Kakoulidis MD, Ramsey Majzoub MD, Claudio Maldonado PhD, Gary Anderson PhD, Lynn Ogden MD, Warren Breidenbach MD, Suzanne Ildstad MD, John Barker MD, PhD. Division of Plastic and Reconstructive Surgery, Institute for Cellular Therapeutics, Department of Surgery, University of Louisville & Christine M. Kleinert Institute. University of Louisville, 511 South Floyd St, 320 MDR Bldg, Louisville, KY, 40292, USA. Tel (502) 852-0166. Introduction: Successful human hand transplants have recently been performed using immunosuppression. However, the risks of the nonspecific immunosuppressants required for graft survival are subject to controversy. Mixed allogeneic chimerism (MAC) induces donorspecific tolerance to solid organ allografts. CTAs add another variable in that the mature donor immune system (bone marrow (BM)) accompanies the graft, potentially resulting in GvHD. The aim of this study was to achieve a GvHD free tolerant state using MAC in a rat hind-limb model. Methods: Group I: naïve WF rats received hind limbs from unmanipulated ACI rats (n ⫽ 6). Group II: host WF rats were irradiated with 950cGy and reconstituted with MHC disparate ACI BM cells (␣-␥␦ T-cell depleted) (n ⫽ 8). Flow cytometry after 28 days confirmed chimerism. These rats then received non-irradiated ACI limbs. Group III: (ACI ⫹ WF) rats, received similar treatment as in Group II but were transplanted with ACI Limbs pretreated with 1050cGy TBI (n ⫽ 8). Results: Group I rejected their limbs in 5.7 ⫾ 1.5 days. Group II: 7/8 (ACI ⫹ WF) rats (⬎85% chimerism) exhibited rejection-free survival of non-irradiated CTAs for 22.4 ⫾ 2.8 days, but developed severe GvHD only after the CTA and died. Group III: 7/8 (ACI ⫹ WF) rats showed no sign of rejection or GvHD at 77 ⫾ 1.8 days. One rat died of infection at 34 days post-op with no sign of GvHD. Notably, this is significantly prolonged beyond the time of onset of GvHD in Group II. Conclusions: Partial conditioning of the host alone led to high levels of MAC without GvHD before CTA. The bone marrow in unmanipulated limbs resulted in GvHD. After transplantation of irradiated limbs, MAC prevented acute rejection and resulted in a GvHD free state. Taken together, these data show that immunocompetent donor cells transferred with the CTA can mediate GvHD and that inactivation of these cells by irradiation could prevent GvHD.
Tolerance to limb allografts in a miniature swine model
12 days of cyclosporine. The aims of this study were 1) to determine if tolerance to allografts could be achieved with the addition of a vascularized skin component, and 2) to evaluate the role of the donor bone marrow following long-term tolerance. Methods: Limb grafts from 4 swine with a 100 cm2 cutaneous paddle were heterotopically transplanted into MHC matched, minor antigen mismatched miniature swine and treated with 12 days of cyclosporine. Biweekly biopsies were performed and evaluated histologically. Pig allelic antigen (PAA), a non-MHC-linked marker, was used to study bone marrow migration in PAA⫹ recipients with PAA⫺ allografts. Swine demonstrating long-term tolerance to their allografts were sacrificed, and the graft marrow was analyzed for the migration of recipient cells into graft marrow space using the PAA marker. Results: All animals accepted the musculoskeletal portion of their grafts by gross inspection and histologic analysis. One animal demonstrated tolerance to the skin (⬎120 days). Three animals sloughed the graft epidermis (days 39, 41, and 50) leaving an intact dermis. The host skin began to re-epithelialize the graft dermis. At sacrifice, the allograft bone marrow harvest yielded viable marrow. All hematopoietic cells identified in the graft marrow were PAA positive and therefore of recipient origin. Conclusions: Tolerance to limb allografts with a skin component can be achieved with a 12-day course of cyclosporine and MHC matching. Immune modulation of the skin component may be necessary for clinical success. Furthermore, recipient hematopoietic cells repopulate the graft marrow space, and tolerance to limb allografts does not appear to depend on the presence of donor bone marrow cells.
