- Email: [email protected]
The growing importance of fat in regenerative medicine
Update
64
TRENDS in Biotechnology
successfully employed for identification of active proteases and for the implication of presumed substrates in a biological context. Coupled with substrate profiling, we expect that continued advances in mass spectrometry and proteomic analysis will result in new methods for use in combination on a protease-specific basis. However, the exciting recent advances in substrate identification highlight candidate substrates that must ultimately be validated in an actual biological setting. Frequently, the process of substrate validation is limited to traditional biochemical techniques and represents the rate-limiting step, requiring a specific inhibitor, antibody or knockout to address the issue. Although outside the scope of this article, the development of a methodology such as RNAi has decreased the time necessary for substrate validation relative to traditional techniques. It has been successfully used to reveal Taspase-1 cleavage of the mixed-lineage leukemia gene product [19]. Similar developments in the biological validation of substrates will complement the recent advances in identification of proteases and their natural substrates and help define the roles of proteases in all physiological processes. Acknowledgements We are grateful to Carly R.K. Loeb for critical review of the manuscript. This work was supported by a University of California President’s Dissertation Year Fellowship (A.B.M) and NIH Grants GM56531 and CA72006 (C.S.C.).
References 1 Rawlings, N.D. et al. (2004). MEROPS: the peptidase database. Nucleic Acids Res. 32 Database issue: D160-D1634 2 Vu, T.K. et al. (1991) Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64, 1057–1068 3 Greenbaum, D. et al. (2000) Epoxide electrophiles as activitydependent cysteine protease profiling and discovery tools. Chem. Biol. 7, 569–581 4 Liu, Y. et al. (1999) Activity-based protein profiling: the serine hydrolases. Proc. Natl. Acad. Sci. U. S. A. 96, 14694–14699
Vol.23 No.2 February 2005
5 Saghatelian, A. et al. (2004) Activity-based probes for the proteomic profiling of metalloproteases. Proc. Natl. Acad. Sci. U. S. A. 101, 10000–10005 6 Matthews, D.J. and Wells, J.A. (1993) Substrate phage: selection of protease substrates by monovalent phage display. Science 260, 1113–1117 7 Greenbaum, D.C. et al. (2002) Small molecule affinity fingerprinting. A tool for enzyme family subclassification, target identification, and inhibitor design. Chem. Biol. 9, 1085–1094 8 Harris, J.L. et al. (2000) Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries. Proc. Natl. Acad. Sci. U. S. A. 97, 7754–7759 9 Barrios, A.M. and Craik, C.S. (2002) Scanning the prime-site substrate specificity of proteolytic enzymes: a novel assay based on ligand-enhanced lanthanide ion fluorescence. Bioorg. Med. Chem. Lett. 12, 3619–3623 10 Salisbury, C.M. et al. (2002) Peptide microarrays for the determination of protease substrate specificity. J. Am. Chem. Soc. 124, 14868–14870 11 Winssinger, N. et al. (2002) Profiling protein function with small molecule microarrays. Proc. Natl. Acad. Sci. U. S. A. 99, 11139–11144 12 Turk, B.E. et al. (2001) Determination of protease cleavage site motifs using mixture-based oriented peptide libraries. Nat. Biotechnol. 19, 661–667 13 Thornberry, N.A. et al. (1997) A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J. Biol. Chem. 272, 17907–17911 14 Zhu, L. et al. (2003) The role of dipeptidyl peptidase IV in the cleavage of glucagon family peptides: in vivo metabolism of pituitary adenylate cyclase activating polypeptide-(1-38). J. Biol. Chem. 278, 22418–22423 15 Marnett, A.B. et al. (2004) Communication between the active sites and dimer interface of a herpesvirus protease revealed by a transitionstate inhibitor. Proc. Natl. Acad. Sci. U. S. A. 101, 6870–6875 16 Lopez-Otin, C. and Overall, C.M. (2002) Protease degradomics: a new challenge for proteomics. Nat. Rev. Mol. Cell Biol. 3, 509–519 17 Tam, E.M. et al. (2004) Membrane protease proteomics: Isotope-coded affinity tag MS identification of undescribed MT1-matrix metalloproteinase substrates. Proc. Natl. Acad. Sci. U. S. A. 101, 6917–6922 18 Bredemeyer, A.J. et al. (2004) A proteomic approach for the discovery of protease substrates. Proc. Natl. Acad. Sci. U. S. A. 101, 11785–11790 19 Hsieh, J.J. et al. (2003) Taspase1: a threonine aspartase required for cleavage of MLL and proper HOX gene expression. Cell 115, 293–303 0167-7799/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2004.12.010
