用化合物C能快速高效的将hESCs和hiPSCs诱导为神经祖细胞

Author contributions: J.Z.: Conception and design, manuscript writing, collection and assembly of data, data analysis and interpretation; P.S.: Collection and assembly of data, data analysis and interpretation; D.L.: Collection and assembly of data, data analysis and interpretation; S.T.: Collection and assembly of data, data analysis and interpretation; E.D.: Conception and design, data analysis and interpretation, financial support; F.W.: Conception and design, financial support, manuscript writing, final approval of manuscript

*Corresponding author: Department of Cell and Developmental Biology and Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 601 S. Goodwin Ave, Urbana, IL -61801, USA. Tel.: 217-333-5972; Fax: 217-244-1648; E -mail:feiwang@https://www.wendangku.net/doc/9518740365.html,; Received May 07, 2010; S TEM C ELLS ?

E MBRYONIC S TEM C ELLS /I NDUCED P LURIPOTENT S TEM C ELLS

High-Efficiency Induction of Neural Conversion in hESCs and hiPSCs with a Single Chemical Inhibitor of TGF-E Superfamily Receptors

Jiaxi Zhou 1,2, Pei Su 1,2, Dong Li 1,2, Stephanie Tsang 1,2, Enkui Duan 3, Fei Wang 1,2,*

1

Department of Cell and Developmental Biology and 2Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. 3State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences (CAS), Beijing 100101, China

Key words. human embryonic stem cells x neural conversion x compound C x TGF-E superfamily receptors x induce

pluripotent stem cells

A BSTRACT

Chemical compounds have emerged as powerful tools for modulating embryonic stem cell (ESC) functions and deriving induced pluripotent stem cells (iPSCs), but documentation of compound-induced efficient directed differentiation in human ESC (hESCs) and human iPSC (hiPSCs) is limited. By screening a collection of chemical compounds, we identified compound C (also denoted as dorsomorphin), a protein kinase inhibitor, as a potent regulator of hESC and hiPSC fate decisions. Compound C suppresses mesoderm, endoderm and trophoectoderm differentiation and induces rapid and high-efficiency neural conversion in both hESCs and hiPSCs (88.7% and 70.4%, respectively). Interestingly, compound C is ineffective in inducing neural conversion in mouse ESCs (mESCs). Large-scale kinase assay revealed that compound C targets at least seven TGF-E superfamily

receptors, including both type I and type II receptors, and thereby blocks both the Activin and BMP signaling pathways in hESCs. Dual inhibition of Activin and BMP signaling accounts for the effects of compound C on hESC differentiation and neural conversion. We also identified muscle segment homeobox gene 2 (MSX2) as a downstream target gene of compound C and a key signaling intermediate of the BMP pathway in hESCs. Our findings provide a single-step cost-effective method for efficient derivation of neural progenitor cells in adherent culture from human pluripotent stem cells. T herefore, it will be uniquely suitable for the production of neural progenitor cells in large scale and should facilitate the use of stem cells in drug screening and regenerative medicine and study of early human neural development. I NTRODUCTION

Human embryonic stem cells (hE SCs) and human induced pluripotent stem cells (hiPSCs) reprogrammed from somatic cells can differentiate into multiple cell types, allowing them to be utilized for biomedical research, drug discovery and cell-based therapies [1, 2]. A deeper understanding of the signaling mechanisms and pathways that maintain

pluripotency and induce direct differentiation of hESCs and iPSCs will shed light on early human development and facilitate the applications of these powerful cellular systems.

Several developmentally important signals have been extensively studied in hESCs. For example, basic FGF (bFGF) supports long-term undifferentiated growth of hESCs [3]. The TGF-? superfamily, comprising TGF-?, Activin,

Nodal and bone morphogenesis proteins (BMPs), has diverse roles in hESCs [4, 5]. TGF-

?/Activin/Nodal was shown to co-operate with FGF signaling to maintain pluripotency of h SCs by controlling NANOG expression. Activation of BMP signaling in hE SCs induces mesoderm and trophoectoderm activities depending on the duration of activation [6-8], while activation of Activin/Nodal pathway can trigger endoderm differentiation [9]. Conversely, inhibition of Activin/Nodal and BMP signaling, alone or in combination, promotes neuroectoderm specification [10-12].

