usmle文件克氏外科学19版chapter

1、 CHAPTER 8REGENERATIVE MEDICINEJason P. Glotzbach, Sae Hee Ko, Geoffrey C. Gurtner,and Michael T. Longakerclinicians with an expanded armamentarium to treat diseased and dysfunctional organs.Embryonic Stem CellsDuring development, two distinct lineages emerge during the transition from morula to bla

2、stocyst, the trophoectoderm and the inner cell mass. Embryonic stem cells (ESCs) are immortal cell lines derived from the inner cell mass of the blastocyst. The two hallmark characteristics of ESCs are their unlimited in vitro self-renewal capacity and their ability to differentiate into all somatic

3、 cell types.1 A number of transcription factors, most prominently Oct4, Sox2, and Nanog, are essential regulators that ensure the maintenance of pluripotency while suppressingRegeneration refers to the restoration of normal tissue and organ architecture and function after injury or disease. Although

4、 numerous complex organisms retain impressive capacity to regenerate limbs and organs throughout adult life, humans have sacrificed regenerative ability for speed and strength of repair. This has allowed us to enjoy remarkable evolutionary success, but it also leads to significant scarring that caus

5、es significant loss of function and aesthetic consequences. It may be possible to improve on the normal recovery from injury and illness by promoting true tissue regeneration instead of repair through fibrosis and scarring. Surgeons have understood these dynamics for decades, but comprehensive tissu

6、e and organ regeneration have remained elusive in clinical practice. The field of regenera- tive medicine is largely focused on stem cells, which are power- ful undifferentiated cells that have the ability to self-renew and give rise to one or more different cell types. As basic scientific research

7、has uncovered the biology of stem cells, translational opportunities for stem cellbased therapies have become increas- ingly plausible. In addition to stem cell biology, the field of regenerative medicine includes the disciplines of tissue engineer- ing and biomaterials, which aim to create molecula

8、r and struc- tural niches to deliver regenerative therapies. This chapter provides an overview of the current status of stem cell biology and tissue engineering research and outlines the future steps required for regenerative medicine to become clinically useful.STEM CELL SOURCESStem cells are defin

9、ed by their capacity to self-renew and dif- ferentiate into multiple functional cell types (Table 8-1). Tradi- tionally, they have been divided into two main groups based on their potential to differentiate (Fig. 8-1). Pluripotent stem cells (embryonic) can differentiate into every cell of the body,

10、 whereas multipotent stem cells (adult) can differentiate into multiple, but not all, cell lineages. In addition to the traditional stem cell classification, a new class of stem cells has recently been describedinduced pluripotent stem (iPS) cellswhich are derived from genetically reprogrammed adult

11、 cells. These diverse cell populations hold much promise to provide researchers and178differentiation.2 The two glycolipid aSSEA3 and SSEA4are operational cell surface markers used to identify humanESCs.3 Since the successful isolation of mouse and human ESCs, their potential for cell replacement th

12、erapy and regenera- tive medicine has been widely acknowledged.4 Both mouse and human ESCs have demonstrated an in vitro capacity to form cardiomyocytes, hematopoietic progenitors, neurons, skel- myocytes, adipocytes, osteocytes, chondrocytes, and pancre- atic islet cells when cultured under specifi

13、c growth factorconditions.5,6However, a number of limitations currently exist regarding the use of human ESCs in regenerative medicine. Although pluripotentiality and unlimited ability for self-renewal make ESCs attractive for cell replacement therapy, these same charac- teristics simultaneously tra

14、nslate into unregulated differentiation and formation of teratomas and teratocarcinomas. These tumors contain differentiated cells that contain all three primary germ layers, as well as undifferentiated pluripotent stem cells. This tendency to form tumors has been observed when ESCs are transplanted

15、 into mice, raising the concern that human ESC based therapy may also lead to unwanted tumor formation.1 Without the elimination of this possibility, the clinical use of ESC-derived tissue will remain limited.In addition, any cell-based therapy must be free of animal contaminants that might contain

