Bài 5: Ổ tế bào gốc

Ổ TẾ BÀO GỐC TS. Trần Hồng Diễm PTN Nghiên cứu và Ứng dụng Tế bào gốc Trường Đại học KHTN - Đại học Quốc Gia Tp. HCM 07/11/2015 1. A position or activity that particularly suits somebody's talents and personality or that somebody can make his or her own. 2. An area of the market specializing in one type of product or service. 3. Place in nature: The role of an organism within its natural environment that determines its relations with other organisms and ensures its survival. 4. A recess in a wall, especially one made to hold a statue. 5. Hollow place: any recess or hollow, e.g. in a rock formation. NICHE a e d c b STEM CELL NICHE (Ổ TẾ BÀO GỐC) Định nghĩa •  Ổ TBG là một không gian chuyên biệt trong mô nơi TBG cư ngụ một khoảng thời gian không xác định và tạo nên các TB con trong quá trình tự làm mới •  Ổ TBG là vi môi trường của các TBG, nơi không chỉ hỗ trợ về mặt vật lí mà số phận và sự tăng sinh TBG cũng được điều hoà tại đây •  Ổ cấu thành đơn vị cơ bản của sinh lý mô, sát nhập các tín hiệu, làm trung gian cho đáp ứng của TBG với nhu cầu cơ thể •  Thành phần chính là các TB xung quanh •  ECM •  Trạng thái tự nhiên lí hoá của môi trường (pH, ion, chất chuyển hoá giống ATP, ) Phân loại (theo Benjamin Ohlstein et.al 2004) •  Ổ đơn giản •  Ổ phức tạp •  Ổ dự trữ Ổ TẾ BÀO GỐC (tt) Thành phần cells have been localized in the hair follicle bulge, but it is not known if they interact [25!!,26!]. Stem cell–stem cell communication is likely in the Drosophila testis, where separate stem cells for germ cells and for somatic cyst cells lie in contact [27]. While coordinated activity of these stem cells has yet to be demonstrated at the single cell level, both stem cell types must divide to generate a new spermatogonial cyst containing one germ cell and two cyst cells. It may be that niches should not be thought of as units of stem cell maintenance, but rather as units of production of specific cellular outputs — spermatogonial cysts, ovarian follicles, intestinal villi, etc. If so niches might be expected to contain whatever stem cells and coordination mechanisms are adequate for the job. Seemingly simple niches may exhibit complex temporal behavior. For example, it may be possible to support new stem cells without maintaining any special, pre-existing stromal architecture (‘empty niches’). Local structures to anchor and maintain new stem cells might simply be induced following stem cell arrival. Finally, some tissues may have the capacity to support stem cells without any anatomical specializations beyond a large expanse of basement membrane. The basement membrane of mammalian epidermis or seminiferous tubule may fall in this category. If stem cells can be supported by spatially uniform signals and non-specific stromal contacts alone, it would follow that niches are sometimes unnecessary for stem cell maintenance or else that they can be extra- ordinarily large. Storage niches A potentially different type of niche, the ‘storage niche’, may contain quiescent stem cells (Figure 1c). The bulge region of the mouse hair follicle currently represents the canonical example of such a niche. During most of the hair cycle and in the absence of wounding, transient epithelial stem cells and melanoblasts in the basal ker- atinocyte layer and the hair follicle matrix support ongoing skin and hair production. Reserve stem cells located in the bulge do not divide during this period and hence can preferentially retain labeled DNA, a trait often associated with stem cells. Following wounding or hair cycle completion, however, sub-populations of bulge stem cells activate, exit the niche, and migrate to the site of damage or stem cell loss [25!!]. Melanoblast progeni- tors are also stored in or just below the bulge [26!]. It is not known whether bulge stem cells comprise distinct sub- types or interact with each other and/or partner cells, or even whether they migrate directly out of the niche or only send their daughters to serve as new transient stem cells. Likewise, the adhesive contacts and molecular signals that mediate their responses have not been char- acterized. Storage niches may simply be normal niches that are located in favorable, damage-resistant regions or theymay contain uniquemechanisms to facilitate the safe maintenance of quiescent cells. Programming daughter cells Niches with active stem cells must contain routes for progeny cells to exit lest they burgeon into tumorous nodules. For example, HSC daughters move away from the osteoblasts of the trabecular bone and toward the center of the marrow, while spermatogonia leave the basal layer and migrate toward the lumen of the seminiferous tubule. We consider a cell to have left the niche when it reaches a location that cannot itself support a stem cell because one or more critical adhesive or