The growing importance of fat in regenerative medicine Brian M. Strem1,2 and Marc H. Hedrick1,2 1 2
Macropore Biosurgery Inc., 6740 Top Gun Street, San Diego, California 92121, USA Departments of Bioengineering and Surgery, University of California, Los Angeles, USA
A recent publication by Michael Longaker and colleagues represents a landmark for the use of adipose tissue as a source of cells for tissue regeneration. The authors investigated the ability of adipose tissuederived cells (ADCs) to regenerate critical size calvarial (superior portion of the skull) defects in mice by using a novel osteoconducive apatite-coated Poly-lactic-coCorresponding author: Strem, B.M. ([email protected]). Available online 13 December 2004 www.sciencedirect.com
glycolic acid (PLGA) scaffold for cell delivery. Direct comparison of this osteogenic ability was performed with bone marrow stromal cells and juvenile calvarialderived osteoblasts.
Introduction Although we and others have previously reported the ability of adipose tissue-derived cells (ADCs) to undergo
Update
TRENDS in Biotechnology
osteogenesis in vitro and to form bone in an in vivo ectopic bone model [1–4], Longaker’s paper is the first to describe the functions of ADCs in an orthotopic site. Adult stem cells have been isolated from many locations in the body. Bone marrow represents a rich source of adult stem cells (BM-MSCs) with much regenerative potential. Adipose tissue represents an even more plentiful reservoir of adult stem cells, similar to BM-MSCs, based on cell surface phenotype and differentiation potential [5,6]. Bone marrow has been more extensively investigated, and thus is better characterized for clinical applications. However, adipose tissue might have advantages over bone marrow, such as: (i) minimal morbidity upon harvest; (ii) clinically relevant stem cell numbers extractable from tissue isolates, potentially removing the need for in vitro propagation; (iii) stem cell frequency is significantly higher in adipose tissue compared with bone marrow (2% vs 0.002%); and (iv) higher proliferation rates than BM-MSCs.
ADC characteristics Adipose tissue has been identified as having differentiation potential extending beyond the osteogenic phenotype. Recently, reports have indicated differentiation in vitro and in vivo towards adipogenic, chondrogenic, myogenic, neurogenic, endothelial, hematopoietic and cardiomyogenic phenotypes [2,7–15]. However, a general misconception associated with ADCs is that the donor must be overweight or obese to have sufficient adipose tissue available for harvest. Adipose tissue harvested from obese and overweight patients has been compared with that harvested from those with normal body mass index (BMI) and results indicate that those with normal BMI have an equal amount of stem cells as overweight or obese patients [16]. We have also investigated the differences between ADCs from patients with and without type II diabetes and these show no differences with the mesenchymal stem cell assays that analyze the ability of cells to form individual colonies that are capable of multilineage differentiation (towards bone and fat) or in their ability to restore blood flow in an experimental model of hind limb ischemia. ADCs support tissue regeneration in several ways. ADCs have been demonstrated by several laboratories to secrete cytokines that support tissue regeneration or minimize tissue damage. Assays performed on the factors secreted by ADCs reveal the presence of multiple angiogenic and anti-apoptotic cytokines [15]. ADCs not only secrete endogenous cytokines, they are also genedelivery vehicles for administering engineered genes that support tissue regeneration. We have shown the ability of ADCs to express transgenes delivered by three distinct viral vector types [16]. We have also demonstrated the ability of ADCs, transduced by an adenovirus encoding the osteogenic factor bone morphogenic protein 2 (BMP-2), to enhance bone formation when transplanted subcutaneously into nude mice [3]. Owing to the enhanced proliferation rate when compared with bone marrow stromal cells, ADCs might be a source of cells capable of enhanced gene delivery. ADCs can be delivered in vivo www.sciencedirect.com
Vol.23 No.2 February 2005
65
using numerous vehicles and could potentially be genedelivery vehicles themselves.