16、pathogens or elicit an immune reaction after transfer to a host. Both mouse cell and human ESC lines are generally grown on a mouse-derived feeder layer of fibroblasts that provides additional factors that promote ESC proliferation as well as inhibit their differentiation. One example of possible an

17、imal product contamination is the demonstration that human ESCs grown on mouse feeder cells express a nonhu- man sialic acid that could elicit a hosts immune response.7stem cell sourcesbioengineering for regenerative medicine clinical applications of stem cellsRegeneRative Medicine Chapter 8 179Conc

18、erns have also been raised over the possible transfer of murine es from feeder layers to human ESCs. Many labo- ratories are working to solve this problem, with some studies demonstrating the ability to culture human ESCs under serum- free defined medium conditions on human cell-derived feeders or u

19、nder feeder-free conditions.8Furthermore, there are significant political and ethical hurdles that hinder further investigations of human ESCs. At this time, the limited number of ESC lines available and the restrictions placed on their use have precluded major progress in ESC-based applications. Al

20、though President Obama in recent months has largely reversed the restrictions put in place by President Bush, alternative solutions are needed to advance cell- based regenerative strategies.ESCiPS cellsMSC/ASCHSCTissue-specific stem cellsMature lineage cellsDifferentiationEmbryonicAdultFIGURE 8-1 Sc

21、hematic of stem cell organization. eScs, derived from the inner cell mass of the blastocyst, have the highest stem cell capacity (pluripotent) and are the least committed to any tissue lineage. adult stem cells such as HScs and MScs are multipotent and are limited to certain tissue lineages, althoug

22、h they remain in a relatively undifferentiated state at rest. tissue-specific stem cells, such as skin follicular bulge cells, are limited to producing a single cell and tissue type (unipotent), although they retain considerable proliferative capacity to regenerate their specific tissue. Mature line

23、age cells, such as mature epithelium, do not have regenerative potential. iPS cells are mature lineage cells or adult stem cells that have been reprogrammed to a state of relative pluripotency and have much of the same regenerative potential as eScs.SECTION I SURgicaL BaSic PRinciPLeSStem cell capac

24、ityNoneUnipotentMultipotentPluripotent table 8-1 Definitions of Stem Cell-related termsterMDeFINItIONtotipotentability to form all cell types and lineages oforganism (e.g., fertilized egg)Pluripotentability to form all lineages of the body (e.g., embryonic stem cells)Multipotentability of adult stem

25、 cells to form multiple celltypes of one lineage (e.g., mesenchymal stem cells)Unipotentcells form one cell type (e.g., follicular bulge skin stem cells)Reprogrammingdedifferentiation into an embryonic state; can be induced by nuclear transfer, genetic manipulation, viral transduction, and related m

26、ethods180 SeCtION I SURgicaL BaSic PRinciPLeSSomatic Cell Nuclear TransferSomatic cell nuclear transfer (SCNT), also referred to as repro- ductive cloning, involves the transfer of nuclei from postnatal somatic cells into an enucleated ovum. Mitotic divisions of thisgenes had more than a fivefold di

27、fference in expression.16 Fur- thermore, chimeras and progeny mice derived from iPS cells had higher than normal rates of tumor formation than those derived from ESCs, which in some cases may have been caused by reac- tivation of the transfected c-Myc oncogene.17 These key differ- ences need to be e

28、lucidated further to define the safety of iPS cell use in regenerative medicine.cell in culture lead to teration of a blastocyst capable ofyielding a whole new organism. Major advances in this fieldcame in 1997 with the production of a normal sheep (Dolly),9 and this procedure has been reproduced in

29、 other mammals, including mice, cattle, pigs, cats, and dogs.10These experimental studies suggest that a similar approach using SCNT might work in humans for therapeutic cloning, whereby human ESCs produced by this approach could be subsequently differentiated into therapeutically useful cells and t