Chemical compounds can be used to control cellular processes and have been applied successfully to the modulation of fate decisions

of stem cells including hE SCs and hiPSCs. Chemical compounds can be synthesized at a large scale and great purity, offer temporal and tunable control of molecular and cellular functions, and has a low cost. E arlier studies showed that the ROCK inhibitor Y-27632 promotes survival of dissociated hE SCs, while the small-molecule valproic acid (VPA) improves the efficiency of hiPSC derivation [13, 14]. Recently, cell-permeable small molecules that direct differentiation of hE SCs into the endoderm or early pancreatic lineage were indentified [15, 16]. Despite these encouraging advances, there is limited documentation about using chemical compounds to efficiently induce differentiation to other lineages such as neuroectoderm in hESCs and hiPSCs.

Here, we report the identification and use of a single chemical compound, compound C/ dorsomorphin, for suppressing endoderm, mesoderm and trophoectoderm differentiation of human pluripotent stem cells and for rapid and highly efficient neural conversion from hE SCs and hiPSCs in defined adherent cultures. We also show that compound C targets at least seven type I and type II TGF-E superfamily receptors and blocks both the Activin and the BMP signaling pathway, which account for compound

C's ability to induce high-efficiency neural conversion.

M ATERIALS AND M ETHODS

Cell Lines and Cell Culture

H1 and H9 hE SCs (WiCell Research Institute)

were routinely maintained under feeder conditions as described [1]. The culture medium

consists of DME M/F12 with 20% knockout

serum replacement (KSR), 1 mM glutamine, 1%

non-essential amino acid, 0.1 mM ?-mercaptoethanol and 4 ng/ml bFGF. KSR is a

defined supplement.For feeder-free cultures,

cells were cultured on plates coated with

Matrigel (BD Biosciences) in the presence of conditioned medium (CM) from mouse embryonic fibroblasts (ME Fs), which replaces

the ME F feeders. Fresh medium was added

every day. For neural conversion of hE SCs,

mTeSR medium (StemCell Technologies) and

DMEM/F12 medium with 20% knockout serum replacement (KSR), 1 mM glutamine, 1% non-

essential amino acid and 0.1 mM ?-mercaptoethanol were used. mTeSR is a defined

feeder-independent medium. The hiPSC line

RND5 (passage 8-10) was from ArunA Biomedical, Inc (Athens, GA) and was maintained under the same conditions as H1 and

H9 hE SCs. Derivation and validation of the

hiPSCs are described: https://www.wendangku.net/doc/9518740365.html,/GeneTargeting/

viPS%20Lentiviral%20Vector%20Kit/. NTera-2 (American Type Culture Collection, ATCC)

cells were cultured according to ATCC recommendations. The medium consists of 10%

fetal bovine serum (FBS, ATCC). For low-

density single-cell culture, cells were dissociated

into single cells with 0.025% trypsin-0.02%

E DTA and plated at the density of 10,000

cells/cm2. W4 mouse E SC (mE SC) line was

from Taconic and was cultured as described

[17].

Western Blotting

For the analysis of proteins except phospho-

Smad2/3, cells (5×106) were lysed directly with

200P l laemmli sample buffer (Biorad). For

detection of phospho-Smad2/3, nuclear fraction

of hESCs was isolated with a NEPER? Nuclear

and Cytoplasmic

E

xtraction Kit (Pierce).

Samples (25 P g) were analyzed by western blot. Dilutions for various antibodies were described in Table S4. The blots were developed using SuperSignal West Pico Chemiluminescent Substrate (Pierce), and signals were quantified with Image J.

EB Formation Assay

H1 or H9 hESCs (5×106

/well) cultured on MEFs

were dissociated as clusters with dispase (1 mg/mL), plated onto Ultra-Low Attachment 6-well plates (Corning) and grown in embryoid body (E B) medium (DME M/F12, 20% FBS, 1 mM glutamine, 1 mM 1% non-essential amino acid and 0.1 mM ?-mercaptoethanol). To change

medium, E Bs and medium were gently transferred into a 15-ml tube and left at RT for 5

min to allow the EBs to sink to the bottom. The medium was gently removed and replaced by fresh medium. E Bs were collected at different time points for further analysis.