Calvarial defect model In this article, Longaker and colleagues further our understanding of whether and how these cells function in vivo. Specifically, they observed that the first evidence of cranial bone formation occurs adjacent to the dura mater, probably indicating a paracrine effect perhaps involving the transforming growth factor-b (TGF-b) family of cytokines. This signaling has been shown to be crucial in calvarial development [17], and the authors postulate that this effect is partially responsible for the differentiation of the adult stem cells directly into mineralproducing osteoblasts, bypassing a cartilage intermediate. Interestingly, our studies of ossification with ADCs show that bone formation can also occur through an endochondral ossification process (B.S. and M.H., unpublished data). This difference might be because of either the anatomical location (calvarium) or the distinctive carrier in which the cells are placed. Because calvarial bone normally develops through intramembranous ossification [18], placing these cells into this niche could epigenetically drive the developmental pattern.
Role of cell carrier Furthermore, in this study, the investigators used Polylactic-co-glycolic acid (PLGA) coated with apatite as the scaffold material. The apatite coating is thought to make the carrier more osteoconductive. This apatite coating might have the additional effect of directing the ADCs to bypass cartilage formation. Kuboki et al. recently reported that hydroxyapatite-coated scaffolds can induce either endochondral or intramembranous ossification. They showed that scaffolds with smaller pore sizes (90–120 mm) mediate bone formation via endochondral ossification whereas the larger pore sized scaffolds (350 mm) drive intramembranous ossification [19]. The scaffolds used in the study by Longaker’s group had pore sizes of 200–300 mm, consistent with intramembranous ossification.
Future potential uses This important study suggests that ADCs could be useful in in vivo tissue engineering strategies for bone. Furthermore, adipose tissue is readily available and easily harvested, which could enable a broader set of clinical applications in the future. For example, small and large animal preclinical data using ADCs have shown significantly improved cardiac function following an acute myocardial infarction (heart attack). One can postulate that in a clinical setting, similar improvements would be observed, which could potentially augment the treatment patients undergo on arrival at a hospital. This is one setting in which regenerative medicine using ADCs could improve the quality of life for a patient and other future applications are currently under investigation.
Update
66
TRENDS in Biotechnology
Vol.23 No.2 February 2005
References 1 Huang, J.I. et al. (2002) Rat extramedullary adipose tissue as a source of osteochondrogenic progenitor cells. Plast. Reconstr. Surg. 109, 1033–1041 2 Zuk, P.A. et al. (2001) Multilineage cells from human adipose tissue: implications for cell- based therapies. Tissue Eng. 7, 211–228 3 Dragoo, J.L. et al. (2003) Bone induction by BMP-2 transduced stem cells derived from human fat. J. Orthop. Res. 21, 622–629 4 Hicok, K.C. et al. (2004) Human adipose-derived adult stem cells produce osteoid in vivo. Tissue Eng. 10, 371–380 5 De Ugarte, D.A. et al. (2003) Comparison of multi-lineage cells from human adipose tissue and bone marrow. Cells Tissues Organs 174, 101–109 6 Zuk, P.A. et al. (2002) Human adipose tissue is a source of multipotent stem cells. Mol. Biol. Cell 13, 4279–4295 7 Ashjian, P.H. et al. (2003) In vitro differentiation of human processed lipoaspirate cells into early neural progenitors. Plast. Reconstr. Surg. 111, 1922–1931 8 Cousin, B. et al. (2003) Reconstitution of lethally irradiated mice by cells isolated from adipose tissue. Biochem. Biophys. Res. Commun. 301, 1016–1022 9 Erickson, G.R. et al. (2002) Chondrogenic potential of adipose tissuederived stromal cells in vitro and in vivo. Biochem. Biophys. Res. Commun. 290, 763–769 10 Mizuno, H. et al. (2002) Myogenic differentiation by human processed lipoaspirate cells. Plast. Reconstr. Surg. 109, 199–209 11 Rangappa, S. et al. (2003) Transformation of adult mesenchymal stem
12
13
14
15 16 17
18
19
cells isolated from the fatty tissue into cardiomyocytes. Ann. Thorac. Surg. 75, 775–779 Safford, K.M. et al. (2002) Neurogenic differentiation of murine and human adipose-derived stromal cells. Biochem. Biophys. Res. Commun. 294, 371–379 Planat-Benard, V. et al. (2004) Plasticity of human adipose lineage cells toward endothelial cells: physiological and therapeutic perspectives. Circulation 109, 656–663 Kang, S.K. et al. (2003) Improvement of neurological deficits by intracerebral transplantation of human adipose tissue-derived stromal cells after cerebral ischemia in rats. Exp. Neurol. 183, 355–366 Rehman, J. et al. (2004) Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells. Circulation 109, 1292–1298 Morizono, K. et al. (2003) Multilineage cells from adipose tissue as gene delivery vehicles. Hum. Gene Ther. 14, 59–66 Opperman, L.A. et al. (1993) Tissue interactions with underlying dura mater inhibit osseous obliteration of developing cranial sutures. Dev. Dyn. 198, 312–322 Rice, D.P. et al. (1997) Detection of gelatinase B expression reveals osteoclastic bone resorption as a feature of early calvarial bone development. Bone 21, 479–486 Kuboki, Y. et al. (2001) Geometry of carriers controlling phenotypic expression in BMP-induced osteogenesis and chondrogenesis. J. Bone Joint Surg. Am. 83–A (Suppl 1), S105–S115