Chondrogenesis of human adipo-derived mesodermal stem cells Jerry I Huang, BS, Marc H Hedrick, MD, H. Peter Lorenz, MD, Min Zhu, MD, Prosper Benhaim, MD. Division of Plastic and Reconstructive Surgery; University of California, Los Angeles 200 UCLA Medical Plaza, Suite #140; Los Angeles, CA 90095-6902, USA; Phone (310) 206-4468 Introduction: The repair of cartilaginous defects remains a significant clinical problem. Human bone marrow contains pluripotent mesenchymal stem cells capable of differentiation down multiple mesodermal lineages, including bone and cartilage. However, bone marrow harvest is painful and yields few cells. The purpose of this study is to determine if human adipose tissue contains a similar population of stem cells with osteochondrogenic potential. Methods: Human liposuction aspirate from five patients were washed and digested, yielding fibroblast-like cells. Cells were psssaged three times in basal media, then induced toward chondrogenesis by placement in high-density micromass cultures supplemented with TGF-1, insulin, transferrin, and ascorbic acid. Chondrogenesis was determined by histologic evaluation at 2, 7, and 14 days with H&E, Alcian blue, and Goldner’s trichrome staining. In addition, immunohistochemistry was performed using antibodies against type II collagen, chondroitin-4-sulfate, and keratan sulfate. Intensity of histologic and immunohistochemistry staining was subjectively graded as: none (⫺); background (⫹/⫺); light (⫹); moderate (⫹⫹); heavy (⫹⫹⫹). Results: Cartilaginous nodules with a perichondral cellular border formed within 48 hours in all treated cultures. Untreated control cells showed no evidence of chondrogenic differentiation. Time Stages of chondrogenic Alcian Trichrome Chondroitin- Keratan Type II point differentiation blue (collagen) 4-sulfate sulfate collagen
David W Mathes, MD, Judy L Bourget, MD, Mark A Randolph, MA, Mario G Solari, BA, Anette, Wu, MD, David H Sachs, MD, PhD, WP Andrew, Lee, MD. Massachusetts General Hospital, Harvard Medical School, Boston, MA, WACC 453, Boston, MA 02114, USA Phone: 617-726-6943
2d Cellular condensation 7d Spheroid formation 14 d Increased extracellular matrix secretion
Introduction: The recent human hand transplants depend on longterm immunosuppression that entails significant risks to the recipients. We have demonstrated long-term tolerance to vascularized musculoskeletal allografts in MHC matched miniature swine treated with only
Conclusions: Human adipose tissue contains mesodermal stem cells capable of in vitro chondrogenesis. This abundant and easily obtained source of cells has considerable potential for future tissue engineering strategies in cartilage repair.
⫹/⫺ ⫹
⫹ ⫹⫹
⫹ ⫹⫹
⫹ ⫹
⫹/⫺ ⫹
⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹
Results: Peak Tensile Wound Strength (Newtons) Treatment Group (n ⴝ 6) Control Normal ⫹ EGF Normal ⫹ EGF & eIF 4E
S47
Surgical Forum Abstracts
7 days
14 days
4.5 ⫾ 1.3 6.5 ⫾ 2.9 8.4 ⫾ 3.6
9.8 ⫾ 3.9 11.1 ⫾ 3.4 18.8 ⫾ 3.5*
* p ⬍ 0.01 compared to control or EGF by ANOVA
Conclusions: Simultaneous biolistic delivery of EGF mRNA with eIF4E mRNA significantly increases wound breaking strength compared to control animals or treatment with EGF mRNA alone without risk of cellular transformation. Further studies of translational activation to augment wound healing are warranted.
Composite tissue allograft (CTA): tolerance induction without graft vs host disease (GvHD) Vijay Gorantla MD, Gustavo Perez-Abadia MD, Kaustubha Prabhune MD, Haldun Orhun MD, Thanos Kakoulidis MD, Ramsey Majzoub MD, Claudio Maldonado PhD, Gary Anderson PhD, Lynn Ogden MD, Warren Breidenbach MD, Suzanne Ildstad MD, John Barker MD, PhD. Division of Plastic and Reconstructive Surgery, Institute for Cellular Therapeutics, Department of Surgery, University of Louisville & Christine M. Kleinert Institute. University of Louisville, 511 South Floyd St, 320 MDR Bldg, Louisville, KY, 40292, USA. Tel (502) 852-0166. Introduction: Successful human hand transplants have recently been performed using immunosuppression. However, the risks of the nonspecific immunosuppressants required for graft survival are subject to controversy. Mixed allogeneic chimerism (MAC) induces donorspecific tolerance to solid organ allografts. CTAs add another variable in that the mature donor immune system (bone marrow (BM)) accompanies the graft, potentially resulting in GvHD. The aim of this study was to achieve a GvHD free tolerant state using MAC in a rat hind-limb model. Methods: Group I: naïve WF rats received hind limbs from unmanipulated ACI rats (n ⫽ 6). Group II: host WF rats were irradiated with 950cGy and reconstituted with MHC disparate ACI BM cells (␣-␥␦ T-cell depleted) (n ⫽ 8). Flow cytometry after 28 days confirmed chimerism. These rats then received non-irradiated ACI limbs. Group III: (ACI ⫹ WF) rats, received similar treatment as in Group II but were transplanted with ACI Limbs pretreated with 1050cGy TBI (n ⫽ 8). Results: Group I rejected their limbs in 5.7 ⫾ 1.5 days. Group II: 7/8 (ACI ⫹ WF) rats (⬎85% chimerism) exhibited rejection-free survival of non-irradiated CTAs for 22.4 ⫾ 2.8 days, but developed severe GvHD only after the CTA and died. Group III: 7/8 (ACI ⫹ WF) rats showed no sign of rejection or GvHD at 77 ⫾ 1.8 days. One rat died of infection at 34 days post-op with no sign of GvHD. Notably, this is significantly prolonged beyond the time of onset of GvHD in Group II. Conclusions: Partial conditioning of the host alone led to high levels of MAC without GvHD before CTA. The bone marrow in unmanipulated limbs resulted in GvHD. After transplantation of irradiated limbs, MAC prevented acute rejection and resulted in a GvHD free state. Taken together, these data show that immunocompetent donor cells transferred with the CTA can mediate GvHD and that inactivation of these cells by irradiation could prevent GvHD.