30、ransplanted back into patients with degenerative diseases. A recent report on primate ESC lines, which were derived from rhesus macaque SCNT blastocysts using adult male skin fibro- blasts as nuclear donors, is an important step in this direction.11 However, similar to human ESCs, SCNT is embroiled

31、inan ethically complex debate about the moral status of created embryo and concerns about obtaining human unfertilized eggs. The technical limitations of this procedure have also dampened early enthusiasm, because several studies have reported less than 10% efficiency in the derivation of SCNT-gener

32、ated ESCs.12 Despite the controversy, SCNT and therapeutic cloning may still be a promising means to generate genetically matched stem cell lines. Long-lasting cell lines from patients with diseases created via SCNT can be used to screen potentially useful drugs or other treatments and may provide r

33、eplacement cells for damaged organs.Induced Pluripotent Stem CellsGiven the complex logistical and ethical considerations sur- rounding donated oocytes for SCNT, alternatives that recapitu- late the reprogramming process in vitro while avoiding the need for oocytes altogether are ultimately preferab

34、le. A groundbreak- ing study in 2006 by Takahashi and Yamanaka13 defined a spe- cific set of transcription factors, Oct4, Sox2, Klf4, and cMyc, that were sufficient to reprogram adult mouse fibroblasts back into a pluripotent state, thus creating ESC-like induced plu- ripotent stem (iPS) cells. Taka

35、hashi and coworkers14 quickly demonstrated that the same combination of transcription factors is sufficient for the pluripotent induction of human cells as well. The ease and reproducibility of generating iPS cells compared with SCNT has raised the hope that iPS cells might fulfill much of the promi

36、se of human ESCs in regenerative medicine.It is widely accepted that mouse and human iPS cells closely resemble molecular and developmental features of blastocyst-derived ESCs.13,15 A number of research groups have shown that iPS cells injected into immunodeficient mice give rise to teratomas compri

37、sing all three embryonic germ layers, similar to ESCs. In addition, when injected into blastocysts, iPS cells generated viable high-contribution chimeras (mice that show major tissue contributions of the injected iPS cells in the host mouse) and contributed to the germline.13,15 Furthermore, using r

38、everse transcription polymerase chain reaction (RT-PCR) assays and immunocytochemistry, studies have shown that iPS cells express key markers of ESCs.However, recent evidence has demonstrated that iPS cells are not identical to ESCs. Global gene expression analysis com- paring iPS cells with human E

39、SCs using microarrays has dem- onstrated that approximately 4% of the over 32,000 analyzedAnother potential complication with teration of iPScells is the use of retroviral and lentiviral vectors to activate the necessary reprogramming transcription factors. Specifically, the viral genome could be in

40、serted near endogenous genes, resulting in gene activation or silencing. This risk of insertional mutagen- esis could lead to uncontrolled modification of th me, with potential development of cancer. Much progress has been made in generating integration-free murine iPS cells, and various recent stud

41、ies using adenoviral, plasmid-based, and recombinant protein-based strategies have reported that viral integration is not required for the reprogramming process.18,19 Even without viral integration, the safety of iPS cells needs to be rigorously tested, because all essential reprogramming factors ar

42、e oncogenesand their overexpression has been linked with cancers.20 The characterization of iPS cells will be enhanced by ongoing improvements in the high-resolution analysis of genomic integ- rity via DNA sequencing technology to identify even minor deletions, inversions, or loss of individual alle

43、les readily.Th ration of iPS cells is likely to create a major impact on regenerative medicine. These iPS cells can be generated from human adipose-derived stem cells (ASCs) in a feeder-free condi- tion with a faster speed and higher efficiency than comparable strategies targeting adult human fibrob

44、lasts.21 Given the ease of isolating a large quantity of ASCs from lipoaspirates, ASCs could be an ideal autologous source of cells for generating individual-specific iPS cells.The therapeutic potential of iPS cells has been demon- strated in several preclinical m s. For example, Wernig and colleagu

