HEP(2006)174:249–282
?Springer-V erlag Berlin Heidelberg2006
Mesenchymal Stem Cells:Isolation,In Vitro Expansion and Characterization
N.Beyer Nardi(u)·L.da Silva Meirelles
Genetics Department,Universidade Federal do Rio Grande do Sul,Av Bento Gon?alves 9500,Porto Alegre RS,CEP91540–000,Brazil
nardi@ufrgs.br
1Introduction (250)
2The Identity of the Mesenchymal Stem Cell (251)
2.1The Colony-Forming Unit-Fibroblast (251)
2.2The Bone Marrow Stroma (252)
2.3The Mesenchymal Stem Cell (253)
2.4Other Cell Populations Related to the Mesenchymal Stem Cell (254)
3Distribution of the Mesenchymal Stem Cell (255)
4Isolation and Culture of Mesenchymal Stem Cells (256)
5Homing and Engraftment of Transplanted Mesenchymal Stem Cells (259)
6Characterization of Mesenchymal Stem Cells (260)
7Differentiation of Mesenchymal Stem Cells (262)
8Applications of Mesenchymal Stem Cells in Cell and Gene Therapy (263)
8.1Study of Cancer Biology (264)
8.2Cell Therapy (264)
8.2.1Fibrosis (265)
8.2.2Cardiovasculogenesis (265)
8.2.3Arteriogenic Effects (266)
8.2.4Immunosuppressive Effects (267)
8.3Mesenchymal Stem Cells and Tissue Engineering (267)
8.4Genetic Therapy (268)
8.4.1Correction of Genetic Disorders (269)
8.4.2Cancer Suppression (270)
9Pharmacologic Aspects of Mesenchymal Stem Cell Biology (271)
10Conclusions (272)
References (274)
250N.Beyer Nardi·L.da Silva Meirelles Abstract Mesenchymal stem cells(MSC),one type of adult stem cell,are easy to isolate, culture,and manipulate in ex vivo culture.These cells have great plasticity and the potential for therapeutic applications,but their properties are poorly understood.MSCs can be found in bone marrow and in many other tissues,and these cells are generally identi?ed through a combination of poorly de?ned physical,phenotypic,and functional properties; consequently,multiple names have been given to these cell populations.Murine MSCs have been directly applied to a wide range of murine models of diseases,where they can act as therapeutic agents per se,or as vehicles for the delivery of therapeutic genes.In addition to their systemic engraftment capabilities,MSCs show great potential for the replacement of damaged tissues such as bone,cartilage,tendon,and ligament.Their pharmacological importance is related to four points:MSCs secrete biologically important molecules,express speci?c receptors,can be genetically manipulated,and are susceptible to molecules that modify their natural behavior.Due to their low frequency and the lack of knowledge on cell surface markers and their location of origin,most information concerning MSCs is derived from in vitro studies.The search for the identity of the mesenchymal stem cell has depended mainly on three culture systems:the CFU-F assay,the analysis of bone marrow stroma,and the cultivation of mesenchymal stem cell lines.Other cell populations,more or less related to the MSC,have also been described.Isolation and culture conditions used to expand these cells rely on the ability of MSCs,although variable,to adhere to plastic surfaces.Whether these conditions selectively favor the expansion of different bone marrow precursors or cause similar cell populations to acquire different phenotypes is not clear. The cell populations could also represent different points of a hierarchy or a continuum of differentiation.These issues reinforce the urgent need for a more comprehensive view of the mesenchymal stem cell identity and characteristics.
Keywords Mesenchymal stem cell·Bone marrow stroma·Differentiation·
Stem cell niche·Cell therapy·Genetic therapy
1
Introduction
Stem cells present in early embryonic stages are pluripotent and can generate all of the cell types found in adult organisms,whereas,adult stem cells exhibit a continuum of plasticity or multipotency.In adult humans,the?rst and one of the best-known stem cells to be described is the hematopoietic stem cell(HSC).A great variety of other stem/precursor cell types have also been described,but much less is known about their origin and maintenance in vivo as organ-speci?c stem cell pools(Nardi2005).
The mesenchymal stem cell(MSC)is one of the most interesting of the adult stem cell types.These cells are easily isolated,cultured,and manipulated ex vivo.MSCs exhibit great plasticity and harbor the potential for therapeutic applications,but these cells are poorly de?ned.This has led to a heterogeneity of names and phenotypes ascribed by different groups to this cell population. MSCs are present in the bone marrow and in many other tissues,and these cells are presently identi?ed through a combination of poorly de?ned physi-cal,phenotypic,and functional properties.A number of recent reviews have adequately described the nature of MSCs(Short et al.2003;Zipori2004;Barry
Mesenchymal Stem Cells:Isolation,In Vitro Expansion and Characterization251 and Murphy2004;Kassem et al.2004;Baksh et al.2004;Javazon et al.2004), and the focus of this review will be to describe experimental approaches for their isolation,in vitro expansion,and characterization.We will also discuss the cellular therapeutic potentials of MSCs and place a special emphasis on the pharmacological prospects of these cells in vitro and in vivo.
2
The Identity of the Mesenchymal Stem Cell
For most cells present in adult organisms,mitosis is accompanied by differ-entiation.Stem cells are de?ned as those cells with the ability to proliferate without differentiating.At the moment,the very existence of a mesenchymal stem cell in vivo is not completely understood,since it is based on indirect evidence derived mainly from the in vitro cultivation of bone marrow and other tissues.This is true,but only to a point,because most types of adult stem cells can only be identi?ed after isolation,which can then be examined through in vitro or in vivo assays to determine if they have the two main characteristics of stem cells:the ability to proliferate and to differentiate into mature cell types.Most of the information for MSCs,which are present at a low frequency,are derived from in vitro studies,due to a lack of information with respect to speci?c surface markers and their location in vivo.In vitro studies, by their very nature,may,however,introduce experimental artifacts(Javazon et al.2004).This possibility is clearly described in several studies,including one reported by Rombouts and Ploemacher(2003),who compared the homing abilities of primary and culture-expanded MSCs in a syngeneic mouse model. Uncultured MSCs demonstrated highly ef?cient homing to bone marrow,but the infusion of immortalized multipotent syngeneic stromal cells,or even pri-mary MSCs that had been cultured for only24h,were rarely if ever seen in the lymphohematopoietic organs.Murine MSCs were also reported to have a de?cient capacity to home to bone marrow by Anjos-Afonso et al.(2004).
The identi?cation of the mesenchymal stem cell has thus far depended on in vitro culture systems,which have provided very heterogeneous information and made the characterization of MSCs even more dif?cult.Three in vitro systems are generally employed to examine these cells:the CFU-F assay,the analysis of the bone marrow stroma,and the cultivation of mesenchymal stem cell lines.Other cell populations,more or less related to the MSC,have also been described.
2.1
The Colony-Forming Unit-Fibroblast
The?rst direct evidence that nonhematopoietic,mesenchymal precursor cells were present in the bone marrow originated from the work conducted in
252N.Beyer Nardi·L.da Silva Meirelles Moscow during the1960s and1970s of Friedenstein and colleagues(reviewed in
Phinney2002).These pioneering experiments involved the incubation of bone
marrow samples in tissue culture?asks.The presence of an adherent fraction
could be seen within a few days,which proved highly heterogeneous.Around
the3rd–5th days,individual foci of two to four?broblasts were observed among
the histiocytes and mononuclear cells,which could differentiate into cells that
could form small deposits of bone or cartilage(Friedenstein et al.1976).These
cells were termed colony forming unit-?broblasts or CFU-F.
During the1980s,several studies showed that cells isolated by the Frieden-
stein method were multipotent and could differentiate into osteoblasts,chon-
droblasts,adipocytes,and even myoblasts(reviewed in Prockop1997).The
frequency of CFU-F in bone marrow suspensions is very different among
species,and the results are in?uenced by the culture conditions(reviewed in
Short et al.2003).Growth factors stimulating the proliferation of CFU-F in-
clude platelet-derived growth factor(PDGF),epidermal growth factor(EGF),
basic?broblast growth factor,transforming growth factor-β,and insulin-like growth factor-1(Gronthos and Simmons1995;Kuznetsov et al.1997a;Baddoo
et al.2003;Bianchi et al.2003).In contrast,cytokines such as interleukin4
(IL-4)and interferon-alpha can inhibit the establishment of CFU-F(Wang
et al.1990;Gronthos and Simmons1995).The formation of CFU-F has been
considered indicative of mesenchymal stem cells,but a direct relationship be-
tween the two has not been clearly established,probably because of the great
heterogeneity in morphology,cell size and differentiation potential observed
among species and between colonies(Javazon et al.2004).
2.2
The Bone Marrow Stroma
Stromal cells,along with extracellular matrix(ECM)components and soluble
regulatory factors,have until recently been thought of as secondary compo-
nents of a microenvironment required for sustained hematopoiesis(Nardi and
Alfonso1999).The stroma,studied both in vitro and in vivo,is composed
of a very heterogeneous population of cells,which includes macrophages,?-
broblasts,adipocytes,and endothelial cells(Dexter et al.1976;Ogawa1993).
Adventitial reticular cells branch through the medullary cavity and provide
a reticular network that supports hematopoietic cells.Marrow adipocytes con-
trol hematopoietic volume,such that impaired hematopoiesis is associated
with increased accumulation of fat inclusions,and accelerated hematopoiesis
is associated with loss of fat vacuoles.These processes determine the space
available for hematopoietic cells(Tavassoli1984).Adipocytes may also act as
a reservoir for lipids needed during proliferation.Macrophages are important
in the clean-up of ineffective erythropoiesis and in the removal of the nuclear
pole that is produced during this process.
Mesenchymal Stem Cells:Isolation,In Vitro Expansion and Characterization253 Stromal cells produce ECM components and both soluble and membrane-associated growth factors to form a dynamic structure that plays an active role in hematopoiesis,i.e.,the hematopoietic stem cell niche.Matrix pro-teins in this microenvironment include?bronectin,collagen,vitronectin,and tenascin,and some of the most relevant soluble factors include stem cell fac-tor(SCF),granulocyte-macrophage colony-stimulating factor(GM-CSF)and the granulocyte colony-stimulating factor(G-CSF).Representative adhesion molecules include members of the integrin superfamily(VLA-1,VLA-2,VLA-3, VLA-4,VLA-5,VLA-6)(reviewed in Whetton and Grahan1999).Physical con-tact among the stromal cells is important for the regulation of this microen-vironment.Although it has been shown that contact is not fundamental for the hematopoietic process to occur(V erfaillie1992),it seems to be related to the quality of the hematopoietic cells produced(Breems et al.1998).The stromal compartment has implications for human health,since abnormalities in the stromal compartment may represent a possible mechanism implicated in aplastic anemia(Kojima1998)and in the abnormal behavior of Ph+cells in chronic myeloid leukemia(Cordero et al.2004).
Based on the well-established generation of multiple types of mesenchymal cell from bone marrow,stromal stem cells were additionally proposed to exist by Owen(1985).Analogous to the hematopoietic system,they proposed that stromal stem cells reside in the bone marrow in their own niche,where the cells were able to self-renew and generate mature conjunctive/stromal cell types.The identity of this stem cell—which is now almost universally termed mesenchymal stem cell—is still,as stated above,poorly understood.
2.3
The Mesenchymal Stem Cell
The lack of consensus about the proper nomenclature needed to describe these cells has resulted in an incorrect,but synonymous use of the terms“marrow stromal cell”and“mesenchymal stem cell.”Actually,stromal cells encompass all cells present in the bone marrow that are not part of the hematopoietic system.MSCs,on the other hand,correspond to that rare cell population that can form other MSCs and generate mature cells of mesenchymal tissues. Protocols involving the isolation of bone marrow cells based on their adherence to plastic surfaces result in the immediate establishment of stromal cell cultures, and not of MSC cultures.A more adequate term for the large number of cell types with the potential to differentiate into mesenchymal tissues would be “mesenchymal progenitor cell”(MPC),which would include cell types from a hierarchy immediately above the pluripotent MSC but intermediate to that represented by mature mesenchymal cell types.