Tolerance to limb allografts in a miniature swine model
12 days of cyclosporine. The aims of this study were 1) to determine if tolerance to allografts could be achieved with the addition of a vascularized skin component, and 2) to evaluate the role of the donor bone marrow following long-term tolerance. Methods: Limb grafts from 4 swine with a 100 cm2 cutaneous paddle were heterotopically transplanted into MHC matched, minor antigen mismatched miniature swine and treated with 12 days of cyclosporine. Biweekly biopsies were performed and evaluated histologically. Pig allelic antigen (PAA), a non-MHC-linked marker, was used to study bone marrow migration in PAA⫹ recipients with PAA⫺ allografts. Swine demonstrating long-term tolerance to their allografts were sacrificed, and the graft marrow was analyzed for the migration of recipient cells into graft marrow space using the PAA marker. Results: All animals accepted the musculoskeletal portion of their grafts by gross inspection and histologic analysis. One animal demonstrated tolerance to the skin (⬎120 days). Three animals sloughed the graft epidermis (days 39, 41, and 50) leaving an intact dermis. The host skin began to re-epithelialize the graft dermis. At sacrifice, the allograft bone marrow harvest yielded viable marrow. All hematopoietic cells identified in the graft marrow were PAA positive and therefore of recipient origin. Conclusions: Tolerance to limb allografts with a skin component can be achieved with a 12-day course of cyclosporine and MHC matching. Immune modulation of the skin component may be necessary for clinical success. Furthermore, recipient hematopoietic cells repopulate the graft marrow space, and tolerance to limb allografts does not appear to depend on the presence of donor bone marrow cells.
Chondrogenesis of human adipo-derived mesodermal stem cells Jerry I Huang, BS, Marc H Hedrick, MD, H. Peter Lorenz, MD, Min Zhu, MD, Prosper Benhaim, MD. Division of Plastic and Reconstructive Surgery; University of California, Los Angeles 200 UCLA Medical Plaza, Suite #140; Los Angeles, CA 90095-6902, USA; Phone (310) 206-4468 Introduction: The repair of cartilaginous defects remains a significant clinical problem. Human bone marrow contains pluripotent mesenchymal stem cells capable of differentiation down multiple mesodermal lineages, including bone and cartilage. However, bone marrow harvest is painful and yields few cells. The purpose of this study is to determine if human adipose tissue contains a similar population of stem cells with osteochondrogenic potential. Methods: Human liposuction aspirate from five patients were washed and digested, yielding fibroblast-like cells. Cells were psssaged three times in basal media, then induced toward chondrogenesis by placement in high-density micromass cultures supplemented with TGF-1, insulin, transferrin, and ascorbic acid. Chondrogenesis was determined by histologic evaluation at 2, 7, and 14 days with H&E, Alcian blue, and Goldner’s trichrome staining. In addition, immunohistochemistry was performed using antibodies against type II collagen, chondroitin-4-sulfate, and keratan sulfate. Intensity of histologic and immunohistochemistry staining was subjectively graded as: none (⫺); background (⫹/⫺); light (⫹); moderate (⫹⫹); heavy (⫹⫹⫹). Results: Cartilaginous nodules with a perichondral cellular border formed within 48 hours in all treated cultures. Untreated control cells showed no evidence of chondrogenic differentiation. Time Stages of chondrogenic Alcian Trichrome Chondroitin- Keratan Type II point differentiation blue (collagen) 4-sulfate sulfate collagen
David W Mathes, MD, Judy L Bourget, MD, Mark A Randolph, MA, Mario G Solari, BA, Anette, Wu, MD, David H Sachs, MD, PhD, WP Andrew, Lee, MD. Massachusetts General Hospital, Harvard Medical School, Boston, MA, WACC 453, Boston, MA 02114, USA Phone: 617-726-6943
2d Cellular condensation 7d Spheroid formation 14 d Increased extracellular matrix secretion
Introduction: The recent human hand transplants depend on longterm immunosuppression that entails significant risks to the recipients. We have demonstrated long-term tolerance to vascularized musculoskeletal allografts in MHC matched miniature swine treated with only
Conclusions: Human adipose tissue contains mesodermal stem cells capable of in vitro chondrogenesis. This abundant and easily obtained source of cells has considerable potential for future tissue engineering strategies in cartilage repair.
⫹/⫺ ⫹
⫹ ⫹⫹
⫹ ⫹⫹
⫹ ⫹
⫹/⫺ ⫹
⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