0167-7799/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2004.12.003
Letters
Protease inhibitor MG132 in cloning: no end to the nightmare Shaorong Gao1, Zhiming Han1, Maki Kihara2, Eli Adashi2 and Keith E. Latham1,3 1
The Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine Division of Reproductive Sciences, Huntsman Cancer Institute, University of Utah Health Sciences Center, 50 North Medical Drive, Salt Lake City, Utah 84132, USA 3 Department of Biochemistry, Temple University School of Medicine, 3307 N. Broad Street, Philadelphia, Pennsylvania 19140 USA 2
Nuclear reprogramming directed by the ooplasm is essential for producing cloned animals by somatic cell nuclear transfer (SCNT). One component of the reprogramming process is the removal of somatic histone H1 variants followed by replacement by the oocyte-specific form, H1FOO [1]. Despite occurrence of this transition in 100% of SCNT constructs, cloned embryo development remains poor and cloned embryos express numerous somatic cell characteristics [2], indicating that reprogramming is slow or incomplete. Nuclear reprogramming might be facilitated by prolonged exposure of the donor nucleus to ooplasm before development is initiated. However a slight delay in activation of the SCNT constructs can be detrimental [2–3]. This probably reflects the ongoing degradation of maternally inherited proteins and mRNAs as the ooplasm ages. Thus, cloning is potentially confounded by an inherent conflict – a desire for extended reprogramming opportunity versus an ongoing deterioration of the supporting ooplasm. Corresponding author: Latham, K.E. ([email protected]). Available online 16 December 2004 www.sciencedirect.com
In a recent issue of Trends in Biotechnology it was suggested that proteasome-mediated protein degradation could contribute to the success or failure of cloning [4]. Protein degradation could have a role in nuclear reprogramming by facilitating removal of proteins from the transplanted nucleus. Inhibiting protease activity could therefore impede this aspect of reprogramming. Alternatively, protease inhibition might slow deterioration of the aging ooplasm, or might provide a means of manipulating the cell cycle. Treating cloned rat constructs with the protease inhibitor MG132 (N-benzyloxycarbonyl-leucylleucyl-leucinal) seemed to improve clone development [5]. This was attributed to a delay in the metaphase to anaphase transition to extend the reprogramming period [6] although MG132 might also inhibit the ongoing deterioration of maternal proteins, thus preserving developmental potential. To test these possibilities, we examined the effects of MG132 on histone H1 switching and clone development in mice. We previously demonstrated that within 1 h of SCNT the ooplasm removes essentially all detectable somatic linker H1 and replaces it with the oocyte-specific
64
TRENDS in Biotechnology
successfully employed for identification of active proteases and for the implication of presumed substrates in a biological context. Coupled with substrate profiling, we expect that continued advances in mass spectrometry and proteomic analysis will result in new methods for use in combination on a protease-specific basis. However, the exciting recent advances in substrate identification highlight candidate substrates that must ultimately be validated in an actual biological setting. Frequently, the process of substrate validation is limited to traditional biochemical techniques and represents the rate-limiting step, requiring a specific inhibitor, antibody or knockout to address the issue. Although outside the scope of this article, the development of a methodology such as RNAi has decreased the time necessary for substrate validation relative to traditional techniques. It has been successfully used to reveal Taspase-1 cleavage of the mixed-lineage leukemia gene product [19]. Similar developments in the biological validation of substrates will complement the recent advances in identification of proteases and their natural substrates and help define the roles of proteases in all physiological processes. Acknowledgements We are grateful to Carly R.K. Loeb for critical review of the manuscript. This work was supported by a University of California President’s Dissertation Year Fellowship (A.B.M) and NIH Grants GM56531 and CA72006 (C.S.C.).