Another point of debate is the fact that the HSC is itself of mesodermic origin,hence a type of MSC.For this reason,some authors prefer the term “nonhematopoietic mesenchymal stem cell.”The fact that these cells,which are
254N.Beyer Nardi·L.da Silva Meirelles described below,may have alternative differentiation pathways that go beyond the normal limits of mesoderm and ectoderm formation renders the term “mesenchymal”inadequate.Probably,the best nomenclature to de?ne this cell type would be“adult nonhematopoietic stem cell,”followed by“plastic-adherent,bone-marrow derived stem cells”.All these concepts,however,are already included when the term“mesenchymal stem cell”is used,and there is
a tendency to accept this terminology,even though it is inadequate.
2.4
Other Cell Populations Related to the Mesenchymal Stem Cell
In addition to the heterogeneity,which characterizes MSC cultures established from various species or in different laboratories,some groups have described cell populations that are very similar to MSCs,but which have a different nomenclature.Bone marrow stromal(stem)cells(BMSSCs),stromal precur-sor cells(SPCs),and recycling stem cells(RS-1,RS-2)are some of these varia-tions(Baksh et al.2004and references therein).More recently,D’Ippolito et al. (2004)described the marrow-isolated adult multilineage inducible(MIAMI) cells which,although isolated from humans,can proliferate extensively with-out showing signs of senescence or loss of differentiation potential(which,as described below,is not usual for human cells).These cells may represent a more primitive subset of bone marrow stem cells.Higher proliferative and differen-tiation potential has also been reported for the multipotential adult progenitor cell(MAPC)described by Catherine V erfaillie’s group(Reyes et al.2001;Reyes and V erfaillie2001).Y oung et al.(2001)described human reserve pluripotent mesenchymal stem cells,present in the connective tissues of skeletal muscle and dermis.
Isolation and culture conditions used by the different groups are also vari-able,and probably represent the main factor responsible for the phenotype and function of the resulting cell populations.Whether these conditions se-lectively favor the expansion of different bone marrow precursors or cause similar cell populations to acquire different phenotypes is not clear.The cell populations could also represent different points in a hierarchy(Caplan1994), and some studies suggest that this second alternative might be more realistic. Lodie et al.(2002),for instance,systematically compared different protocols used to isolate/expand human bone marrow adherent cells and concluded that the cell populations isolated by these various techniques are virtually indistinguishable.We have recently observed that the maintenance of MSC lines generated according to established protocols(Meirelles and Nardi2003) but grown under MAPC conditions for4weeks induced changes in the im-munophenotypic pro?le of the cells to become more similar to that of MAPCs (N.Nardi and L.Meirelles,unpublished results).In either case and as reported for hematopoietic stem cells(Pranke et al.2001),these results demonstrate that the mesenchymal stem cell compartment is heterogeneous and that cultivation
Mesenchymal Stem Cells:Isolation,In Vitro Expansion and Characterization255 conditions can alter some of their basic properties.These points reinforce the urgent need for a more comprehensive view of the mesenchymal stem cell identity and its characteristics.
3
Distribution of the Mesenchymal Stem Cell
Although very poorly understood,the interaction of MSCs with their niche is as essential for their existence and function as it is for any other of the adult stem cells(Watt and Hogan2000;Fuchs et al.2004).The primary source of MSCs in adult individuals is the bone marrow,where they are immersed in the stroma(Pittenger et al.1999).They are present at a low frequency in bone marrow,and recent studies employing the CFU-F assay suggest that in humans there is one MSC per34,000nucleated cells(Wexler et al.2003).In mice,the frequency was estimated to be one for11,300–27,000nucleated cells (Meirelles and Nardi2003).Once again,the heterogeneity of this microen-vironment hampers the unraveling of its components and their relationship, and basic questions remain unanswered.What is the niche for the MSC?Do hematopoietic and mesenchymal stem cells share the same niche and exchange signals to drive proliferation and differentiation?
MSCs have been found in several other tissues and in ontogeny(Table1).In mice,they were isolated from the brain,thymus,liver,spleen,kidney,muscle, and lungs of adult mice(L.Meirelles and N.Nardi,unpublished results),and other MSC-related populations such as MAPCs have been observed in different organs as well(Jiang et al.2002).This distribution could be explained by different scenarios:(a)adult tissues contain independent reservoirs of similar Table1Distribution of MSCs in different organs/tissues and ontogeny stages
Site Species Ontogeny stage Reference
Adipose tissue Human Post-natal Zuk et al.2001,2002
Adipose tissue Mouse Post-natal Safford et al.2002
Pancreas Human Fetal Hu et al.2003
Bone marrow Human Fetal Campagnoli et al.2001
Liver Human Fetal Campagnoli et al.2001
Blood Human Fetal Campagnoli et al.2001
Tendon Mouse Postnatal Salingcarnboriboon et al.2003 Synovial membrane Mouse Postnatal de Bari et al.2003
Amniotic liquid Human Fetal in’t Anker et al.2003
Peripheral blood Human Postnatal Zvai?er et al.2000;Kuwana et al.2003 Umbilical cord blood Human Fetal/postnatal Alfonso et al.2000
256N.Beyer Nardi·L.da Silva Meirelles stem cells,whose characteristic traits are determined by signals released by each niche;(b)MSCs exist as a reservoir in one speci?c location,from which they circulate through the organism to colonize different tissues/organs;or (c)MSCs originate from cell populations belonging to blood vessels,and are, as a consequence,present through the whole organism.Since Bianco and Cossu(1999)suggested that MSCs originate from marrow pericytes,this third possibility has received experimental support(reviewed in Short et al.2003). The issue is,however,still unclear.
4
Isolation and Culture of Mesenchymal Stem Cells
Few adult stem cell populations can be unequivocally identi?ed,and isolation of these cells requires in vitro or in vivo experimentation and characterization based on immunophenotypic or functional traits.Hematopoietic stem cells, for instance,can be enriched through the selection of cells expressing surface markers such as CD34in humans and Sca-1in mice,or by their ability to ex-clude the DNA-binding dye Hoechst33342(Goodell et al.1996).Mesenchymal stem cells lack clearly de?ned surface markers,so that the most widely used approach to isolate them relies on their ability to adhere to plastic surfaces (Wakitani et al.1995;MacKay et al.1998;Makino et al.1999;Muraglia et al. 2000).
For the selective isolation of bone marrow MSCs,total cells are washed, counted,resuspended in culture medium,and plated in six-well tissue culture dishes at approximately1.94×106cells/cm2.Nonadherent cells are removed 24–72h later by changing the medium.After1week,a heterogeneous culture develops,which is generally referred to as bone marrow stroma(Fig.1a). Maintenance of the culture with a twice-weekly medium change and removal of nonadherent cells results,after2or3weeks,in a relatively homogeneous culture of morphologically and immunophenotypically similar mesenchymal stem cells(Fig.1b,c).Our experience shows that the identi?cation of MSCs depends on the availability of a good inverted microscope with phase contrast, since they are dif?cult to visualize otherwise.
Cultures can be maintained for variable periods,depending on the species and organ of origin,by passaging and subculturing the adherent cells which are detached by trypsinization.Although Dulbecco’s modi?ed Eagle’s medium (DMEM)is frequently employed for the culture of MSCs,other media have also been shown to be appropriate(reviewed by Otto and Rao2004).The presence of HEPES buffer is also important in our experience.
In an attempt to further enrich the frequency of MSCs in the initial cell population,other methods have been developed,such as the immunodepletion of hematopoietic contaminants identi?ed,for instance,by the molecules CD34, CD45,and CD11b(Kopen et al.1999;Badoo et al.2003;Ortiz et al.2003).
Mesenchymal Stem Cells:Isolation,In Vitro Expansion and Characterization257
Fig.1a BALB/c bone marrow cells cultured for1week in DMEM with10%FCS generate het-erogeneous cell populations,referred to as bone marrow stromal cells(×100).Maintenance of the adherent cell population results in a homogeneous culture of mesenchymal stem cells of a?at-type morphology(b,×400;c,×100)
Other techniques involved cell size-based enrichment,involving the?ltration of bone marrow cells through a3-μm seive(Hung et al.2002;Tuli et al.2003), or changing plating densities(Colter et al.2000;Sekiya et al.2002).Since none of these approaches results in the establishment of homogeneous cell cultures, the development of ef?cient—and particularly reproducible—methods for the
258N.Beyer Nardi·L.da Silva Meirelles isolation and expansion of MSCs remains an important goal of this research
?eld.It is possible that only when the true origin and nature of MSCs is better
understood will we be able to con?dently work with them in vitro.
Following removal of nonadherent cells1–4days after the establishment
of the culture,cells are maintained with periodic passages until a relatively
homogeneous population is established.Culture media may vary,but the
most frequently used are Dulbecco’s modi?ed Eagle’s medium(DMEM)and α-minimum essential medium(reviewed in Otto and Rao2004).The batch of fetal calf serum employed to cultivate these cells may introduce phenotypic
variations,which show that unknown factors in?uence the selection and ex-
pansion of these cells.The addition of speci?c growth factors is also important
in de?ning the?nal characteristics of MSC cultures,and these are probably
the main reasons for the heterogeneity observed in the mesenchymal stem
cell types described in the literature.The growth of murine MAPCs,for in-
stance,depends on the supplementation of leukemia inhibitory factor(LIF)
and the use of?bronectin-coated surfaces(Jiang et al.2002).The persistence
of hematopoietic contaminants,shown by the presence of CD45+and CD11b+
cells in the cultures,has also been reported(Phinney et al.1999).
Ideal culture conditions would maintain mesenchymal stem cells with(a)
phenotypic and functional characteristics similar to those exhibited in their
original niche,(b)inde?nite proliferation,and(c)a capacity to differentiate
into multiple lineages.Since the in situ characteristics of MSCs are not known,
efforts have concentrated in the last two objectives.The self-renewal potential
of MSCs is not de?nitely established and can vary greatly according to the
methodology used and the species(Bianco et al.2001),but cells can be expected
to expand for at least40population doublings(PDs)before their growth
rate decreases signi?cantly,as seen with human MSCs(Bruder et al.1997).
Supplementation of growth factors can also modify these results.Fibroblast
growth factor-2(FGF-2),for instance,was shown to increase the lifespan of
human MSCs to more than70PDs(Bianchi et al.2003).Murine MSCs,on
the other hand,show apparently unlimited in vitro growth capacity(Meirelles
and Nardi2003and unpublished observations).The high self-renewal capacity
shown by murine MSCs without evidence of replicative senescence is probably
related to that of rat oligodendrocyte precursor cells(Tang et al.2001).
Cell seeding density may also in?uence the expansion capacity of mes-
enchymal stem cells.Human MSCs,for instance,expand to much higher PDs
when plated at low density than at high density,with an increase of total cells
from60-to2,000-fold(Colter et al.2000).On the other hand,the establish-
ment of long-term cultures of murine mesenchymal stem cells is dependent
on a minimal cell density of2×106bone marrow cells/cm2(Meirelles and
Nardi2003).Long-term culture and high cell density are also determinants of
loss of differentiation potential for human cells,another indication that the
conditions for the in vitro maintenance of MSCs differ from those provided by
their natural microenvironment.