Induction of Neural Differentiation

For neural differentiation, H1 or H9 hE SCs were cultured on Matrigel with mTeSR medium to eliminate variations introduced by the ME F feeders. hE SCs were gently dissociated into single-cell suspension with 1 mg/mL accutase for 5–7 min at 37oC. The dissociated cells were plated on Matrigel at high density (106/cm 2) and cultured to confluency in mTeSR medium before

induction of differentiation. For compound C-induced neural conversion, mTeSR medium was

replaced with DMEM/F12 containing 20% KSR,

1 mM glutamine, 1% non-essential amino acid and 0.1 mM ?-mercaptoethanol. This condition was used for all treatment groups. Medium was replaced every day. For early neural patterning, SHH (100 ng/mL) was incubated with

compound C-induced neural progenitor cells for 5 d. For late neural terminal differentiation/maturation, SHH-treated cells were gently dissociated into small clusters with dispase and plated on laminin-coated plates. Cells were cultured in neural differentiation medium, which consists of neural basal medium, 1x N2 supplement, purmorphamine (1 μM), ascorbic acid (200 μM), BDNF (20 ng/mL), GDNF (20 ng/mL), SHH (100 ng/mL) and FGF8 (100 ng/mL), for 2 weeks before collected for analysis.

Luciferase Assay

The ID-120-luc reporter construct for BMP signaling activity, kindly provided by Dr. Renhe Xu (University of Connecticut), was described [18]. To measure the reporter activity, H1 hESCs were plated on Matrigel-coated 24-well plates

before transfection. The ID-120-luc plasmid was co-transfected into cells with the Rellina plasmid

(Promega), and 24 h after transfection, medium was replaced with hESC growth medium without

bFGF. BMP-4 (10 ng/mL) in combination with different concentrations of compound C was

administered to cells. The cells were lysed 24 h after the addition of BMP-4 and compound C by using lysis buffer provided with a dual-luciferase assay kit (Promega). The luciferase activity was

measured with LUMIstar plate reader (BMG Labtech). The luciferase-based Activin reporter plasmid (pARE-GL3, [19]) was kindly provided by Dr. Yisrael Sidis (Partners Healthcare). To measure the reporter activity, H1 hESCs was co-transfected with 0.25 μg pARE -GL3, 0.25 μg FAST-1 (forkhead activin signal transducer-1), and Rellina plasmid. Activin A (100 ng/mL) and various concentrations of SB421542 or compound C were added 24 h after transfection.

The luciferase activity was measure as described above.Additional experimental procedures and associated references are available in the Supporting Information. R ESULTS Compound C Modulates Differentiation Activities in hESCs

We recently screened a collection of chemical compounds, identified and reported mTOR as a key positive regulator of pluripotency [17]. In the current study, we screened for compounds that prevent hE SC differentiation. We cultured two hE SC lines with distinct genotypes [WA01 (H1), XY; WA09 (H9), XX] [1]in the absence

of bFGF and conditioned medium (CM) produced by mouse embryonic fibroblasts (ME Fs), both required to support hE SC self-renewal and pluripotency [1, 20]. Human E SCs grown without bFGF and CM continued to

proliferate at a slower rate, but began to differentiate from the center of the colonies and gradually lost the expression of pluripotency markers OCT-4 and SOX2 as described [17] (Fig. 1A). We applied the collection of chemical compounds and inhibitors (Table S1) to hE SCs cultured in the absence of bFGF and CM. We found that compound C (1 P M), a kinase inhibitor, supported the growth of hE SCs as compact colonies, reminiscent of control cells cultured with bFGF and CM (Fig. S1A). In addition to preventing the differentiation morphology, compound C greatly attenuated the reduction of OCT-4, SOX2 and NANOG proteins (Fig. 1A, Fig. S1B). The supportive effects of compound C on OCT-4, NANOG and SOX2 expression were further confirmed at the transcription level: the mRNA levels of POU5F1(the gene encoding OCT-4), SOX2 and NANOG in compound C-treated hESCs were significantly higher than in cells without the treatment (Fig. 1B). Interestingly, the rescuing effect of compound C on NANOG expression was weaker than POU5F1 and SOX2. Furthermore, compound C markedly prevented the differentiation morphology and down-regulation of pluripotency markers in a pluripotent human embryonal carcinoma cell line, NTera-2 cells (Supporting Information) (Fig. S1C and S1D), suggesting a conserved effect in human pluripotent stem cells.