References 1 Rawlings, N.D. et al. (2004). MEROPS: the peptidase database. Nucleic Acids Res. 32 Database issue: D160-D1634 2 Vu, T.K. et al. (1991) Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64, 1057–1068 3 Greenbaum, D. et al. (2000) Epoxide electrophiles as activitydependent cysteine protease profiling and discovery tools. Chem. Biol. 7, 569–581 4 Liu, Y. et al. (1999) Activity-based protein profiling: the serine hydrolases. Proc. Natl. Acad. Sci. U. S. A. 96, 14694–14699
Vol.23 No.2 February 2005
5 Saghatelian, A. et al. (2004) Activity-based probes for the proteomic profiling of metalloproteases. Proc. Natl. Acad. Sci. U. S. A. 101, 10000–10005 6 Matthews, D.J. and Wells, J.A. (1993) Substrate phage: selection of protease substrates by monovalent phage display. Science 260, 1113–1117 7 Greenbaum, D.C. et al. (2002) Small molecule affinity fingerprinting. A tool for enzyme family subclassification, target identification, and inhibitor design. Chem. Biol. 9, 1085–1094 8 Harris, J.L. et al. (2000) Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries. Proc. Natl. Acad. Sci. U. S. A. 97, 7754–7759 9 Barrios, A.M. and Craik, C.S. (2002) Scanning the prime-site substrate specificity of proteolytic enzymes: a novel assay based on ligand-enhanced lanthanide ion fluorescence. Bioorg. Med. Chem. Lett. 12, 3619–3623 10 Salisbury, C.M. et al. (2002) Peptide microarrays for the determination of protease substrate specificity. J. Am. Chem. Soc. 124, 14868–14870 11 Winssinger, N. et al. (2002) Profiling protein function with small molecule microarrays. Proc. Natl. Acad. Sci. U. S. A. 99, 11139–11144 12 Turk, B.E. et al. (2001) Determination of protease cleavage site motifs using mixture-based oriented peptide libraries. Nat. Biotechnol. 19, 661–667 13 Thornberry, N.A. et al. (1997) A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J. Biol. Chem. 272, 17907–17911 14 Zhu, L. et al. (2003) The role of dipeptidyl peptidase IV in the cleavage of glucagon family peptides: in vivo metabolism of pituitary adenylate cyclase activating polypeptide-(1-38). J. Biol. Chem. 278, 22418–22423 15 Marnett, A.B. et al. (2004) Communication between the active sites and dimer interface of a herpesvirus protease revealed by a transitionstate inhibitor. Proc. Natl. Acad. Sci. U. S. A. 101, 6870–6875 16 Lopez-Otin, C. and Overall, C.M. (2002) Protease degradomics: a new challenge for proteomics. Nat. Rev. Mol. Cell Biol. 3, 509–519 17 Tam, E.M. et al. (2004) Membrane protease proteomics: Isotope-coded affinity tag MS identification of undescribed MT1-matrix metalloproteinase substrates. Proc. Natl. Acad. Sci. U. S. A. 101, 6917–6922 18 Bredemeyer, A.J. et al. (2004) A proteomic approach for the discovery of protease substrates. Proc. Natl. Acad. Sci. U. S. A. 101, 11785–11790 19 Hsieh, J.J. et al. (2003) Taspase1: a threonine aspartase required for cleavage of MLL and proper HOX gene expression. Cell 115, 293–303 0167-7799/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2004.12.010
The growing importance of fat in regenerative medicine Brian M. Strem1,2 and Marc H. Hedrick1,2 1 2
Macropore Biosurgery Inc., 6740 Top Gun Street, San Diego, California 92121, USA Departments of Bioengineering and Surgery, University of California, Los Angeles, USA
A recent publication by Michael Longaker and colleagues represents a landmark for the use of adipose tissue as a source of cells for tissue regeneration. The authors investigated the ability of adipose tissuederived cells (ADCs) to regenerate critical size calvarial (superior portion of the skull) defects in mice by using a novel osteoconducive apatite-coated Poly-lactic-coCorresponding author: Strem, B.M. ([email protected]). Available online 13 December 2004 www.sciencedirect.com