Mesenchymal Stem Cells:Isolation,In Vitro Expansion and Characterization259 5
Homing and Engraftment of Transplanted Mesenchymal Stem Cells
In vivo tracking of implanted MSCs is very important,because the success of cell and gene therapy protocols with these cells depends on their engraftment abilities.The analysis of MSC engraftment is also related to the exact nature of the grafted cells.Heterogeneous populations of adherent cells derived from mouse bone marrow engraft in multiple organs after systemic infusion(Pereira et al.1995,1998),and although there is strong evidence showing that the grafted cells are comprised of mainly MSCs,engraftment of other cell types can not be excluded.
More recently,studies using a well characterized and relatively homoge-neous cell population comprised of murine bone marrow devoid of hematopoi-etic cells,were consistent with a successful in vivo engraftment of candidate MSCs in the central nervous system(Kopen et al.1999;McBride et al.2003). This cell population,in contrast to other murine MSCs,was not expandable in vitro(Baddoo et al.2003;Meirelles and Nardi2003;Gojo et al.2003;Fang et al. 2004),and interspecies experiments were used to study MSC systemic engraft-ment in mice.For instance,when human MSCs were injected intraperitoneally into13-day-old mouse embryos in uterus,multiorgan engraftment was de-tected8weeks after birth by real-time PCR(McBride et al.2003).The sites analyzed included femur,heart,brain,liver,kidney,spleen,and lungs,where the highest level of human DNA was detected.
When cells obtained from bleomycin-resistant BALB/c mice,essentially as described by Kopen et al.(1999),were injected systemically into bleomycin-sensitive C57BL/6mice,engraftment in the lungs was considerably higher in animals with lung injury than in animals without injury.Tissue damage, therefore,enhances MSC engraftment(Ortiz et al.2003);however,murine MSC engraftment has also been demonstrated in noninjured animals.Gojo et al.(2003)reported the engraftment of in vitro-proliferative murine MSCs in the heart,lung,spleen,stomach,small intestine,and skeletal muscle of non-injured mice,where they differentiated locally into cardiomyocytes,vascular endothelial cells,and possibly vascular luminal cells.Selective sorting of an adherent fraction of passage two or three bone marrow cultures yielded non-hematopoietic cells that engrafted into several organs after systemic infusion (Anjos-Afonso et al.2004).Engraftment in some organs was infrequent(brain, bone marrow),but in others(liver,lung,kidney),it exhibited higher levels of engrafted MSCs,and the presence of donor cells in circulating blood was also observed.
The deposition of MSCs in the lungs may represent a signi?cant hurdle for engraftment therapies that employ systemic delivery of MSCs.This approach caused some animals to develop?brosis and subsequent breathing dif?culties. Gao et al.(2001)observed this phenomenon following systemic infusion of MSCs in rats,perhaps because the MSC diameter was larger than that of lung
260N.Beyer Nardi·L.da Silva Meirelles capillaries(20–24μm vs10–15μm,respectively).The use of the vasodilator sodium nitroprusside at the time of injection,however,reduced entrapment of MSCs in the lungs.Murine MSCs,when detached from the dish,are also 20–25μm wide(L.Meirelles,unpublished results),and if administered in large doses are likely to be trapped in lung capillaries before reaching important organs such as the brain and bone marrow.
In2002,two groups(Ying et al.2002;Terada et al.2002)showed that stem cells can fuse with other cells in vitro and can acquire the characteristics of these cells,thus raising the possibility that the stem cell contribution to target tissues might be due to cell fusion rather than to(trans)differentiation.Fusion was also demonstrated between MSCs and epithelial cells in vitro(Spees et al. 2002);however,chromosomal analysis of xenografts indicate that fusion is not the principle mechanism responsible for the MSC contribution to multiple tissues in vivo(Pochampally et al.2004;Sato et al.2005).In a study describing the role of MSCs in the generation of gastric cancer in a mouse model,for example,it was observed that the percentage of tetraploid cells in affected and unaffected animals remained at the same levels(Houghton et al.2004;see Sect.8.1for further information).
While the experimental study of MSC engraftment in humans is elusive, a recent paper describing microchimerism,possibly due to circulating fetal mesenchymal stem cells in pregnancy,in bone marrow and bone of women decades after giving birth to male fetuses has provided some insights into human MSC engraftment properties in vivo(O’Donoghue et al.2004).A com-bined approach including immunocytochemistry,FISH,and PCR using rib sections and cultured MSCs derived from rib bone marrow was used.The results strongly suggest that circulating MSCs present in fetal blood(Campag-noli et al.2001)crossed the placenta during pregnancy,entered the maternal circulation(O’Donoghue et al.2003)and grafted with maternal bone and bone marrow.Khosrotehrani et al.(2004)also reported the detection of grafted male fetal cells in thyroid,cervix,intestine,liver,and lymph nodes when analyzing biopsy material of women who have had male pregnancies.The male cells were shown to express liver,hematopoietic,or epithelial markers,indicating tissue-speci?c incorporation.
Although fetal stem cells entering the maternal circulation include other cell populations,the results of experiments in animals suggest that MSCs are the main cell type that can engraft in maternal tissues.Human adult MSCs, therefore,can be expected to have multisite engraftment capabilities as well.
6
Characterization of Mesenchymal Stem Cells
Cultured MSCs have been extensively analyzed both morphologically and with respect to surface and molecular markers.None of these characteristics,how-
Mesenchymal Stem Cells:Isolation,In Vitro Expansion and Characterization261 ever,is speci?c enough to adequately de?ne this cell type,and mesenchymal stem cells are still operationally de?ned by the ultimate criteria used for iden-tifying stem cells:prolonged proliferation and the potential to originate differ-entiated cell types.To abandon the term“operational MSC,”it is necessary to show that these cells contribute to the formation of mesenchymal tissues after in vivo infusion(V erfaillie2002).
A great number of surface markers have been described for committed mesenchymal progenitors(Otto and Rao2004),and Deans and Moseley(2000) have compiled a long list of candidate markers,including CD44,CD29,and CD90,to de?ne human MSCs.The expression of CD34is not clearly de?ned in murine MSCs,but the marker is known to be absent from human and rat cells.More speci?c antigens such as Stro-1,SH2,SH3,and SH4are also important markers for MSCs(reviewed in Barry and Murphy2004),which are also positive for MHC-1and Sca-1.None of these markers,however,seems to be a reliable parameter for the analysis of culture purity,since on the one hand even long-term cultures may exhibit some heterogeneity(maybe due to cell cycle-related marker expression)and,on the other,functionally different cultures may have similar immunophenotypic pro?les.
Relatively little attention has been given to the morphology of the MSCs originating in culture.Two types of morphology can be observed—large,?at cells or elongated,?broblastoid cells.The derivation of two types of adherent cell cultures from cord blood has already been pointed out by our group (Alfonso et al.2000).The functional signi?cance of these differences remains to be established.
In an earlier report(Meirelles and Nardi2003),we described the isolation and long-term culture of murine MSCs without the need for any other medium supplementation than fetal calf serum.The cells exhibited a constant?at-type morphology(see Fig.1),even when originating from other tissues such as spleen,lungs and brain(not published).In most publications,it is dif?cult to adequately assess cell morphology,but a review of the literature shows that in many cases the cells maintained a?attened shape,while others exhibited an elongated,?broblastic phenotype(Table2).The morphology of MAPCs also seems to be relatively?at(Reyes et al.2001).
Still little is known about the pro?le of gene expression in mesenchymal stem cells.Tremain et al.(2001),in a study that also emphasized the heterogeneity of MSC cultures,reported over2,000expressed transcripts in a clone that originated from a stromal cell culture.In two recent studies,gene expression of bone marrow(Silva et al.2003)and cord blood-derived MSCs(Panepucci et al.2004;Jeong et al.2005)was analyzed by serial analysis of gene expression (SAGE).A great number of genes were identi?ed in the cultured cells,and an important contribution of extracellular protein products,adhesion molecules, cell motility,TGF-beta signaling,growth factor receptors,DNA repair,protein folding,and ubiquination as part of their transcriptome was observed.
262N.Beyer Nardi·L.da Silva Meirelles Table2Reports of?at or elongated/?broblastic morphology of cultured mesenchymal stem cells from different origins
Morphology Species Reference
Flat Mouse Meirelles and Nardi2003(Fig.2)
Human Azizi et al.1998(Fig.2a)
Human D’Ippolito et al.2004(Fig.1)
Human Gronthos et al.2003(Fig.2G)
Human Stute et al.2004(Fig.6B)
Human Hung et al.2004(Fig.1)
Rat Azizi et al.1998(Fig.2c)
Rat Davani et al.2003(Fig.1A)
Rat Kobayashi et al.2004(Fig.1)
Fibroblastic Human Azizi et al.1998(Fig.2b)
Human Ko?et al.2000(Fig.1)
Human Campagnoli et al.2001(Fig.1)
Human Pittenger et al.1999(Fig.1)
Human Pittenger and Martin2004(Fig.1)
Other Human Seshi et al.2000(Fig.1)
7
Differentiation of Mesenchymal Stem Cells
Although considered nondifferentiated cells,MSCs are nevertheless capable of performing at least one specialized function:they support hematopoiesis. This function is generally attributed to the bone marrow stroma,which is frequently confused with MSCs.Apparently homogeneous cultures of MSCs support hematopoietic stem cells with greater ef?ciency than conventionally established bone marrow stroma(Meirelles and Nardi2003).
The in vitro differentiation of MSCs into several lineages is easily achieved. Representative examples of the protocols employed in a number of studies are shown in Table3.Determination of the phenotype of differentiated cells depends on morphological,immunophenotypic,and functional criteria.The differentiation of osteoblasts,for instance,is determined by upregulation of alkaline phosphatase activity and deposition of a mineralized extracellular matrix in the culture plates that can be detected with Alizarin Red or other stains.Adipocytes are easily identi?ed by their morphology and staining with Oil Red O.For identi?cation of myocytes or neuronal cells,immunocytochem-istry is performed with antibodies speci?c for antigens such as myosin and dystrophin,or Tau and GFAP,respectively.
Mesenchymal Stem Cells:Isolation,In Vitro Expansion and Characterization263 Table3Examples of the culture protocols used for inducing in vitro differentiation of MSCs Tissue Species Culture medium complement Reference
Bone Mouse10–8M dexamethasone,5μg/ml ascorbic
acid2-phosphate and10mMβ-glycero-
phosphate Meirelles and Nardi 2003
Cartilage Human Transforming growth factor-β3in serum-
free medium,added to three-dimensional
cultures
Pittenger et al.1999
Fat Human1-methyl-3-isobutylxanthine,dexam-
ethasone,insulin,and indomethacin
Pittenger et al.1999
Mouse108M dexamethasone and5μg/ml insulin Meirelles and Nardi
2003
Neuron-like Human Isobutylmethylxanthine and dibutyryl
cyclic AMP
Deng et al.2001
Mouse50ng/ml of basic?broblast growth factor (bFGF)and20ng/ml of epidermal growth
factor(EGF)Anjos-Afonso et al. 2004
Muscle Mouse Amphotericin B Phinney et al.1999 Rat5-azacytidine Wakitani et al.1995
Pig5-azacytidine Moscoso et al.2005 In vitro cultured MSCs show great heterogeneity in their differentiation potential.Although the analysis of established MSC cultures show them to be pluripotent,with a tri-lineage(osteo/chondro/adipo,De Ugarte et al.2003)or even higher(Anjos-Afonso et al.2004)differentiation potential,clonal assays have shown that only one-third of the MSC clones derived from established cultures are pluripotent(Pittenger et al.1999;Muraglia et al.2000).Within es-tablished cultures,thus,a minority of cells seem to be pluripotent,with most of them having bi-or only uni-lineage differentiation capacity(Digirolamo et al. 1999).Models have been proposed to explain these results(Baksh et al.2004), and it is possible that cultures are composed of a mixture of cells with different differentiation potentials.A small portion may correspond to authentic stem cells,whereas most may be committed to more differentiated phenotypes.