Compound C profoundly influenced the differentiation activities in h E

SCs. It significantly reduced the expression of markers for mesoderm (MESP1,T and MIXL1),

endoderm (GATA4,GATA6) and trophoectoderm

(CDX2 and CGB7) in H1 cells cultivated in the absence of bFGF and CM (Fig. 1C), but caused the expression of neural ectoderm markers SOX1

and NEUROD1to moderately increase (Fig. 1C).

The effect of compound C on h E

SC differentiation was further verified with the embryoid body (EB) formation assay. During EB formation, expression of differentiation markers was consistently up-regulated in untreated control cells (Fig. 1D and Fig. S1E) [17]. In this assay, compound C treatment markedly prevented the up-regulation of markers for endoderm, mesoderm and trophoectoderm, but enhanced the expression of neuroectoderm markers after E B induction (Fig. 1D and Fig. S1E). Compound C treatment had no effects on the size or the number of EBs (Fig. S1F). High-Efficiency Induction of Neural Conversion by Compound C

Because compound C potently suppressed endoderm, mesoderm and trophoectoderm but enhanced neural ectoderm activities in hE SCs, we asked whether it could be applied to the induction of neural conversion. To test this, we developed differentiation conditions for hE SCs using adherent cell culture, which is more consistent and easier to manipulate than the EB condition. Cells dissociated by accutase (1 mg/mL) were plated on Matrigel and continuously cultured to confluency in mTeSR medium, which could eliminate variations introduced by the ME F feeders. Differentiation was initialized by replacing mTeSR with DM E

M/F12 medium containing knock-out serum replacement (KSR) (but lacking bFGF and CM) and compound C (1 μM). Another small-molecule inhibitor, SB431542, which inhibits type I TGF-E receptors and reportedly promotes neural conversion in hESCs [12], was used with or without compound C. We used PAX6, a neural progenitor marker, to monitor early neural conversion. Human E SCs, when

plated as single cells in the absence of bFGF and CM, began to exhibit differentiation morphology by d 3, and the differentiation was more apparent

by d 5~d 6 with the majority of cells spread out and losing the expression of OCT-4 (Fig. 2A and Fig. S2A). Interestingly, cells treated with compound C grew as a compact uniform

monolayer, in contrast to untreated cells, which often showed areas with disassociated cells and cell clumps (Fig. S2A). Immunofluorescence studies revealed that PAX6 protein was nearly

uniformly expressed in compound C-treated cells (Fig. 2A, middle panel), whereas much fewer untreated cells exhibited anti-PAX6 fluorescence (Fig. 2A, upper panel). Other early neural progenitor markers such as SOX1 and SOX2 were also highly expressed in compound C-treated cells (Fig. 2A, middle panel). Western blot analysis confirmed increased expression of PAX6 and SOX2 proteins (Fig. 2B). Furthermore, western blot analysis also showed that expression of Nestin protein, a neural stem cell marker, was elevated in compound C-treated cells (Fig. 2B). In contrast, addition of SB431542 caused only a slight increase of PAX6 protein and failed to induce further increase of PAX6, Nestin and SOX2 proteins in compound C-treated cells (Fig. 2A and 2B). The expression of pluripotency marker OCT-4 was almost undetectable 7 d after differentiation induction (data not shown). In keeping with the increased level of protein, a gradual increase of PAX6mRNA was also observed during the differentiation process, which lasted for at least 10 d (Fig. S2B).

To determine the fraction of PAX6+

cells, we analyzed the differentiated cells by flow cytometry 7 d to 10 d after compound C treatment. Remarkably, the treatment yielded a high percentage of PAX6+ cells (88.7±2.5%, Fig. 2C and Fig. S2C). There was no significant difference between d 7 to d 10 cells (data not shown). Interestingly, the dual treatment of compound C and SB431542 failed to increase the percentage of PAX6+ cells (80.3%±10.3%) (Fig. 2C and Fig. S2C). Furthermore, while Noggin treatment alone moderately increased the percentage of PAX6+

cells (36.9±1.6%), combined treatment of SB431542 and Noggin significantly increased the percentage (84.7±3.9%) (Fig. 2C and Fig. S2C), consistent with a previous report [10]. The percentage of PAX6+ cells acquired with compound C was slightly higher than with combined treatment of Noggin and SB431542 (Fig. 2C). Thus, the use of compound C, mTeSR medium and KSR-containing DME M/F12 medium enabled us to develop a single-step cost-effective procedure for high-efficiency neural conversion in hE SCs in defined adherent cultures.