glycolic acid (PLGA) scaffold for cell delivery. Direct comparison of this osteogenic ability was performed with bone marrow stromal cells and juvenile calvarialderived osteoblasts.
Introduction Although we and others have previously reported the ability of adipose tissue-derived cells (ADCs) to undergo
Update
TRENDS in Biotechnology
osteogenesis in vitro and to form bone in an in vivo ectopic bone model [1–4], Longaker’s paper is the first to describe the functions of ADCs in an orthotopic site. Adult stem cells have been isolated from many locations in the body. Bone marrow represents a rich source of adult stem cells (BM-MSCs) with much regenerative potential. Adipose tissue represents an even more plentiful reservoir of adult stem cells, similar to BM-MSCs, based on cell surface phenotype and differentiation potential [5,6]. Bone marrow has been more extensively investigated, and thus is better characterized for clinical applications. However, adipose tissue might have advantages over bone marrow, such as: (i) minimal morbidity upon harvest; (ii) clinically relevant stem cell numbers extractable from tissue isolates, potentially removing the need for in vitro propagation; (iii) stem cell frequency is significantly higher in adipose tissue compared with bone marrow (2% vs 0.002%); and (iv) higher proliferation rates than BM-MSCs.
ADC characteristics Adipose tissue has been identified as having differentiation potential extending beyond the osteogenic phenotype. Recently, reports have indicated differentiation in vitro and in vivo towards adipogenic, chondrogenic, myogenic, neurogenic, endothelial, hematopoietic and cardiomyogenic phenotypes [2,7–15]. However, a general misconception associated with ADCs is that the donor must be overweight or obese to have sufficient adipose tissue available for harvest. Adipose tissue harvested from obese and overweight patients has been compared with that harvested from those with normal body mass index (BMI) and results indicate that those with normal BMI have an equal amount of stem cells as overweight or obese patients [16]. We have also investigated the differences between ADCs from patients with and without type II diabetes and these show no differences with the mesenchymal stem cell assays that analyze the ability of cells to form individual colonies that are capable of multilineage differentiation (towards bone and fat) or in their ability to restore blood flow in an experimental model of hind limb ischemia. ADCs support tissue regeneration in several ways. ADCs have been demonstrated by several laboratories to secrete cytokines that support tissue regeneration or minimize tissue damage. Assays performed on the factors secreted by ADCs reveal the presence of multiple angiogenic and anti-apoptotic cytokines [15]. ADCs not only secrete endogenous cytokines, they are also genedelivery vehicles for administering engineered genes that support tissue regeneration. We have shown the ability of ADCs to express transgenes delivered by three distinct viral vector types [16]. We have also demonstrated the ability of ADCs, transduced by an adenovirus encoding the osteogenic factor bone morphogenic protein 2 (BMP-2), to enhance bone formation when transplanted subcutaneously into nude mice [3]. Owing to the enhanced proliferation rate when compared with bone marrow stromal cells, ADCs might be a source of cells capable of enhanced gene delivery. ADCs can be delivered in vivo www.sciencedirect.com
Vol.23 No.2 February 2005
65
using numerous vehicles and could potentially be genedelivery vehicles themselves.