8
Applications of Mesenchymal Stem Cells in Cell and Gene Therapy Although human MSCs can be immortalized through genetic modi?cation using expression vectors carrying the catalytic subunit of human telomerase (Mihara et al.2003),the study of murine MSCs in vitro is more attractive since they represent an unmodi?ed natural population.They can also be promptly
264N.Beyer Nardi·L.da Silva Meirelles obtained by researchers who do not have access to a source of human cells or do not have adequate facilities for their manipulation.More importantly, murine MSCs can be directly applied to a wide range of murine models of diseases,where they can act as therapeutic agents per se or as vehicles for the delivery of therapeutic genes.Finally,MSCs obtained from rats,rabbits,pigs, and sheep will also be useful for the development of engineered tissues using autologous cells.
8.1
Study of Cancer Biology
The sustained proliferation of murine MSCs provides an interesting model for the evaluation of genetic and epigenetic factors involved in the maintenance of stemness,as well as the components responsible for the generation of tumors. Murine MSC self-renewal is not linked to neoplasia:experiments in which mice received intravenous or intraperitoneal MSC infusion do not develop donor-derived tumors(L.Meirelles and N.Nardi,unpublished results).Unraveling the genetic determinants of self-renewal may lead to the identi?cation of can-didate genes involved in tumorigenesis and to the development of drugs that can act speci?cally on their products.The fusion of murine MSCs and nonpro-liferative cells,for instance,would help with the mapping of genes involved in proliferation to speci?c chromosomes and chromosomal regions.
While studying the behavior of marrow-derived cells in a mouse model of gastric cancer induced by Helicobacter pylori,cells bearing the marker TFF2could be associated with the predominant cell type present in this can-cer(Houghton et al.2004).In vitro studies using puri?ed marrow-derived hematopoietic stem cells or MSCs exposed to cancerous tissue extracts showed that MSCs,but not HSCs,acquired expression of TFF2.The contribution of MSCs to tumor formation by cell fusion was ruled out by comparing the ploidy of stomach cells from infected and noninfected mice:the number of tetraploid cells in both groups did not differ signi?cantly.Another recent study described the involvement of stem cells in human brain tumors(Singh et al.2004).These results are consistent with an emerging view of cancer as a stem cell disorder, rather than a disease con?ned to fully or partially differentiated cells.
Besides their potential use for the study of basic cancer biology,modi?ed MSCs may prove to be ef?cient antitumoral agents.Human MSCs have been shown to incorporate into tumor stroma(Studeny et al.2002),a potentially useful tool for the delivery of gene products directly to tumors(see below). 8.2
Cell Therapy
Mesenchymal stem cells may participate in cell therapy protocols through two mechanisms.First,MSCs may contribute physically to injured sites when
Mesenchymal Stem Cells:Isolation,In Vitro Expansion and Characterization265
administered locally or systemically.Second,MSCs may have a supportive role through means of secreted factors.Examples of these applications are given below.
8.2.1
Fibrosis
As mentioned earlier,murine MSCs administered systemically to mice sub-jected to lung injury show superior lung engraftment rates relative to unin-jured animals.Furthermore,MSC treatments performed immediately after antibiotic challenge reduces the?brotic and in?ammatory effects of the lesion signi?cantly more than that in animals receiving the cells7days after the chal-lenge(Ortiz et al.2003).An earlier work,using a poorly characterized murine MSC population,showed that infusion of the cells in mice subjected to lung injury by bleomycin showed enhanced reproducibility of engraftment(Kotton et al.2001).The injected cells were found to engraft as type I pneumocytes, but not type II pneumocytes.Similar results were reported by Ortiz et al. (2003).In the case of bleomycin-induced lung injury,this indicated that the main cellular contribution from the plastic-adherent fraction of bone marrow can be attributed to MSCs.This information may be valuable for future ther-apies aiming to reduce lung?brosis in humans by autologous bone marrow transplantation.
MSCs have also been used to treat liver?brosis.Fang et al.(2004)depleted murine bone marrow from CD45+,GlyA+,and CD34+cells to obtain adher-ent Flk+cells that are expandable in vitro for more https://www.doczj.com/doc/894851708.html,ing a murine model of tetrachloride-induced liver injury,they showed in the an-imals receiving MSCs systemically immediately after the challenge,but not 1week later,that the?brotic effects caused by the lesion were reduced.The presence of albumin-producing cells that exhibit donor-derived markers was also detected,although at a low frequency.
8.2.2
Cardiovasculogenesis
In a study originally designed to assess the contribution of murine MSCs to the cardiac tissue,Gojo et al.(2003)showed that a5-azacytidine-responsive, CD34low/–c-kit+CD140a+Sca-1high clone transduced with an EGFP construct contributed to several sites when implanted in vivo.After injection into the ventricular myocardium,EGFP+cardiomyocytes were detected,along with EGFP+CD31+cells lining the vessels surrounding the site of injection.The number of endothelial cells and cardiomyocytes grafted in the ventricle was estimated to be,respectively,1,625and751week after injection,and275 and253months later.When MSCs were infused systemically through the inferior vena cava,the cells engrafted predominantly in the lungs1week after
266N.Beyer Nardi·L.da Silva Meirelles administration.Four weeks after the injection,the number of EGFP+cells in the lungs declined considerably.The grafted cells lacked CD31expression, indicating that they had formed pericytes or smooth muscle cells.In addition to these results,EGFP+cells were found in the brain,thymus,uterus,and kidney.Engraftment in the stomach and small intestine was observed,and the number of donor-derived cells seemed to increase over time.When high cell numbers were implanted in the muscle,liver,or spleen,ectopic bone formation was observed,in contrast to the muscular and vascular fates adopted by cells delivered in low quantities.This?nding cautions against experimental protocols involving the injection of large doses of MSCs directly in heart muscle,since they might differentiate into tissues other than those expected.
The use of puri?ed MSCs to treat human heart diseases has not yet come into practice,despite the reported successes of blood or bone marrow-derived mononuclear cells(Assmus et al.2002;Perin et al.2003).Although studies demonstrating that bone marrow-derived c-kit+lin–cells,a cellular fraction putatively enriched in HSCs,regenerate mouse infarcted myocardium(Orlic et al.2001)these?ndings been challenged by others(Murry et al.2004;Baslam et al.2004).The plastic-adherent fraction of bone marrow has also improved cardiac performance in a rat model of heart infarction(Olivares et al.2004).The CD34–CD45–cells collected for these latter experiments most likely comprised MSCs.Histological analyses indicated that the main contribution of the cells to the infarcted zone occurred through the formation of new myocardium and blood vessels.
Taken together,these studies indicate that the cell type most likely involved in cardiac regeneration is the MSC,possibly due more to its arteriogenic effects (see the next section)than to its cardiomyogenic properties.
8.2.3
Arteriogenic Effects
Kinnaird et al.(2004)induced unilateral hind limb ischemia in mice to demon-strate that murine bone marrow,devoid of CD34+CD45+cells and delivered in situ24h after lesion,improved limb function despite little long-term cell engraftment.They found that medium conditioned by these cells contained several potent arteriogenic cytokines,such as basic?broblast growth fac-tor(bFGF),vascular endothelial growth factor(VEGF),placental growth fac-tor(PlGF),and monocyte chemoattractant protein-1(MCP-1).Animals that received the cells after injury contained donor-marked cells surrounded by bFGF and VEGF positive cells,indicating that the transplanted MSCs ex-pressed these cytokines in situ.These?ndings support the hypothesis that factors secreted by MSCs have a signi?cant role in limb recovery.Interest-ingly,MCP-1,which is secreted by cells present in the vascular wall,has been shown to recruit circulating monocytes that can differentiate into endothelial cells(Fujiyama et al.2003).The arteriogenic effects of MSCs may thus involve
Mesenchymal Stem Cells:Isolation,In Vitro Expansion and Characterization267 the recruitment of circulating cells through the secretion of chemoattractant factors.
8.2.4
Immunosuppressive Effects
The subcutaneous co-injection of primary murine MSCs or of an embry-onic mouse mesenchymal stem cell line(CH310T1/2)with a melanoma cell line(B16)was shown to favor tumor growth(Djouad et al.2003).In vitro experiments in which activated murine splenocytes were co-cultured with CH310T1/2cells in a transwell culture system indicated that soluble factors secreted by MSCs inhibit CD8+T cell proliferation.Even though the primary murine MSCs were not well characterized,the use of CH310T1/2validated the hypothesis that MCSs favored tumor growth,since the results observed in vivo were similar for both cell types.These results indicated that the im-munosuppressive effects should be considered whenever MSC transplantation takes place.Krampera et al.(2003)demonstrated even more clearly the im-munosuppressive effects of MSCs by showing that culture-expanded murine MSCs are capable of inhibiting both na?ve and memory antigen-speci?c T cell activation.The authors used a mixed lymphocyte reaction system in dose-dependent experiments,and determined that T cell inhibition was transient and independent of MHC antigen-presenting cells or CD4+CD25+regulatory T cells.In contrast,the study by Djouad et al.(2003)showed that the MSC immunosuppressive effect required cell contact.Whether or not cell contact is required,it is clear that MSCs have immunomodulatory capabilities.Treat-ment of acute graft-versus-host disease in a human subject using third-party haploidentical MSCs can be taken as proof of this concept(Le Blanc et al.2004).
The mechanisms involved in MSC-mediated immunosuppression are cur-rently being investigated.Glennie et al.(2004)reported that MSCs suppress T cell effector function transiently,but the cells do not block activation.On the other hand,they induce an irreversible proliferation arrest not only in CD4+ and CD8+T cells,but also in B cells,by downregulating cyclin-D2expression. The interactions of MSCs and immune cells may have future implications not only for the knowledge of MSC biology,but also for the understanding of immune system homeostasis.
8.3
Mesenchymal Stem Cells and Tissue Engineering
In addition to systemic engraftment capabilities,MSCs show great potential for the replacement of damaged tissues such as bone,cartilage,tendon,and lig-ament.Although bone is capable of regeneration,the three other tissues often develop?brous scar tissues when injured,which usually renders them unable to function https://www.doczj.com/doc/894851708.html,rge bone defects,however,do not heal spontaneously,
268N.Beyer Nardi·L.da Silva Meirelles Table4Examples of tissues engineered with the use of MSCs
Engineered tissue Methods Reference
Respiratory mucosa Human MSCs co-cultured with normal human
bronchial epithelial cells,in vitro Le Visage et al. 2004
Cartilage Human MSCs cultured under chondrogenic con-
ditions on3D scaffold,in vitro Chen et al. 2004a;Li et al. 2005
Bone Critical-size cranial defect created in rabbit,re-
paired with BMP-2-expressing rabbit MSCs em-
bedded in alginate Chang et al. 2004
Full-thickness mandibular defects created in pigs, and repaired with autologous MSCs previously seeded in poly-DL-lactic-coglycolic acid scaffolds and kept in osteo-inductive medium Abukawa et al. 2004
Cardiac pacemakers Human MSCs transfected with a cardiac pace-
maker gene(mHCN2).Functional results ob-
served in vitro by co-culture with neonatal rat
ventricular myocytes,and in vivo by subepicar-
dial injection into the canine left ventricular wall Potapova et al. 2004
suggesting that MSC-based reconstitution may be feasible.The application of MSCs in the engineering of new tissue is dependent on the use of an appro-priate scaffold to maintain an adequate three-dimensional distribution and on the use of speci?c molecules to drive their differentiation into cells that can restore the tissue-speci?c matrix(Huang et al.2004).
The use of murine MSCs for tissue engineering is limited,due to the small size of the mouse.However,murine MSCs along with MSCs from other species are interesting candidates for the study of cell interactions with novel bioma-terials,and the study of new molecules on differentiation.Examples of the use of MSCs for tissue engineering in larger animal models can be found in the literature,some of which are listed in Table4.