signaling factors is no longer present. Even before it has done so, the daughter cell may begin to differentiate. Thus, niches are likely to contain specific structural features and The stem cell niche: theme and variations Ohlstein, Kai, Decotto and Spradling 695 Figure 1 Adherens junction Partner cell Stem cell Daughter cell (a) Simple niche (c) Storage niche (b) Complex niche Current Opinion in Cell Biology Proposed niche types. (a) Simple niche. A stem cell (red) is associated with a permanent partner cell (green) via an adherens junction (blue). The stem cells divides asymmetrically to give rise to another stem cell and a differentiating daughter cell (orange). (b) Complex niche. Two (or more) different stem cells (red and pink) are supported by one or more partner cells (green). Their activity is coordinately regulated to generate multiple product cells (orange and yellow) by niche regulatory signals. (c) Storage niche. Quiescent stem cells are maintained in a niche until activated by external signals to divide and migrate (arrows). www.sciencedirect.com Current Opinion in Cell Biology 2004, 16:693–699 Curr Opin Cell Biol. 2004 Dec;16(6):693-9. •  trong đó TBG liên kết với TB cạnh bên bằng khe nối liền (adherent junction), TBG sẽ phân chia bất đối xứng tạo nên 1 TBG khác và 1 TB chị em biệt hoá •  Ổ đơn giản được cho là đóng vai trò chủ yếu đảm bảo nguồn cung cấp TB tiền thân ổn định và lâu dài giúp thay thế TBG bị mất, chẳng hạn: -  Sự thay thế TBG bị mất ở buồng trứng Drosophila bởi phân bào của 1 TBG liền kề duy trì SL TBG invivo -  Ở tinh hoàn chuột rat, khi TB mầm bị mất, ổ TBG của con chuột này hỗ trợ TB ‘donor’ ghép vào và thay thế TBG bị mất Ổ đơn giản •  trong đó hai hay nhiều hơn hai TBG khác nhau được hỗ trợ bởi 1 hay nhiều TB cạnh bên. •  Hoạt động của chúng được đồng điều hoà để tạo nên nhiều TB khác nhau bởi các tín hiệu điều hoà ổ •  Ổ không được xem là đơn vị duy trì TBG mà là đơn vị tạo nên những output TB chuyên biệt như túi nguyên bào tinh, nang buồng trứng, lông nhung ruột, Ổ phức tạp Ổ dự trữ •  TBG ở trạng thái ‘im lặng’ được duy trì trong ổ cho tới khi được hoá hoá bởi tín hiệu ngoại bào để phân chia và di cư •  Có thể hiểu đây là những ổ TBG bình thường định vị ở những vùng ‘an toàn’ có những cơ chế riêng thuận lợi cho sự duy trì an toàn TB ở trạng thái im lặng •  Trong điều kiện bình thường không tổn thương, các TBG ở trạng thái im lặmg không phân chia do đó DNA và các tính trạng được bảo tồn •  Trong điều kiện bị tổn thương, các TBG này sẽ rời khỏi ổ và di cư đến vị trí tổn thương hay nơi TB bị mất •  Ở GĐ ấu trùng, DTC cung cấp vi môi trường hay ‘ổ’ để duy trì GSC •  Trong suốt quá trình phát triển, DTC cần thiết cho GSC tạo nên mô dòng mầm trưởng thành •  Ở cơ thể trưởng thành, DTC giúp duy trì dòng mầm •  DTC sử dụng tín hiệu Notch (bảo tồn ở ĐV đa bào) để duy trì GSC thông qua duy trì chức năng FBF1 và FBF2, ức chế chức năng kích thích sự biệt hoá của gen Gld -> kiểm soát tính tự làm mới của GSC GSC niche C. elegans Drosophila 5. Stem Cell Niches 44 address essential questions regarding how stem cells interact with their surrounding microenvironment. In Drosophila, the ability to generate clones of cells that are genetically distinct from neighboring cells allows both lineage tracing and analysis of the effects of lethal mutations during late stages of the life cycle, when lethality would already have occurred in a entirely mutant animal. Lineage tracing by clonal marking analysis has led to the identifica- tion of GSCs in both the male and the female germ lines in vivo, within their normal environment. These genetically marked GSCs can be observed to continually produce a series of differentiating germ cells. Clonal analysis also allows the generation of mutant GSCs in an otherwise wild-type animal, allowing the analysis of a specific gene’s function on stem cell maintenance, self-renewal, and survival. In Drosophila, both male and female GSCs normally divide with invariant asymmetry, producing precisely one daughter stem cell and one daughter cell that will initiate differentiation. In both the ovary and the testis, GSCs are in intimate contact with surrounding support cells that provide critical self-renewal signals, maintenance signals, or both, thereby constituting a stem cell niche. Oriented division of stem cells is important for placing one daughter cell within the niche while displacing the other daughter cell destined to initiate differentiation outside of the germ-line stem cell niche. GERM-LINE STEM CELL NICHE IN THE DROSOPHILA OVARY The adult Drosophila ovary consists of approximately 15 ovarioles, each with a specialized structure, the germarium, at the most anterior tip (Figure 5-1A). Two to three GSCs lie at the anterior tip of the germarium, close to several groups of differentiated somatic cell