Compound C also induced high-efficiency neural conversion in hiPSCs. We used hiPSCs derived from human fetal lung fibroblasts (IMR-90) for neural induction by following the same procedure developed for h E

SCs. Immunofluorescence and western blot analyses revealed a drastic increase of PAX6 and SOX2 proteins in cells treated with compound C (1 P M, 7 d) (Fig. 2D and 2E ). Similar to hE SCs, compound C treatment of hiPSCs induced a high population of PAX6+ cells (70.4%±5.5%) (Fig. 2C). These results demonstrate that compound C alone induces high-efficiency neural conversion in hE SCs and hiPSCs and could be used as a powerful tool in large-scale generation of neural progenitors for therapeutic applications.

The terminal differentiation potential of PAX6+cells derived with compound C was confirmed. Immunofluorescence of Neuron specific class ????-tubulin (TUJ1), a pan-neuronal marker, was nearly uniformly expressed in cells after 2-week

neural maturation (Fig. 2F, I). In contrast, TUJ1 immunofluorescence was not detected in undifferentiated hESCs or compound C-induced PAX6+cells (data not shown). In addition, a fraction of the cells also expressed Nur-related factor 1 (NURR1), a dopaminegic neuron marker (Fig. 2F, II). Thus, the PAX6+neural progenitors derived by the use of compound C have the potential to undergo terminal differentiation and to produce neuronal subtypes. It was reported that compound C/dorsomorphin

can promote cardiomyogenesis in mouse E SCs (mESCs) [21], prompting us to examine whether it could exert the same effect in hE SCs. However, we found that compound C failed to induce cardiomyogenesis in h E

SCs (Supplementary Text; Fig. S3). In addition, treatment of mESCs with compound C failed to induce early neural conversion (Supplementary Text; Fig. S3). The differential effects of

compound C in mESCs and hESCs might be due to their differences in species, origin of

development or other undefined characteristics. Some known signaling pathways mediating self-renewal and differentiation of mE SCs are not conserved in hESCs [22, 23].

Compound C Targets Several Families of Protein Kinases

Compound C is widely used as a ATP-competitive inhibitor of AMP-activated kinase (AMPK), a multimeric protein complex that regulates cellular and organismal metabolism [24, 25], but recently its specificity has been questioned [26, 27]. In keeping with this, compound C has been shown to also target the type I BMP receptors (ALK2, ALK3 and ALK6) and likely other protein kinases [27, 28]. To gain a broader, more comprehensive view of the potential targets of compound C, we assessed its effects on a panel of kinases (402) in a large-scale in-vitro kinase assay, as described [29]. This assay identified far more targets of compound C than previously recognized (Fig. 3A and Table S2). Notably, compound C robustly inhibited many other members of TGF-

E superfamily receptors in addition to ALK2, ALK3 and ALK6 and of the SNF/AMPK family kinases in addition to AMPK D1 and D2 (Fig. 3A). Furthermore, compound C potently inhibited PDG

F receptors (PDGFR D and PDGFR E), VEGFR receptors [VEGFR1 (FLT1) and VEGFR2 (KDR)], FLT3 and KIT (Fig. 3A). To determine if AMPK inhibition was responsible for the effects of compound C on hESCs, we abolished the function of AMPK by depleting the ?1 and ?2 subunits. Surprisingly, depletion of either ?1 or ?2 subunit failed to support the undifferentiated growth and pluripotency of hE SCs and instead markedly decreased the levels of SOX2 protein (Fig. S4). Furthermore, AICAR, an AMPK activator, failed to rescue the phenotypic changes caused by compound C in hE SCs. Based on these results, we concluded that AMPK inhibition was not responsible for the effect of compound C. Inhibition of PDGF receptors in hE SCs also failed to mimic the effects of compound C. Treatment of hESCs with PDGF receptor kinase inhibitor V slightly decreased cell proliferation (data not shown).