8.4
Genetic Therapy
Genetic diseases can be generally classi?ed into two categories:those caused by genetic alterations leading to loss of protein/gene function and those caused by a gain of function mutation.Other factors are involved such as the restriction of a disease phenotype to a speci?c organ as opposed to the whole body.When loss of function is the cause,introduction of genetic constructs expressing the missing product may be suf?cient to revert or suppress the disease phenotype. When the disease involves gain of function,however,the insertion of vectors
普通高中生物学课程标准（2017年最新修订版） 普通高中生物学课标研制组 目录 一、课程性质与基本理 念 ..................................................................... .. (1) （一）课程性 质 ..................................................................... .. (1) （二）基本理 念 ..................................................................... .. (1) 二、学科核心素养与课程目 标 ..................................................................... . (2) （一）学科核心素 养 ..................................................................... . (2) （二）课程目 标 ..................................................................... (3)
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2017年干细胞存储行业研究报告 因为具有潜在的再生能力，干细胞自其诞生之日起，就被认为可能是革命性的医疗技术。2007年和2012年的诺贝尔生理或医学奖，分别授予了与干细胞有关的发现。 2011年开始，干细胞概念再次被市场热炒。根据Market Research及Transparency Market Research预计，到2018年全球干细胞储存市场容量将增长至181.6亿美元。 干细胞存储业务的想象空间虽然不及干细胞治疗，但依然是现阶段许多干细胞公司的重要业务。对投资机构来说，一方面存储业务充沛的现金流可以缓解研发费用的压力，一方面细胞分离等技术能力的高低也能侧面反映公司的技术水平，对于分析标的的风险和潜力意义重大，值得持续关注和研究。 一、概述 干细胞是具有自我更新、高度增殖、多项分化潜能及良好组织相容性等特点的细胞群体。因为具有潜在的再生能力，干细胞自其诞生之日起，就被认为可能是革命性的医疗技术。2007年和2012年的诺贝尔生理或医学奖，分别授予了“在利用胚胎干细胞引入特异性基因修饰的原理上的发现”和“发现成熟细胞可被重写成多功能细胞，细胞核重编程技术（iPS细胞）”两项与干细胞有关的发现。 2011年开始，干细胞概念再次被市场热炒。在A股市场上，以中源协和为代表的企业当年股价飞涨。 存储业务涉及的主要是造血干细胞和间充质干细胞，前者主要来源于骨髓、外周血、脐带血、胎盘等，后者主要来源于骨髓、脐带、脂肪、脐带血、羊膜、胎盘等，进而形成了脐带血造血干细胞等细分种类。目前干细胞存储的主要市场仍在新生儿相关的业务，随着分离等技术的成熟和临床研究的深入，新生儿干细胞存储种类正在从脐带血造血干细胞转向胎盘、脐带等来源的间充质干细胞。
干细胞与组织工程 随着生命科学的飞速发展，目前组织工程、干细胞研究已经成为21世纪生命科学研究的焦点和前沿领域。组织工程研究涉及种子细胞、生物支架材料以及组织构建等众多研究方向．干细胞研究则有望解决组织工程研究中的种子细胞来源问题，可能成为组织工程研究中的理想种子细胞。 一“组织工程”的概念 1 “组织工程”的产生和发展 组织、器官的损伤或功能障碍是人类健康所面临的主要危害之一，也是人类疾病和死亡的最主要原因。据美国的一份资料显示，每年有数以百万计的美国人患有各种组织、器官的损伤或功能障碍，每年需进行800万次手术进行修复，年住院日在4000万～9000万之间，年耗资超过400亿美元。 随看现代外科学的发展，人类对组织、器官缺损的治疗有了很大的进步，但仍然存在许多问题。目前临床常用的治疗方法有三种： 1.自体组织移植、 2.异体组织移植、 3.人工合成组织代用品 组织工程是近年来正在兴起的一门新兴学科,1984年， Wolter首先提出“组织工程”（Tissue Engineering）一词。1987年，美国国家科学基金会于正式提出和确定“组织工程”一词，开辟了组织工程学研究的新纪元。它是应用生命科学和工程学的原理与技术，在正确认识哺乳动物的正常及病理两种状态下结构与功能关系的基础上，研究、开发用于修复、维护、促进人体各种组织或器官损伤后的功能和形态生物替代物的科学。 从事组织工程研究的科学家们利用细胞生物学、分予生物学以及材料科学等学科的最新技术，像工厂生产零部件一样，针对患音组织或器官缺失情况，利用构成组织或器官的基本单位——细胞以及为细胞生存提供空间的支架材料，在体内外培育出所需的人体组织或器官．需要多少就培育多少．量体裁衣制备完成后再给患者安装上去。 组织工程研究的核心是建立由细胞和生物材料构成的三维空间复合体。这一三维的空间结构为细胞提供了获取营养、气体交换、排泄废物和生长代谢的场所，
柳宗元驳复仇议翻译集团公司文件内部编码：（TTT-UUTT-MMYB-URTTY-ITTLTY-
臣伏见天后时①，有同州下邽人徐元庆者②，父爽为县尉赵师韫所杀③，卒能手刃父仇，束身归罪④。当时谏臣陈子昂建议诛之而旌其闾⑤，且请“编之于令，永为国典。”臣窃独过之⑥。 注释： ①伏见：旧时臣下对君主有所陈述时的表敬之辞，可译为知道，了解。天后：即武则天（624-705），名武瞾。690年，废睿宗李旦自立为皇帝，改洛阳为神都，建立武周王朝，在位十六年。705年，武则天病笃，宰相张柬之发动兵变，迫使武氏退位，史称神龙革命。中宗李哲复位，恢复唐朝。 ②同州：唐代州名，今陕西渭南市大荔县一带地区。下邽（guī）：县名，今陕西省渭南县。徐元庆：当时某驿馆的服务人员，徐元庆替父报仇，谋杀官员赵师蕴案是武则天时轰动一时的谋杀案。 ③县尉：县令的属官，专司当地的治安工作。或称御史大夫。 ④卒：最后，最终。束身归罪：自首。 ⑤陈子昂：（661—702），字伯玉。武后时曾任右拾遗，为谏诤之官。旌（jīng）：表彰。闾：里巷的大门。 ⑥过：错误，失当。 译文：微臣知道则天皇后时，同州下邽县有个叫徐元庆的人，他的父亲徐爽被县尉赵师韫杀害，他最后能亲手杀掉他父亲的仇人，并且自己捆绑着身体到官府自首。当时的谏官陈子昂建议将他处以死罪，同时在他的家乡表彰他的行为，并请朝廷将这种处理方式“编入法令，永远作为国家的法律制度”。臣私下认为，这样做是不对的。
臣闻礼之大本，以防乱也。若曰无为贼虐①，凡为子者杀无赦。刑之大本，亦以防乱也，若曰无为贼虐，凡为治者杀无赦。其本则合，其用则异。旌与诛莫得而并焉。诛其可旌，兹谓滥，黩②刑甚矣。旌其可诛，兹谓僣③，坏礼甚矣。果以是示于天下，传于后代，趋义者不知所向，违害者不知所立，以是为典可乎？盖圣人之制，穷理以定赏罚，本情以正褒贬，统于一而已矣。 注释： ①贼虐：残害，践踏 ②黩（dú）刑：滥用刑法。黩，轻率。 ③僭（jiàn）：越过，超出本分。 译文： 臣听说，礼的根本作用是为了防止人们作乱。意思是说，不要让礼受到践踏，凡是作儿子的，为报父仇而杀了人，就必须处死，不能予以赦免。刑法的根本作用也是为了防止人们作乱。意思是说，不能让刑受到践踏，凡是当官的错杀了人，也必须处死，不能予以赦免。礼和刑的根本目的是一致的，但是实际应用却不同。表彰和处死是不能同施一人的。处死可以表彰的人，这就叫乱杀，就是滥用刑法太过分了。表彰应当处死的人，这就是过失，破坏礼制太严重了。如果以这种处理方式昭示天下，并传给后代，那么，追求正义的人就不知道前进的方向，躲避刑罚的人就不能辨别立身之道，以此作为法则行吗？圣人制定礼法，是透彻地探究事理来制定赏罚，根据事实来确定奖惩，不过是把礼和刑二者结合在一起罢了。 向使刺谳①其诚伪，考正其曲直，原始而求其端②，则刑礼之用，判然离矣。何者？若元庆之父，不陷于公罪，师韫之诛，独以其私怨，奋其吏气，虐于非辜；州牧不知罪，刑官不知问，上下蒙冒③，吁号不闻。而元庆能以戴天④为大耻，枕戈⑤为得礼，处心积虑，