types, including the terminal fila- ment, cap cells, and inner germarial sheath cells (Figure 5-1A, Figure 5-1B). When a female GSC divides, the daughter cell that lies closer to the terminal filament and cap cells retains stem cell identity; the daughter cell that is displaced away from the cap cells initiates differentiation as a cystoblast. The cystoblast and its progeny undergo four rounds of cell divi- Figure 5-1. Germ-line stem cell niches in the Drosophila ovary and testis. (A) Schematic of a Drosophila germarium, which houses the germ-line stem cells (GSCs), anterior to the left and posterior to the right. The terminal filament, cap, and inner sheath cells express molecules important for the maintenance and self-renewal of female GSCs and comprise the stem cell niche. GSCs undergo asymmetric cell division, producing one daughter cell that will retain stem cell identity and one daughter cell, a cystoblast, that will initiate differentiation. As these divisions take place, the more mature cysts are displaced toward the posterior of the germarium. Cyst encapsulation by the somatic stem cell (SSC) derivatives occurs in region 2A–2B. Mature encapsulated cysts budding from the germarium make up region 3. (B) In the immunofluorescence image of a Drosophila germarium, germ cells are labeled with an antibody to the germ cell-specific protein, Vasa. Antibodies to the membrane protein a-spectrin label the somatic cells within the germarium, as well as a vesiculated, cytoplasmic ball-shaped structure known as the spectrosome in GSCs (arrow) and cystoblasts. (C) Schematic of the early steps in Drosophila spermatogenesis. GSCs sur- round and are in contact with a cluster of postmitotic, somatic cells known as the apical hub. The hub cells are a primary component of the male GSC niche. Each GSC is surrounded by two somatic stem cells, the cyst progenitor cells. The GSC undergoes asymmetric cell division, generating one daughter cell that will retain stem cell identity and one daughter cell, a gonialblast, which then undergoes four rounds of cell division with incomplete cytokinesis to produce 16 spermatogonia. The gonialblast is surrounded by cyst cells, which ensure spermatogonial differentiation. (D) In the immunofluorescence image of the apical tip of a Drosophila testis, the germ cells are labeled with an antibody to Vasa, and the somatic hub is labeled with an antibody to the membrane-associated protein, Fasciclin III. Eight GSCs (arrowheads) surround the apical hub. Stem Cell Niches , Essensial of stem cell biology (2006), page 43-54 ovary testis R E P O R T S that it remained a cystoblast, two lacZ+ stem cells were present at the tip (Fig. 2D). These stem cells were connected by an elongated fu- some, indicating that they were recently divided sister cells in early interphase (4); the fusome was oriented in an unusual manner, perpendic- ular to the anteriorlposterior (dp) axis (10). These observations suggest a specific model for GSC replacement (Fig. 2E). After one GSC is lost, its neighboring stem cell divides perpen- dicular to d p axis, causing a daughter cell to occupy the environment recently vacated by the departed GSC. For this mechanism to work, the environment at the site of the lost GSC must be capable of programming the incoming cell to become a GSC rather than a cystoblast. Our observations indicate that it is capable of doing so, and hence that GSCs reside in a true stem cell niche. The ability of the ovariole tip to act as a stem cell niche is likely to be biologically im- portant. Females produce eggs for months, de- spite the 4- to 5-week half-life of an individual stem cell (11). To investigate whether stem cell replacement occurs normally, we measured the number of stem cells and somatic niche cells in aging females (Fig. 3). During the first 5 weeks of adult life the average number of GSCs per germarium declined from about 2.5 to 2.0 (Fig. 3A), significantly less than the 50% reduction expected in the absence of replacement (P < 0.01). Replacement stem cells must function efficiently because the rate of stem cell loss does not increase with age (11,12). Some of the ovarioles that did lose a stem cell started with three GSCs, because the number of such ova- rioles declined over the same period. A CB G S C ~ - & h * CPC' IGs7-- c' v B EF\ / GSC A )CB TF 4 i RF ' CPC 7 -&--'"" One of the three somatic cell types, cap cells, interacted with stem cells in a manner that suggested they play a role in niche function. Over the 36-day period, the number of cap cells and GSCs remained closely correlated at about 2.5 cap cells per GSC (Fig. 3A). Moreover, GSCs were observed to always make special contacts with cap cells that characteristically align with the 'dp axis of the ovariole. The GSC's hsome remains adjacent to the GSCI cap cell interface dwing.most