45、es have demonstrated that neurons derived from repro- grammed fibroblasts could alleviate the disease phenotype in a rat m of Parkinsons disease.22 Using a humanized sickle cell anemia mouse m , Hanna and associates23 have shown that the genetic defect could be corrected using transplantation of hem

46、atopoietic stem cells (HSCs) derived from iPS cells (derived from fibroblasts of those mice) that had homologous recombina- tion of an intact wild-type -globin gene. Although these early preclinical studies are very promising, iPS cell technology will require further refinement before clinical appli

47、cations can be feasible.FStem CellsAlthough less prominently discussed, fstem cells representanother source for a regenerative building block with clinicalpotential. F stem cells can be derived from f blood, liver, bone marrow, amniotic fluid, and placenta, and are rich in a population of stem cells

48、 that proliferate more rapidly and exhibit greater multipotentiality than adult stem cells.24,25 F stem cells have been found to expand in culture for at least 20 passages, and their capacity for adipogenic, osteo- genic, and chondrogenic differentiation has been demon- strated under appropriate cul

49、ture conditions.26 In addition, transplantation into a xenogeneic sheep m has shown the ability of these cells to engraft and undergo site-specific tissue differentiation.RegeneRative Medicine Chapter 8 181Despite these promising findings, however, significant debate has been raised over the issue o

50、f using cells from fetuses and the attendant risks associated with intrauterine procedures. Nonetheless, f stem cells may still provide a novel means whereby future autogenous in utero cellular and genetic thera- pies can be devised.Adult Stem CellsOnce embryonic development has completed, humans an

51、d other complex organisms lose their cache of embryonic stem cells. During adult life, the regenerative capacity of tissues and organs is maintained by adult stem cells, which reside in mature tissues and in general repositories throughout bone marrow and adipose tissue. Unlike embryonic stem cells

52、and induced plu- ripotent stem cells, adult stem cells are multipotent; they can differentiate into some but not all tissue lineages and are typi- cally confined to a certain tissue type and microenvironment, usually termed a stem cell niche.27 The most studied and best characterized adult stem cell

53、 types is the hematopoietic stem cell, which has served as the experimental paradigm for basic studies into the biology of adult stem cell biology.28 Recently, much insight has been gained into the organization and function of mesenchymal stem cells and adipose stromal cells, which have shown consid

54、erable promise for the field of regenerative medicine.Tissue-Specific Stem CellsGiven the frequent cellular turnover and significant regenerative capacity of epithelial organs such as the cornea, small intestine, and skin,29 it is not surprising that these tissues harbor robust resident stem cell po

55、pulations. However, resident stem cells have also been isolated from organ systems that were thought to have little or no regenerative capacity, such as cardiac tissue30 and neural tissue,31 suggesting that most or all mature mammalian tissues and organs have corresponding stem cell populations that

56、 play some role in local tissue homeostasis and organ regenera- tion. These tissue-specific resident multipotent stem cells are characterized by profound self-renewal capacity, which allows them to maintain lifelong homeostasis of mature tissues in the absence of disease or injury.Although a thoroug

57、h discussion of each tissue-specific stem cell type is beyond the scope of this chapter, a limited description of a few cell types that are most relevant to surgeons is war- ranted. In the skin, stem cells reside in two general niches, along the hair follicles in the bulge region deep to the sebaceo

58、us glands and in the deep interfollicular epidermis.32 The follicular bulge cells proliferate and form the hair shaft as it grows and may contribute to epidermal regeneration after trauma or injury. The deep interfollicular epidermal cells migrate upward to replenish the layers of the epidermis duri

59、ng normal homeostasis of the epidermis, a process that replaces all skin cells every 3 to 4 weeks. In the small intestine, a group of proliferative cells resides at the base of the crypts and send differentiating cells upward to repop- ulate the mature gut epithelium, with rapid turnover every 4 to 5 days. It is clear that intestine-specific stem cells exist, but theIn the hea