《中学生物教学论-期末总结》（刘恩山版） 绪论 1.要胜任当代中学生物学教师的工作，一个人不仅要有坚实的生物学专业知识和实验技能为基础，同时还要具有哪些专业知识及技能？ 答：（1）理解中学生物学课程的性质和价值 （2）理解生物科学和技术的本质和特征 （3）掌握学生的学习规律和学习特点 （4）能够使用多样化的教学方法 （5）能够利用多种评价方式来反映学生的进步 （6）专业素养的持续发展 2.怎样做一名合格的中学生物学教师？ 3.课程改革的焦点聚集在“面向全体学生”和“倡导探究性学习”。 4.“面向全体学生”带来的变化是课程内容加大了灵活性和选择性，使不同地区、不同学校的学生能学到更适合他们的需求和条件的内容 5.“倡导探究性学习”则大大增加了课程中对于过程技能的要求和探究活动（或解决问题）的内容，并对教师指导学生进行生物学研究的能力和引导发现的教学技能提出了新的要求 第一章 1.中学生物学课程的性质和地位 答：性质：学科课程；科学课程；技术课程。地位：必修课程 2.生物学课程的价值 答：（1）培养学生的生物科学素养 （2）为学生终生的学习和发展打下基础 （3）为学生步入社会、择业和确定进一步学习的专业方向提供帮助； 3.具有生物科学素养的人应具有正确的科学态度、价值观和世界观；有一定的科学探究能力，理解生物学的核心概念和原理；具有良好的思维习惯和理解力，能够应用生物学的知识和方法去面对现实生活中与生物学相关的问题。 4.课程标准 课程标准是由教育部颁布的带有指令性的、重要的国家课程文件，是国家对基础教育课程的基本规范和要求。《基础课程改革纲要（试行）》指出：课程标准是教材编写、教学、评估和考试命题的依据，是国家管理和评价课程的基础。 国家课程标准体现了国家对不同阶段的学生在知识与技能、过程与方法、态度情感与价值观等方面的要求。生物课程标准则是具体规定了中学生物学课程的性质、目标和内容标准，并提出了教学评价及编写教材等方面的建议。它是我国基础教育阶段生物学课程的基本规范和质量要求，是每个中学生物学教材编写人员、生物教师和教育管理者开展工作的依据和准绳。 5.标准和大纲的异同是什么？ 标准和大纲的共同之处是它们都是教育部颁布的指令性的课程文件“编教材、教学、评估、命题”的依据，也就是常说的“4个依据”的作用。 课程标准有一些与大纲不同的特点： （1）课程标准主要描述了学生在某一阶段学习后的学习成果，而大纲则强调的是具体的学习内容 （2）生物课程标准是国家制定的初中、高中阶段共同的、统一的基本要求，不是最高要求，
干细胞行业分析报告 贵州省科技风险投资管理中心 贵州省科技风险投资有限公司 贵州鼎信博成投资管理有限公司
目录 一、干细胞概述 (3) （一）产品定义 (3) （二）干细胞产品分类 (3) 二、干细胞行业及发展概况 (5) （一）国际行业发展概况 (5) （二）国内行业发展概况 (6) （三）干细胞行业特点 (8) 三、国内外干细胞临床研究的概况 (10) （一）国外干细胞临床研究现状 (10) （二）国内干细胞临床研究现状 (10) 四、干细胞市场现状分析 (11) 五、干细胞市场供需分析 (16) 六、涉及干细胞技术研究与应用的上市公司基介绍 (21) 七、干细胞行业产业链及投资价值分析 (22) 附件一：2011年最新国外干细胞研发企业及其相关产品 (25) 附件二：干细胞产业政策研究 (33) 附件三：人类干细胞之伦理原则与监管政策 (35)
一、干细胞概述 （一）产品定义 干细胞(stem cells，SC)是一类具有自我复制能力(self-renewing)的多潜能细胞，在一定条件下，它可以分化成多种功能细胞。干细胞(Stem Cell)是一种未充分分化，尚不成熟的细胞，具有再生各种组织器官和人体的潜在功能，改变了人类疾病的应对方法，医学界称为“万用细胞”。 （二）干细胞产品分类 1、干细胞具有自我更新能力（Self-renewing），能够产生高度分化的功能细胞。干细胞按照生存阶段分为以下五类： （1）胚胎干细胞 胚胎干细胞当受精卵分裂发育成囊胚时，内层细胞团（Inner Cell Mass）的细胞即为胚胎干细胞。胚胎干细胞具有全能性，可以自我更新并具有分化为体内所有组织的能力。 （2）成体干细胞 成年动物的许多组织和器官，比如表皮和造血系统，具有修复和再生的能力。成体干细胞在其中起着关键的作用。在特定条件下，成体干细胞或者产生新的干细胞，或者按一定的程序分化，形成新的功能细胞，从而使组织和器官保持生长和衰退的动态平衡。
附件1： 863 计划生物和医药技术领域 “干细胞与组织工程”重大项目 课题申请指南 一、指南说明 “十一五”期间，本重大项目以替代、修复或再造人体各种组织器官的“再生医学”为主线，以干细胞与克隆技术、组织工程技术、组织器官代用品和再生医学相关评价体系为重点，利用多学科、多技术交叉合作的关键技术和资源平台，建立具有一定规模的高水平研究、生产和应用基地，形成我国再生医学工程研究开发技术体系，研制具有自主知识产权的系列干细胞与组织工程产品及代用品，建立、完善和细化相应的技术标准、准入规范和相关伦理学指导原则，培育和带动新兴产业，加快实现部分干细胞、组织工程产品和组织器官代用品的产业化，从而使我国再生医学研究和应用的整体水平进入世界先进行列，部分关键技术和产品达到国际领先水平。 本重大项目的任务分解及主要任务指标为：完成1-2 种重要退行性疾病的治疗性克隆临床前研究，力争获得第一个治疗性克隆的临床批 文；完成3-5 种重要疾病干细胞治疗的临床前研究，获得SFDA或卫生 行政主管部门的临床批文，力争1-2种治疗技术或产品 完成临床试验，获得相应证书；初步建立1－2 个国家级人类（疾病）胚胎干细胞库，建立干细胞库有关的硬件标准和管理规范；4-6 种具有自主知识产权的系列组织工程产品获得SFDA临床批文，力争 2-3 项完成临床试验并获得许可证；开发3-5 项具有自主知识产权的新型组织器官代用品，力争获得生产许可证；建立3-5 种针对干细胞和组织工程应用的灵长类动物疾病与评价模型；申请专利200 项左右，其中
10项左右为国际PCT专利，力争10%获得授权。 上述任务分解除个别保密课题外，所有课题均通过公开发布课题申请指南落实任务。 此次发布的是本重大项目课题申请指南，拟安排的主要内容包括干细胞与治疗性克隆、组织工程技术与产品研制、组织器官代用品研发及灵长类动物疾病与评价模型等，拟支持的经费为 2 亿元人民币。课题支持强度依据所承担任务和完成指标而定，课题支持年限为5 年，但依据中期评估进行滚动支持。 二、指南内容 课题 1 、帕金森病或卢迦雷氏病的治疗性克隆研究与应用研究目标：完成治疗性克隆技术及治疗性克隆干细胞的临床前研究及有效性和安全性评价，向SFDA申报临床批文，争取在治疗性克隆领域有所突破。此课题是重大项目总体目标中的重要组成部分和亮点，也是体现本项目水平的重要指标之一。 主要研究内容及考核指标：针对帕金森病或卢迦雷氏病等神经系统退行性疾病，建立治疗性克隆胚胎干细胞系，建立治疗性克隆临床应用评价体系和相关检测手段，完成治疗性克隆技术及治疗性克隆干细胞的有效性和安全性评价等临床前研究，申报SFDA临床批文，争取率先将1－2 种治疗性克隆技术应用到临床。申请30 项左右国家专利及部分PCT力争10%获得授权。 课题支持年限：五年。 课题经费来源及构成：课题拟支持的国拨总经费为1500 万元人民币。作为战略高技术类课题，以国拨经费为主，课题申请单位配套相应的人员经费。 课题2、我国人类（疾病）胚胎干细胞库的建立与应用 研究目标：有计划、有组织、有目标、有分工地利用好、保护好我
驳复仇议阅读题及答案 《驳复仇议》出自于唐代文学家柳宗元的一篇驳论性的奏议，这篇奏议阐述了"调"即"和谐"在处理社会矛盾中的重要作用。以下是我给你推荐的驳复仇议阅读题及参考答案，希望对你有帮助! 《驳复仇议》阅读原文 臣伏见天后①时，有同州下邦人徐元庆者，父爽为县吏赵师韫所杀，卒能手刃父仇，束身归罪。当时谏臣陈予昂建议诛之而旌其闾，且请编之于令，永为国典。臣窃独过之。 臣闻礼之大本，以防乱也。若日无为贼虐，凡为子者杀无赦。刑之大本，亦以防乱也。若曰无为贼虐，凡为理者杀无赦。其本则合，其用则异，旌与诛莫得而并焉。诛其可旌，兹谓滥;黩刑甚矣。旌其可诛，兹谓僭;坏礼甚矣。果以是示于天下，传于后代，趋义者不知所向，违害者不知所立，以是为典，可乎? 盖圣人之制，穷理以定赏罚，本情以正褒贬，统于一而已矣。向使刺谳②其诚伪，考正其曲直，原始而求其端，则刑礼之用，判然离矣。何者?若元庆之父，不陷于公罪，师韫之诛，独以其私怨，奋其吏气，虐于非辜，州牧不知罪，刑官不知问，上下蒙冒，吁号不闻;而元庆能以戴天为大耻，枕戈为得礼，处心积虑，以冲仇人之胸，介然自克，即死无憾，是守礼而行义也。执事者宜有惭色，将谢之不暇，而又何诛焉? 其或元庆之父，不
免于罪，师韫之诛，不愆于法，是非死于吏也，是死于法也。法其可仇乎?仇天子之法，而戕奉法之吏，是悖骜而凌上也。执而诛之，所以正邦典，而又何旌焉? 且其议曰：人必有子，子必有亲，亲亲相仇，其乱谁救?是惑于礼也甚矣。礼之所谓仇者，盖其冤抑沉痛，而号无告也;非谓抵罪触法，陷于大戮。而曰彼杀之，我乃杀之，不议曲直，暴寡胁弱而已。其非经背圣，不亦甚哉!《周礼》：调人，掌司万人之仇。凡杀人而义者，令勿仇;仇之则死。有反杀者，邦国交仇之。又安得亲亲相仇也?《春秋公羊传》曰：父不受诛，子复仇可也。父受诛，子复仇，此推刃③之道，复仇不除害。今若取此以断两下相杀，则合于礼矣。 且夫不忘仇，孝也;不爱死，义也。元庆能不越于礼，服孝死义，是必达理而闻道者也。夫达理闻道之人，岂其以王法为敌仇者哉?议者反以为戮，黩刑坏礼，其不可以为典，明矣。 请下臣议附于令，有断斯狱者，不宜以前议从事。谨议。 【注】①天后：武则天。②刺谳：刺，探寻;谳，议罪。 ③推刃：往来项杀。 《驳复仇议》阅读题目 4.对下列句子中加点的词的解释，不正确的一项是(3分) A.臣窃独过之过：认为不对 B.旌其可诛，兹谓僭僭：僭越 C.不愆于法愆：罪过
驳《复仇议》阅读答案附翻译 驳《复仇议》 [唐]柳宗元 臣伏见天后①时，有同州下邦人徐元庆者，父爽为县吏赵师韫所杀，卒能手刃父仇，束身归罪。当时谏臣陈予昂建议诛之而旌其闾，且请编之于令，永为国典。臣窃独过之。 臣闻礼之大本，以防乱也。若日无为贼虐，凡为子者杀无赦。刑之大本，亦以防乱也。若曰无为贼虐，凡为理者杀无赦。其本则合，其用则异，旌与诛莫得而并焉。诛其可旌，兹谓滥；黩刑甚矣。旌其可诛，兹谓僭；坏礼甚矣。果以是示于天下，传于后代，趋义者不知所向，违害者不知所立，以是为典，可乎？ 盖圣人之制，穷理以定赏罚，本情以正褒贬，统于一而已矣。向使刺谳②其诚伪，考正其曲直，原始而求其端，则刑礼之用，判然离矣。何者？若元庆之父，不陷于公罪，师韫之诛，独以其私怨，奋其吏气，虐于非辜，州牧不知罪，刑官不知问，上下蒙冒，吁号不闻；而元庆能以戴天为大耻，枕戈为得礼，处心积虑，以冲仇人之胸，介然自克，即死无憾，是守礼而行义也。执事者宜有惭色，将谢之不暇，而又何诛焉? 其或元庆之父，不免于罪，师韫之诛，不愆于法，是非死于吏也，是死于法也。法其可仇乎？仇天子之法，而戕奉法之吏，是悖骜而凌上也。执而诛之，所以正邦典，而又何旌焉？ 且其议曰：人必有子，子必有亲，亲亲相仇，其乱谁救？是惑于礼也甚矣。礼之所谓仇者，盖其冤抑沉痛，而号无告也；非谓抵罪触法，陷于大戮。而曰彼杀之，我乃杀之，不议曲直，暴寡胁弱而已。其非经背圣，不亦甚哉!《周礼》：调人，掌司万人之仇。凡杀人而义者，令勿仇；仇之则死。有反杀者，邦国交仇之。又安得亲亲相仇也？《春秋公羊传》曰：父不受诛，子复仇可也。父受诛，子复仇，此推刃③之道，复仇不除害。今若取此以断两下相杀，则合于礼矣。 且夫不忘仇，孝也；不爱死，义也。元庆能不越于礼，服孝死义，是必达理而闻道者也。夫达理闻道之人，岂其以王法为敌仇者哉？议者反以为戮，黩刑坏礼，其不可以为典，明矣。 请下臣议附于令，有断斯狱者，不宜以前议从事。谨议。 【注】①天后：武则天。②刺谳：刺，探寻；谳，议罪。③推刃：往来项杀。 4．对下列句子中加点的词的解释，不正确的一项是 A．臣窃独过之过：认为不对 B．旌其可诛，兹谓僭僭：僭越 C．不愆于法愆：罪过 D．不宜以前议从事从事：处置 5．下列各组句子中，加点词的意义和用法相同的一组是 A．旌与诛莫得而并焉臣与将军戮力而攻秦。 B．而又何诛焉王问：何以知之？ C．我乃杀之今其智乃反不能及 D．是必达理而闻道者也虽一龙发机，而七首不动。 6．下列对原文有关内容的概括和分析，不正确的一项是 A．徐元庆杀了父亲的仇人后投案认罪。陈于昂建议，先处死徐元庆，再在他的家乡表彰他，并把这个案例编入法律文书中。作者认为，陈子昂的建议是错误的。 B．作者认为，礼与刑根本作用一致，但在实际运用中有区别，不能对同一个人既施死刑又行褒奖。自相矛盾的做法，公之于众，只会让天下人无所适从。