of the cell cycle. In contrast, the behavior of inner sheath cells and terminal filament cells did not correlate closely with GSCs. As germaria aged, terminal filament cells decreased in number from an average of 9.2 (3 days) to 5.0 (36 days) (Fig. 3A) and changed from a linear to a ball-like arrangement (Fig. 3, B to D) (19). Likewise, the relative number of inner germarium sheath (IGS) cells and GSCs varied (Fig. 3E). How- ever, the number of IGS cells was closely cor- related with the number of differentiating germ cells (r = 0.88). A functional connection be- tween IGS cells and germ cell cysts has been previously suggested, because ovariole tips that develop without germ cells lack IGS cells (I I). To investigate the role of IGS cells in adult geharia we studied females carrying a hs-barn transgene, whose stem cells can be induced to differentiate (20). Over the course of several days after heat shock, GSCs were lost and all germ line cysts completed development and left the germarium. Such germaria also lost all IGS cells, further indicating that developing germ cells control IGS cell number (Fig. 3, F and G). In contrast, terminal filament and cap cells did not change in the absence of germ cells. Somat- ic cell divisions continued in their vicinity as in germaria that form in the absence of germ cells (21). Despite their presence near the GSC niche, these dividing somatic cells did not be- come GSCs. Because the number of cap cells correlates closely with the number of GSCs, we inves- tigated whether they might function by pref- erentially sending a dpp signal. Suitable an- tibodies to Dpp are unavailable, so we used whole-mount in situ hybridization to deter- mine which cells at the ovariole tip express dpp mRNA. These experiments detected low levels of dpp mRNA in both cap cells and inner sheathcells, as well as higher levels in prefollicle cells farther posterior in the ger- marium. No dpp mRNA was seen in terminal filament cells or in any germ line cells, in- cluding GSCs (Fig. 4A). These results show that cap cells are one of several cell types located near the GSCs that express dpp. Moreover, it does not appear to be the ab- sence of contact with a dpp-expressing cell that causes the posterior stem cell daughter to differentiate as a cystoblast. Our studies suggest a working model for a GSC niche (Fig. 4B). We propose that cap cells are critical to the formation, maintenance, and regulation of the GSC niche. Cap cells and terminal filament cells form a characteristic structure with sufficient internal surface area to Fig. 1. Germarium structure and stem cells. (A) Diagram of a Drosophila germarium in cross section indicating germ Line stem cells (CSCs, red), differentiating germ cells (pink), terminal filament cells (TF, brown), cap cells (CPC, green), inner germarium sheath cells (ICS, or- ange), somatic follicle cells (FC, blue), and fu- somes (yellow). Fusome shape correlates with germ cell stage. (B) Asymmetric location of stem cell and cystoblast relative to somatic cells. C, germ line cyst; CB, cystoblast; EF, elon- gated stem cell fusome; RF, round stem cell fusome. Fig. 2. A niche at the ovariolar tip can replace lost stem cells. (A) Generation of marked shn mutant GSC clones. All cells (ovals) express arm-lacZ marker (red), except shn mutant clones generated by FRT-mediated recombination as shown. (B to D). Cermaria with a recently lost shn GSC, analyzed 7 days after a heat shock to induce recombination, display two CSCs (numbered), indicating replacement. For details of arm-lacZ marker (anti-IacZ, red), germ cell fusomes, and somatic cell membranes (anti-Hts, green), see (78). The Lost stem cell has differentiated into a young 16-cell cyst (B and C, dotted ovals), but is still a cystoblast (large dotted circle) in (D), indicating a recent loss. A new cell (2) occupies the position of the lost GSC and is still connected to the remaining wild-type GSC (1) by an extended fusome. (E) An explanatory model for GSC replacement. A GSC differentiates and moves away from the cap cells (Left). The other GSC divides perpendicular to the alp axis (center). Both daughters become GSCs, whereas the lost CSC is now a four-cell cyst (right). Bar (B), 10 pm. www.sciencemag.org SCIENCE VOL 290 13 OCTOBER 2000 R E P O R T S that it remained a cystoblast, two lacZ+ stem cells were present at the tip (Fig. 2D). These stem cells were connected by an elongated fu- some, indicating that they were recently divided sister cells in early interphase (4); the fusome was oriented in an unusual manner, perpendic- ular to the anteriorlposterior (dp) axis (10). These observations suggest a specific model for GSC replacement (Fig. 2E). After one GSC is lost, its neighboring stem cell divides perpen- dicular to d p axis, causing a daughter cell to occupy the env