Calvarial defect model In this article, Longaker and colleagues further our understanding of whether and how these cells function in vivo. Specifically, they observed that the first evidence of cranial bone formation occurs adjacent to the dura mater, probably indicating a paracrine effect perhaps involving the transforming growth factor-b (TGF-b) family of cytokines. This signaling has been shown to be crucial in calvarial development [17], and the authors postulate that this effect is partially responsible for the differentiation of the adult stem cells directly into mineralproducing osteoblasts, bypassing a cartilage intermediate. Interestingly, our studies of ossification with ADCs show that bone formation can also occur through an endochondral ossification process (B.S. and M.H., unpublished data). This difference might be because of either the anatomical location (calvarium) or the distinctive carrier in which the cells are placed. Because calvarial bone normally develops through intramembranous ossification [18], placing these cells into this niche could epigenetically drive the developmental pattern.
Role of cell carrier Furthermore, in this study, the investigators used Polylactic-co-glycolic acid (PLGA) coated with apatite as the scaffold material. The apatite coating is thought to make the carrier more osteoconductive. This apatite coating might have the additional effect of directing the ADCs to bypass cartilage formation. Kuboki et al. recently reported that hydroxyapatite-coated scaffolds can induce either endochondral or intramembranous ossification. They showed that scaffolds with smaller pore sizes (90–120 mm) mediate bone formation via endochondral ossification whereas the larger pore sized scaffolds (350 mm) drive intramembranous ossification [19]. The scaffolds used in the study by Longaker’s group had pore sizes of 200–300 mm, consistent with intramembranous ossification.
Future potential uses This important study suggests that ADCs could be useful in in vivo tissue engineering strategies for bone. Furthermore, adipose tissue is readily available and easily harvested, which could enable a broader set of clinical applications in the future. For example, small and large animal preclinical data using ADCs have shown significantly improved cardiac function following an acute myocardial infarction (heart attack). One can postulate that in a clinical setting, similar improvements would be observed, which could potentially augment the treatment patients undergo on arrival at a hospital. This is one setting in which regenerative medicine using ADCs could improve the quality of life for a patient and other future applications are currently under investigation.
Update
66
TRENDS in Biotechnology
Vol.23 No.2 February 2005
References 1 Huang, J.I. et al. (2002) Rat extramedullary adipose tissue as a source of osteochondrogenic progenitor cells. Plast. Reconstr. Surg. 109, 1033–1041 2 Zuk, P.A. et al. (2001) Multilineage cells from human adipose tissue: implications for cell- based therapies. Tissue Eng. 7, 211–228 3 Dragoo, J.L. et al. (2003) Bone induction by BMP-2 transduced stem cells derived from human fat. J. Orthop. Res. 21, 622–629 4 Hicok, K.C. et al. (2004) Human adipose-derived adult stem cells produce osteoid in vivo. Tissue Eng. 10, 371–380 5 De Ugarte, D.A. et al. (2003) Comparison of multi-lineage cells from human adipose tissue and bone marrow. Cells Tissues Organs 174, 101–109 6 Zuk, P.A. et al. (2002) Human adipose tissue is a source of multipotent stem cells. Mol. Biol. Cell 13, 4279–4295 7 Ashjian, P.H. et al. (2003) In vitro differentiation of human processed lipoaspirate cells into early neural progenitors. Plast. Reconstr. Surg. 111, 1922–1931 8 Cousin, B. et al. (2003) Reconstitution of lethally irradiated mice by cells isolated from adipose tissue. Biochem. Biophys. Res. Commun. 301, 1016–1022 9 Erickson, G.R. et al. (2002) Chondrogenic potential of adipose tissuederived stromal cells in vitro and in vivo. Biochem. Biophys. Res. Commun. 290, 763–769 10 Mizuno, H. et al. (2002) Myogenic differentiation by human processed lipoaspirate cells. Plast. Reconstr. Surg. 109, 199–209 11 Rangappa, S. et al. (2003) Transformation of adult mesenchymal stem