重点句翻译 一、《诗经》 1、王事靡盬，不遑启处；忧心孔疾，我行不来 2、昔我往矣，杨柳依依；今我来思，雨雪霏霏 二、《郑伯克段于鄢》 1、制，岩邑也，虢叔死焉，亻它邑唯命 2、都城过百雉，国之害也 3、姜氏何厌之有，不如早为之所，无使滋蔓，蔓，难图也 4、多行不义，必自毙，子姑待之 5、国不堪贰，君将若之何 6、厚将得众，不义不暱，厚将崩 7、大叔完聚，缮甲兵，具卒乘，将袭郑 8、公赐之食，食舍肉 9、君何患焉，若阙地及泉，隧而相见，其谁曰不然 10、孝子不匮，永锡尔类，君将不堪 11、今京不度，非制也，君将不堪 三、《燕昭王求士》 1、先趋而后患，先问而后嘿，则什己者至 2、此古服道致士之法也 3、燕国殷富，士卒乐佚轻战 4、燕兵独追北，人至临淄，尽取齐宝，烧其宫室宗庙 5、诎指而事之，北面而受学，则百己者至 6、今王诚欲致士，先从隗始，隗且见事，况贤于隗者乎，岂远千里哉 7、冯几据杖，眄视指使，则厮役之人至 四、《管晏列传》 1、仓廪实而知礼节，衣食足而知荣辱，上服度则六亲固 2、下令如流水之原，令顺民心 3、吾闻君子诎于不知己而信于知己者 4、通货积财，富国强兵，与俗同好恶 5、将顺其美，匡救其恶，故上下能相亲也 6、少时常与鲍叔牙游，鲍叔知其贤，管仲贫困，常欺鲍叔，鲍叔终善遇之，不以为言 7、吾尝三仕三见逐于君，鲍叔不以为我不肖，知我不遭时也 8、知我不羞小节而耻功名不显于天下也 1
9、知与之为政，政之宝也 10、其在朝，君语及之，即危言；语不及之，即危行 五、《苏武传》 1、武帝嘉其义，乃遣武以中郎将使持节送匈奴使留在汉者，因厚赂单于，答其善意 2、见犯乃死，重负国 3、复举剑拟之，武不动 4、空以身膏草野，谁复知之 5、武既至海上，廪食不至，掘野鼠去草实而食之 6、张胜闻之，恐前语发，以状语武 7、单于壮其节，朝夕遣人候问武，而收系张胜 8、凿地为坎，置煴火，覆武其上，蹈其背以出血 9、且单于信女，使决人死生，不平心持正，反欲斗两主，观祸败 10、天雨雪，武卧啮雪，与旃毛并咽之，数日不死 11、自分已死久矣，王必欲降武，请毕今日之欢，效死于前 12、收族陵家，为世大戮，陵尚复何顾乎 六、《先秦诸子语录》 1、其身正，不令而行；其身不正，虽令不从 2、老吾老以及人之老，幼吾幼以及人之幼，天下可运于掌 3、彼窃钩者诛，窃国者为诸侯，诸侯之门，而仁义存焉 七、《谏逐客书》 1、是以泰山不让土壤，故能成其大；河海不择细流，故能就其深；王者不却众庶，故能明其德 2、此所谓“藉寇兵而赍盗粮”者也 3、臣闻吏议逐客，窃以为过矣 4、今乃弃黔首以资敌国，却宾客以业诸侯 八、《驳复仇议》 1、其本则合，其用则异，旌与诛莫得而并焉 2、向使刺谳其诚伪，烤正其曲直，原始而求其端，则刑礼之用，判然离矣 3、元庆能不越于礼，服孝死义，是必达理而闻道者也 4、执事者宜有惭色，将谢之不暇，而又何诛焉 5、盖圣人之制，穷理以定赏罚，本情以正褒贬，终于一而已矣 6、仇天子之法，而戕奉法之吏，是悖骜而凌上也，是非死于吏也，是死于法也，法其可仇乎 九、《留侯论》
精品文档 2011版初中生物学课程标准解读 莱西市城关中学张春松 1．生物学课程标准修订的背景 （1）国际上科学教育发展迅速。如：2011年7月美国颁布了《国家科学教育框架》，并即将颁布《美国下一代科学教育标准》。 （2）国内对学生学习科学的研究结果支持了科学教育的发展；（3）2011版初中生物学课程标准有标志性的进步，可以说与全球同步。 2.我国初中生物学课标修订要点 2.1. 修订工作的基本思路 （1）在课程宗旨、课程理念、课程目标、课程内容框架及学生学习方式等重大方向、思路和框架等方面保持稳定； （2）执行基于研究和证据的课程决策程序，确保《生物学课程标准》的高质量； （3）基于研究成果修改或补充，新课标有以下四方面的变化变化：–课程性质 –内容标准中对适合各年级学生学习和理解的生物学重要概念 –教学建议 –评价建议 2.2. 原《生物学课程标准》中对于课程性质的表述： 生物科学是自然科学中的基础学科之一，是研究生命现象和生命精品
文档． 精品文档活动规律的一门科学。它是农林、医药卫生、环境保护及其 他有关应从定性到定量的发展用科学的基础。生物科学经历了从现象到本质、过程，并与工程技术相结合，对社会、经济和人类生活产生越来越大以提高学生的影响。义务教育阶段的生物学课程是国家统一规定的、生物科学素养为主要目的的学科课程，是科学教育的重 要领域之一。新《生物学课程标准》中对于课程性质的表述增加的内容：它不仅是一个结论丰生物科学有着与其他自然科学相同的性质。也包括了人类认识自然现象和规律的一些特有的思维富的知识体系，方式和探究过程。生物科学的发展需要许多人的共同努力和不断探索。这些是生物学课程性质的重要决定因素。其精要义务教育阶段的生物学课程是自然科学领域的学科课程，它既要让学生理反映自然科学的本质。是展示生物科学的基本内容，又要让学生领悟生物学家在研究过程中所持有解基础的生物学知识，的观点以及解决问题的思 路和方法。获生物学课程期待学生主动地参与学习过程，在亲历提出问题、取信息、寻找证据、检验假设、发现规律等过程中习得生物学知识，养成理性思维的习惯，形成积极的科学态度，发展终生学习的能力。其学习成果是学习生物学课程是每个未来公民不可或缺的教育经历，公民素养的基本组成。特别指出，生物学作为一门自然科学，它属于理科，教学中必须要加强理解，防止死记硬背。 2.3.《生物学课程标准》中重要概念的表述： 精品文档． 精品文档 50个重要概念，其中包括8个核心概念）（10个一级主题共
目录 一、干细胞行业的“万用功能“及发展前景 (1) 二、干细胞行业相关政策分析 (2) 三、干细胞抗衰老作用机理 (2) 1.骨髓间充质干细胞 (3) 2.脐带血干细胞 (4) 3.脂肪干细胞 (5) 4.胚胎干细胞 (5) 四、干细胞产业链分析 (6) 五、干细胞国内相关企业分析 (7) 1.中源协和：中国干细胞产业链的整合者，全面布局上下游 (7) 2.北科生物：干细胞技术全球领先，有望成为中国的“苹果” (8) 3.金卫医疗：通过CCBC股权间接经营干细胞存储业务 (9) 4.冠昊生物：依托技术优势进军干细胞治疗领域 (10)
图表目录 图表 1：干细胞治疗应用方向 (3) 图表 2：干细胞抗衰老行业相关政策 (4) 图表 3：随着年龄的增长，骨髓中干细胞数目急剧下降 (5) 图表 4：小鼠骨髓间充质干细胞具有抗衰老作用 (6) 图表 5：脐带血干细胞可以促进细胞增殖，修复受伤组织 (6) 图表 6：肌肉注射胚胎干细胞后各系统疗效（临床改善指数） (7) 图表 7：干细胞产业链 (8) 图表 8：北科生物发展历程 (11) 图表 9：金卫医疗发展历程 (12)
一、干细胞行业的“万用功能“及发展前景 干细胞是具有自我复制和多向分化潜能的原始细胞，是机体的起源细胞，是形成人体各种组织器官的原始细胞。在一定条件下，它可以分化成多种功能细胞或组织器官，医学界称其为“万用细胞”，对应干细胞治疗具有极大的潜力。干细胞按发育状态分为胚胎干细胞、成体干细胞。按分化潜能分为全能干细胞、多能干细胞、单能干细胞。 干细胞治疗则是把健康的干细胞移植到病人或自己体内，以达到修复病变细胞或重建功能正常的细胞和组织的目的。即干细胞治疗从细胞层上治疗疾病，相较很多传统治疗方法具有无可比拟的优点： （1）安全：低毒性（或无毒性）； （2）在尚未完全了解疾病发病的确切机理前也可以应用； （3）治疗材料来源充足； （4）治疗范围广阔； （5）是最好的免疫治疗和基因治疗载体； （6）传统疗法认为是“不治之症”的疾病，又有了新的疗法和新的希望。 因此干细胞治疗作为一种比较优势突出的新型治疗，临床上应用的领域包括治疗心血管疾病、神经系统疾病、血液病、肝病、肾病、糖尿病、骨关节疾病等，现在比较成熟的如应用骨髓移植治疗白血病等恶性血液病，随着未来越来越多临床试验的成功，产业发展前景将十分广阔。 图表 1：干细胞治疗应用方向 资料来源：银联信
干细胞技术及发展 摘要：干细胞技术是生物技术领域最具有发展前景和后劲的前沿技术，其已成为世界高新技术的新亮点，势将导致一场医学和生物学革命。本文主要介绍干细胞及干细胞的相关技术与发展方向。 关键词：干细胞干细胞技术发展方向 1 概述 干细胞技术，又称为再生医疗技术，是指通过对于干细胞进行分离、体外培养、定向诱导、甚至基因修饰等过程，在体外繁育出全新的、正常的甚至更年轻的细胞、组织或器官，并最终通过细胞组织或器官的移植实现对临床疾病的治疗。 干细胞技术最显著的作用就是：能再造一种全新的、正常的甚至更年轻的细胞、组织或器官。由此人们可以用自身或他人的干细胞和干细胞衍生组织、器官替代病变或衰老的组织、器官，并可以广泛涉及用于治疗传统医学方法难以医治的多种顽症，诸如白血病、早老性痴呆、帕金森氏病、糖尿病、中风和脊柱损伤等一系列目前尚不能治愈的疾病。从理论上说，应用干细胞技术能治疗各种疾病，且其较很多传统治疗方法具有无可比拟的优点。 2 干细胞技术 干细胞有诱人的临床应用前景，因此干细胞技术的研究开发自然成为当今世界生物医药领域的一个热点研究方向。干细胞技术涉及许多方面，包括干细胞的鉴别、分离纯化、分裂增殖、诱导分化、冷冻保存、解冻使用等。还包括干细胞培养液、干细胞再生医学生物材料、干细胞产品保存包装材料等相关技术。以下着重简述来自不同发育阶段和不同组织的干细胞的相关技术。 2.1 干细胞 在细胞的分化过程中，细胞往往由于高度分化而完全失去了再分裂的能力，最终衰老死亡。机体在发展适应过程中为了祢补这一不足，保留了一部分未分化的原始细胞即干细胞。一旦生理需要，这些干细胞可按照发育途径通过分裂而产生分化细胞。 2.1.1干细胞的特点 （1）干细胞本身不是处于分化途径的终端。 （2）干细胞能无限的增殖分裂。 （3）干细胞可连续分裂几代，也可在较长时间内处于静止状态。