Compound C Inhibits Both Activin And BMP Signaling in hESCs

Two type II receptors, ActRIIA and ActRIIB, mediate Activin signaling [30] and were markedly inhibited by compound C in vitro (Fig. 3A), leading us to assess whether compound C targeted this pathway in hE SCs. We found that compound C inhibited the activity of a luciferase-based Activin reporter system (pARE-GL3, [19]) in a dose-dependent manner (Fig. 3B). At a dose that effectively modulated hESC differentiation (1 P M), compound C consistently reduced the Activin reporter activity triggered by the addition of Activin A (100 ng/mL), to a similar extent to low concentration of SB431542 (250 nM) (Fig. 3B), which inhibits type I receptors including ALK4, ALK5 and ALK7 and consequently blocks the Activin signaling pathway, without affecting BMP signaling [31].

A higher dose of compound C (10 P M) was required to completely prevent the Activin reporter activity. In keeping with reduced reporter activity, compound C (1 P M) significantly attenuated the increase in the nuclear phospho-Smad2/3 levels in h

E

SCs induced by Activin A (Fig. 3C). Thus, compound C potently inhibited the Activin pathway in hE SCs. Interestingly, inhibition of Activin signaling by compound C was observed in mouse pulmonary artery smooth muscle cells (PASMCs) at higher concentrations [28]. The variability might be attributed to the differences in cell types or other undefined characteristics.

Compound C also blocked BMP signaling in hE SCs. Compound C inhibited a luciferase-based inhibitor of DNA binding (ID) reporter activity (Fig. S5A). It reduced BMP-4-induced activation (phosphorylation) of Smad1/5/8 in hE SCs (Fig. S5B). Thus, compound C potently inhibited the BMP-4 signaling pathway in hE SCs. These results are consistent with the

ability of compound C to inhibit both type I (ALK2, ALK3 and ALK6) and type II BMP receptors (ActRIIA and ActRIIB) in vitro. The effects of compound C on BMP signaling in hE SCs confirm and extend previous studies in other model systems [28].

Compound C Prevents Differentiation Activities Induced By Activin A and BMP-4

It has been well established that activation of Activin signaling triggers endoderm activities in E SCs [9, 32]. Furthermore, Activin A can directly act to down-regulate PAX6 expression [33]. In RPMI medium containing low serum, Activin A induces efficient differentiation of hE SCs to definitive endoderm [9]. We asked whether compound C blocked Activin A - induced endoderm activities. hE SCs exhibited reduced survival in RPMI medium after the addition of compound C, prompting us to use a custom mTeSR medium instead, which lacks bFGF and TGF-E and thus causes cells to differentiate. Compound C did not cause apoptosis of cells cultured in the custom mTeSR medium. As expected, the addition of Activin A (100 ng/mL) induced up-regulation of endoderm markers including SOX17,GSC, and FOXA2 in hESCs (Fig. 3D). The presence of compound C

(1 μM) reduced the up-regulation (Fig. 3D).

E xpectedly, SB431542, at a dose commonly used to inhibit Activin signaling (10 P M) [31], nearly completely prevented the up-regulation of the endoderm markers (Fig. 3D). In contrast, Noggin, a secreted protein that antagonizes BMP signaling, exerted little effects (Fig. 3D). Thus, compound C decreased Activin A-induced endoderm activities in hE SCs, separately from its actions on BMP receptors.

BMP-4 induced mesoderm or trophoectoderm activities in hE SCs, depending upon the length of stimulation [7, 8]. We thus treated hE SCs with BMP-4 (20 ng/mL) for 24 h and 6 d, with or without compound C (1 μM). Short-term BMP-4 stimulation (24 h) induced a rapid up-regulation of mesoderm markers, including T, MESP1and MIXL1 (Fig.S5C). Compound C nearly completely abolished the up-regulation (Fig. S5C). In addition, compound C prevented the up-regulation of CDX2 and down-regulation

of pluripotency markers in hE SCs after long-term exposure to BMP-4 (Supporting Information) (Fig. S5D and S5

E

). Thus, compound C inhibited BMP-4-induced mesoderm and trophoectoderm activities in hESCs.