12
13
14
15 16 17
18
19
cells isolated from the fatty tissue into cardiomyocytes. Ann. Thorac. Surg. 75, 775–779 Safford, K.M. et al. (2002) Neurogenic differentiation of murine and human adipose-derived stromal cells. Biochem. Biophys. Res. Commun. 294, 371–379 Planat-Benard, V. et al. (2004) Plasticity of human adipose lineage cells toward endothelial cells: physiological and therapeutic perspectives. Circulation 109, 656–663 Kang, S.K. et al. (2003) Improvement of neurological deficits by intracerebral transplantation of human adipose tissue-derived stromal cells after cerebral ischemia in rats. Exp. Neurol. 183, 355–366 Rehman, J. et al. (2004) Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells. Circulation 109, 1292–1298 Morizono, K. et al. (2003) Multilineage cells from adipose tissue as gene delivery vehicles. Hum. Gene Ther. 14, 59–66 Opperman, L.A. et al. (1993) Tissue interactions with underlying dura mater inhibit osseous obliteration of developing cranial sutures. Dev. Dyn. 198, 312–322 Rice, D.P. et al. (1997) Detection of gelatinase B expression reveals osteoclastic bone resorption as a feature of early calvarial bone development. Bone 21, 479–486 Kuboki, Y. et al. (2001) Geometry of carriers controlling phenotypic expression in BMP-induced osteogenesis and chondrogenesis. J. Bone Joint Surg. Am. 83–A (Suppl 1), S105–S115
0167-7799/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2004.12.003
Letters
Protease inhibitor MG132 in cloning: no end to the nightmare Shaorong Gao1, Zhiming Han1, Maki Kihara2, Eli Adashi2 and Keith E. Latham1,3 1
The Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine Division of Reproductive Sciences, Huntsman Cancer Institute, University of Utah Health Sciences Center, 50 North Medical Drive, Salt Lake City, Utah 84132, USA 3 Department of Biochemistry, Temple University School of Medicine, 3307 N. Broad Street, Philadelphia, Pennsylvania 19140 USA 2
Nuclear reprogramming directed by the ooplasm is essential for producing cloned animals by somatic cell nuclear transfer (SCNT). One component of the reprogramming process is the removal of somatic histone H1 variants followed by replacement by the oocyte-specific form, H1FOO [1]. Despite occurrence of this transition in 100% of SCNT constructs, cloned embryo development remains poor and cloned embryos express numerous somatic cell characteristics [2], indicating that reprogramming is slow or incomplete. Nuclear reprogramming might be facilitated by prolonged exposure of the donor nucleus to ooplasm before development is initiated. However a slight delay in activation of the SCNT constructs can be detrimental [2–3]. This probably reflects the ongoing degradation of maternally inherited proteins and mRNAs as the ooplasm ages. Thus, cloning is potentially confounded by an inherent conflict – a desire for extended reprogramming opportunity versus an ongoing deterioration of the supporting ooplasm. Corresponding author: Latham, K.E. ([email protected]). Available online 16 December 2004 www.sciencedirect.com
In a recent issue of Trends in Biotechnology it was suggested that proteasome-mediated protein degradation could contribute to the success or failure of cloning [4]. Protein degradation could have a role in nuclear reprogramming by facilitating removal of proteins from the transplanted nucleus. Inhibiting protease activity could therefore impede this aspect of reprogramming. Alternatively, protease inhibition might slow deterioration of the aging ooplasm, or might provide a means of manipulating the cell cycle. Treating cloned rat constructs with the protease inhibitor MG132 (N-benzyloxycarbonyl-leucylleucyl-leucinal) seemed to improve clone development [5]. This was attributed to a delay in the metaphase to anaphase transition to extend the reprogramming period [6] although MG132 might also inhibit the ongoing deterioration of maternal proteins, thus preserving developmental potential. To test these possibilities, we examined the effects of MG132 on histone H1 switching and clone development in mice. We previously demonstrated that within 1 h of SCNT the ooplasm removes essentially all detectable somatic linker H1 and replaces it with the oocyte-specific