《中学生物学教学论》 1：简答题：举例说明什么是"前科学概念”。 参考答案： 许多学生在进入课堂之前已经思考过一些在生活中所见到的生物学现象，并形成了一些想法来解释身边发生的现象，这就是"前科学概念”。学生的前科学概念有些与科学家的认识接近或相同，但大多数是科学界不能接受的结论，有人也将其称为"错误概念或迷思概念”。例如人们腐肉生蛆的认识。 2：简答题：请你介绍一个中学生物学教育相关的网站，并对其进行评价。 参考答案： 提供一个中学生物学教育相关的网站的网址；主要栏目、特色；简单评价。 3：简述概念图在教学中的用途。 参考答案： （1）作为教的工具，主要是用于组织课程内容； （2）作为学的工具； （3）作为评价工具。 4：简答题：你是如何理解教学目标与评价之间的关系的。 参考答案：教学目标是制定评价标准的基础；评价是围绕着目标展开，否则就是无效的评价。 5：简答题：简要介绍导入的基本结构。 参考答案：引起注意；激起动机；组织引导；建立联系。 6：简答题：举例说明建构主义学习观的某些合理性。
参考答案： （1）强调学习者的经验（举例证明该观点的合理性）； （2）注重以学习者为中心（举例证明该观点的合理性）； （3）创造冲突的真实的学习情境（举例证明该观点的合理性）； （4）注重互动的学习方式（举例证明该观点的合理性）。 7：简答题：简述初中生物学新课程倡导的课程理念。 参考答案：面向全体学生；提高生物科学素养；倡导探究性学习。 8：简答题：简述中学生物学实验的类型。 参考答案： （1）从生物学学科特点看，生物学实验可分为：形态学实验、解剖学实验、生理学实验和分类学实验等。 （2）从教学活动的特点看，可以将生物学实验分为：观察实验、验证性实验、探究性实验及设计和制作实验等。 9：[多选题]教态的变化包括（） A：声音的变化B：停顿C：目光接触D：面部表情变化E：身体移动F：头手的动作变化 参考答案：ABCDEF 10：[单选题]讲授"光合作用”这部分内容时，按布鲁姆的教育目标分类，属于理解水平的问题是（）。 A：叶绿体中含有哪两类色素B：光反应的场所C：光合作用的实质D：作物栽培要合理密植参考答案：C 11：[多选题]下列属于"模型”的特点的是（）。
驳复仇议 臣伏见天后时①，有同州下邽人徐元庆者②，父爽为县尉赵师韫所杀③，卒能手刃父仇，束身归罪④。当时谏臣陈子昂建议诛之而旌其闾⑤，且请“编之于令，永为国典。”臣窃独过之⑥。 注释： ①伏见：旧时臣下对君主有所陈述时的表敬之辞，可译为知道，了解。天后：即武则天（624-705），名武瞾。690年，废睿宗李旦自立为皇帝，改洛阳为神都，建立武周王朝，在位十六年。705年，武则天病笃，宰相张柬之发动兵变，迫使武氏退位，史称神龙革命。中宗李哲复位，恢复唐朝。 ②同州：唐代州名，今陕西渭南市大荔县一带地区。下邽（guī）：县名，今陕西省渭南县。徐元庆：当时某驿馆的服务人员，徐元庆替父报仇，谋杀官员赵师蕴案是武则天时轰动一时的谋杀案。 ③县尉：县令的属官，专司当地的治安工作。或称御史大夫。 ④卒：最后，最终。束身归罪：自首。 ⑤陈子昂：（661—702），字伯玉。武后时曾任右拾遗，为谏诤之官。旌（j īng）：表彰。闾：里巷的大门。 ⑥过：错误，失当。 译文：微臣知道则天皇后时，同州下邽县有个叫徐元庆的人，他的父亲徐爽被县尉赵师韫杀害，他最后能亲手杀掉他父亲的仇人，并且自己捆绑着身体到官府自首。当时的谏官陈子昂建议将他处以死罪，同时在他的家乡表彰他的行为，并请朝廷将这种处理方式“编入法令，永远作为国家的法律制度”。臣私下认为，这样做是不对的。 臣闻礼之大本，以防乱也。若曰无为贼虐①，凡为子者杀无赦。刑之大本，亦以防乱也，若曰无为贼虐，凡为治者杀无赦。其本则合，其用则异。旌与诛莫得而并焉。诛其可旌，兹谓滥，黩②刑甚矣。旌其可诛，兹谓僣③，坏礼甚矣。果以是示于天下，传于后代，趋义者不知所向，违害者不知所立，以是为典可乎盖圣人之制，穷理以定赏罚，本情以正褒贬，统于一而已矣。 注释： ①贼虐：残害，践踏 ②黩（dú）刑：滥用刑法。黩，轻率。 ③僭（jiàn）：越过，超出本分。
驳《复仇议》|柳宗元|附译文翻译|文言文阅读题在线测试（附答案） 驳《复仇议》|柳宗元|附译文翻译|文言文阅读题 驳《复仇议》 [唐]柳宗元 臣伏见天后①时，有同州下邦人徐元庆者，父爽为县吏赵师韫所杀，卒能手刃父仇，束身归罪。当时谏臣陈予昂建议诛之而旌其闾，且请“编之于令，永为国典”。臣窃独过之。 臣闻礼之大本，以防乱也。若日无为贼虐，凡为子者杀无赦。刑之大本，亦以防乱也。若曰无为贼虐，凡为理者杀无赦。其本则合，其用则异，旌与诛莫得而并焉。诛其可旌，兹谓滥；黩刑甚矣。旌其可诛，兹谓僭；坏礼甚矣。果以是示于天下，传于后代，趋义者不知所向，违害者不知所立，以是为典，可乎？ 盖圣人之制，穷理以定赏罚，本情以正褒贬，统于一而已矣。向使刺谳②其诚伪，考正其曲直，原始而求其端，则刑礼之用，判然离矣。何者？若元庆之父，不陷于公罪，师韫之诛，独以其私怨，奋其吏气，虐于非辜，州牧不知罪，刑官不知问，上下蒙冒，吁号不闻；而元庆能以戴天为大耻，枕戈为得礼，处心积虑，以冲仇人之胸，介然自克，即死无憾，是守礼而行义也。执事者宜有惭色，将谢之不暇，而又何诛焉?其或元庆之父，不免于罪，师韫之诛，不愆于法，是非死于吏也，是死于法也。法其可仇乎？仇天子之法，而戕奉法之吏，是悖骜而凌上也。执而诛之，所以正邦典，而又何旌焉？ 且其议曰：“人必有子，子必有亲，亲亲相仇，其乱谁救？”是惑于礼也甚矣。礼之所谓仇者，盖其冤抑沉痛，而号无告也；非谓抵罪触法，陷于大戮。而曰“彼杀之，我乃杀之”，不议曲直，暴寡胁弱而已。其非经背圣，不亦甚哉!《周礼》：“调人，掌司万人之仇。凡杀人而义者，令勿仇；仇之则死。有反杀者，邦国交仇之。”又安得亲亲相仇也？《春秋公羊传》曰：“父不受诛，子复仇可也。父受诛，子复仇，此推刃③之道，复仇不除害。”今若取此以断两下相杀，则合于礼矣。 且夫不忘仇，孝也；不爱死，义也。元庆能不越于礼，服孝死义，是必达理而闻道者也。夫达理闻道之人，岂其以王法为敌仇者哉？议者反以为戮，黩刑坏礼，其不可以为典，明矣。 请下臣议附于令，有断斯狱者，不宜以前议从事。谨议。 【注】①天后：武则天。②刺谳：刺，探寻；谳，议罪。③推刃：往来项杀。 柳宗元《驳复仇议》参考译文 我（从记载上）看到天后在位时，有个同州下圭人，叫徐元庆，父亲徐爽被县尉赵师韫杀害，他终于能够亲手杀死父亲的仇人，再自缚其身投案认罪。当时谏臣陈子昂建议，处死徐元庆，然后再在他的家乡表彰他，并且请求将这一案例编入律令，永远作为国家的法典。我个人认为他的建议是不对的。 我听说“礼”的最根本作用，用来防止动乱。如“礼”说，不要做凶残暴虐的事，凡是做儿子的杀害无辜之人，要处以死刑而不能赦免。“刑”的最根本作用，也是用来防止动乱的，如“刑”规定，不要做凶残暴虐的事，凡是做官的杀害无辜之人，要处以死刑而不能赦免。它们的根本作用一致，但运用起来有差异，表彰和诛杀是不能同时运用到一个人身上的。处死当
第一章中学生物学课程 本章學習目標： １．概述中学生物学课程的性质和价值。 ２．利用生物学课程标准指导教学。 ３．阐明中学生物学课程的目的。 ４．说明教科书的作用。 生物学课程是基础教育中学阶段重要的学科课程。随着时代的前进，生物 科学技术的迅速发展及其对人类社会产生的日益广泛而深入的影响，人们更加关注生物学课程在基础教育中的地位和作用，科学教育家和生物学教育专家们也在不断思考如何使中学生物学课程跟上科学技术进步和时代的步伐。在过去的几十年间，这些因素都有力地推动了世界范围的中学生物学课程改革。 生物学课程的不断改革，给生物学教师（包括实习教师）提出了许多新的挑 战和新的要求。在对教师的诸多要求之中，一个基本要求就是生物学教师要能够正确认识和理解中学生物学课程，包括对课程的性质、课程的价值、课程目标以及课程标准和教材等方面的认识。本章将基于我国第八次课程改革的成果，就中学生物学课程的这些基本问题展开讨论和介绍。 第一节中学生物学课程的性质、价值和地位 生物学教师对生物学课程性质、价值的认识是教师专业素养的基本要求之 一，这种认识会直接影响教师对自身教学任务的理解和教学行为的调整。因此，生物学教师要对生物学课程性质、价值及其在基础教育中的地位有深入的理解，并能随着新课程的推进，不断地思考这个问题。 第一章中学生物学课程·５·
一、生物学课程的性质和地位 中学生物学课程从课程性质来说属于学科课程。在我国大陆地区，初中阶 段的生物学课程是国家统一规定的，以提高学生生物学素养为主要宗旨的必修课程；高中生物学课程是在义务教育基础上，适应高中学生身心发展特点和规划人生、终生发展的需要，以进一步提高学生生物科学素养为主要目的的科学课程，包括必修和选修两部分内容。中学生物学课程是科学教育中的一门重要学科。 学科的性质影响学科课程的性质。生物学课程是学科课程，这是生物学课 程的一个基本性质。因此，生物学课程要体现科学的本质和特征。每一个生物学教育工作者，不论是教材编写人员、生物学教师，还是教研人员，都应在实现生 物学课程的过程中注意把握并向学生展示课程的这一性质。 科学不仅是一个内容丰富的知识体系，它也是人类认识自然世界的一些特 殊途径和方法。由于有了这些对生命世界准确地提出问题及获取较为可靠答案的方法，如观察、提出问题、定量化、求证和思考，人类对自身和环境的认识日益 深入、全面、更加可靠。这些方法反映出自然科学与其他领域认识模式的不同，也体现了科学本质和特征最基本的部分。生物学课程作为学科课程，不仅要传播科学的事实和概念，更要体现科学是一个探究的过程。（参见第二章第一节）。生物学课程具有技术课程的性质。技术是推动人类文明的强大动力，技术 增强了人们改变世界的能力，它在许多方面表现为生产力。科学和技术有密切的关系，科学家将科学和技术称为“一个硬币的两个侧面”。在当今世界中，生物 科学和技术的发展已经充分地表现出了这些特点。中学生物学课程在注意到科学课程性质的同时，也注意到了技术的本质和特征，其中生物技术的内容在不断增加。一个具有生物科学素养的人，应该对生物技术的特征有一些基本的认识。这种认识包括生物科学与技术的关系、生物技术与社会的关系、生物技术的基本原理。在中学生物学课程中加强生物技术，是我国生物学课程在进入２１世纪