The large-scale in vitro kinase assay showed that compound C targeted all three type I and two type II BMP receptors, which might explain its potent inhibition of BMP signaling activities in hESCs. To test this possibility, we compared the effects of depleting a single or multiple BMP receptors. Indeed, depletion of ALK2, ALK3 or ALK6 alone failed to affect BMP4-induced signals (Fig. S6A). In contrast, depletion of all three receptors nearly abolished BMP-4-induced signals including Smad1/5/8 phosphorylation, and up-regulation of the early BMP-4 responsive gene ID1and the differentiation markers T, MESP1 and CDX2 (Fig. S6B). Moreover, addition of compound C to the receptor-depleted cells caused no additional effects on Smad1/5/8 phosphorylation (Fig. 3E). Depletion of two type

I receptors also attenuated up-regulation of target genes, although the effects were less prominent than those from depleting all three (Fig. S6B). Furthermore, in cells induced to spontaneously differentiate, mRNA levels of mesoderm and trophoectoderm differentiation markers T and CGB7were reduced in cells with triple depletion, whereas the decrease of the pluripotency and neuroectoderm markers such as POU5F1,NANOG and SOX1was attenuated (Fig. S6C). These results suggest that inhibition

of at least two receptors is necessary for robust inhibition of BMP signals. Together, the ability

to target multiple TGF-E family receptors, dual inhibition of Activin and BMP signaling and suppression of Activin- and BMP-induced endoderm, mesoderm and trophoectoderm activities provide mechanistic explanations for the strong effects of compound C in hESCs.

MSX2 as A Compound C's Target Gene and a Key Intermediate of BMP Signaling in hESCs To identify potential target genes and signaling pathway(s) of compound C in hE SCs, we used Affymetrix-based whole-genome microarrays to compare global gene-expression profiles of differentiating hE SCs (induced by bFGF and CM removal) with and without compound C treatment. Focusing on early times (12 and 24 h) after treatment, we sought to identify early responsive gene(s) and/or signaling pathway(s). Microarray analyses of cells at 12 and 24 h identified a small number of genes with 1.5-fold altered expression (Table S3). Interestingly, a subset of transcription factors, including ID2, muscle segment homeobox gene 2 (MSX2) and distal-less homeobox 2 (DLX2), were down-regulated 12 and 24 h after compound C treatment (Table S3, highlighted in yellow). Among those, IDs are shown as downstream genes of the BMP signaling in hE SCs and mE SCs [7, 34]. Real-time PCR confirmed and extended these findings: transcription of the ID family members (ID1-ID3) and MSX family members (MSX1 and MSX2) were markedly suppressed after compound C treatment (Fig. 4A).

Despite the established roles of MSX family transcription factors in development, their functions have not been depicted in hESCs. We explored their potential functions in mediating BMP signaling pathway. BMP-4 stimulation rapidly up-regulated the mRNA levels of MSX1 and MSX2 in hE SCs (as early as 30 min post-stimulation) (Fig. 4B). Concomitant to the increase in mRNA, MSX2 protein levels were also elevated after BMP-4 stimulation (Fig. 4C). In contrast, BMP-4 stimulation failed to alter levels of MSX1 protein, and in addition, mRNA levels of MSX1were significantly lower than those of MSX2 in hESCs.

Depleting MSX2 prevented the morphological changes induced by BMP-4 in hESCs (Fig. 4D). Western blot analysis showed that MSX2 depletion also attenuated the down-regulation of OCT-4 and SOX2 (Fig. 4E). Interestingly, the rescuing effect of MSX2 depletion on OCT-4 expression was more prominent than SOX2. In addition, down-regulation of mRNA levels of POU5F1, SOX2 and NANOG was much slower

in MSX2-depleted cells than control cells after BMP-4 stimulation. These results indicate that MSX2 acts as a key component of BMP signaling in hE SCs. Future experiments will dissect the potential role of the DLX family members in regulating BMP signals or other signaling pathways, pluripotency and differentiation in hESCs.

D ISCUSSION

Developing conditions and factors for efficient directed differentiation of human pluripotent stem cells such as hESCs and hiPSCs is essential

for their potential applications for the study of early human development, drug discovery and the treatment of diseases. Two strategies have been explored. One is to manipulate developmental pathways via the use of growth factors and their antagonists, and the other is to identify chemical compounds to direct the differentiation activities. The benefits of utilizing compounds to control stem cell fate include the elimination of animal products, better temporal and tunable control, ease of production and significant cost reduction for materials. Despite these benefits, there is no documentation to date

for compounds that induce efficient differentiation from hESCs and hiPSCs to neural progenitors. Here we identified compound C, which potently suppresses mesoderm, endoderm and trophoectoderm activities and can be applied

to high-efficiency neural conversion in hE SCs and hiPSCs. Our study provides the first example for the efficient derivation of neural progenitor cells from human pluripotent stem cells with a single chemical compound.

We found that compound C can induce nearly 90% of hE SCs to form neuroectoderm in adherent cultures. Why is this compound so potent in inducing neural conversion? Our results suggest the following mechanisms. Compound C inhibits at least seven type I and

type II TGF-E superfamily receptors, including two Activin receptors and five BMP receptors, thus effectively preventing Activin and BMP signals. Indeed, our experiments show that depletion of at least two BMP receptors is necessary for robust inhibition of BMP-induced signaling activities. Furthermore, dual inhibition

of the BMP and the Activin pathway is necessary for efficient neural conversion in hESCs. In this scenario, inhibition of Activin and BMP signaling suppresses endoderm, mesoderm and trophoectoderm activities, thereby

facilitating h

E SC differentiation into the

neuroectoderm lineage. This notion is supported by our observation and a recent report that inhibition of BMP and Activin signaling alone improves the efficiency of neural induction, but combined inhibition (with Noggin and SB431542) leads to high-efficiency neural conversion (Fig. 2) [10]. Therefore, our results identified TGF-E superfamily receptors and the Activin and BMP signaling pathways as the main biological targets in hESCs. The ability to target multiple TGF-E family receptors, dual inhibition of Activin and BMP signaling and suppression of Activin- and BMP-induced endoderm, mesoderm and trophoectoderm activities could account for the effects of compound C on differentiation and neural conversion in hESCs.

Neural progenitors and more specified functional neural subtypes were derived previously from hESCs by using EBs as the initial step [35, 36]. While the EB-based procedures are effective and flexible in producing desired differentiated cell types, they often involve multiple steps, produce multiple differentiation lineages and require subsequent isolation/enrichment of specific differentiated cells. These limitations could hinder the acquisition of efficient neural conversion and the generation of large-scale neural precursors. In our study, we applied a single chemical compound to hESCs and hiPSCs cultivated in adherent cultures and achieved rapid, high-efficiency neural conversion in a single step. Compared with the Noggin - based protocol [10], our method produces a slightly higher efficacy of induction and also offers some other potential benefits in the long term. First, compound C costs significantly less than Noggin, thereby markedly reducing the cost for deriving neural progenitor cells from hESCs and hiPSCs, especially on a large-scale. Second, utilization of the defined medium mTeSR [37] prior to the induction of neural conversion eliminates the variability introduced by the ME Fs or ME F-CM. Furthermore, xeno-free TeSR2 and KSR media can be used if more clinically-compatible culture conditions are desired. Third, compound C can be conveniently delivered and removed and is therefore highly suited for therapeutic applications. Thus, our method is highly suitable for large-scale efficient derivation of potentially therapy-grade neural precursors from hE SCs and hiPSCs and should facilitate the utilization of these cells for the study of early human neural development, drug discovery, tissue repair and regeneration. Compound C had long been used as a specific inhibitor against AMPK [24, 25]. It was recently found to inhibit the type I BMP receptors ALK2, ALK3 and ALK6 and induce dorsalization in zebrafish (thus denoted as dorsomorphin) [28]. Our large-scale proteomic analysis confirmed these known targets but discovered far more additional targets, including many other members of the TGF-E superfamily receptors, of the SNF/AMPK family and PDGF receptors. This approach served as a starting point, enabling us to identify the targets and pathways of compound C in hE SCs. Our findings might explain the variability in previous studies with compound C and could provide a platform for further analysis of its potential targets in various model systems. They also suggest that experimentation with kinase inhibitors should be evaluated critically under the specific cellular and biological context. As such, our results outline a strategy that may be utilized in future studies with kinase inhibitors.

A CKNOWLEDGMENTS

We thank Dr. Renhe Xu for providing the ID-120-luc reporter construct and Dr. Yisrael Sidis for providing the pARE-GL3 construct, Jenny Drnevich for analyzing the microarray data and members of the Wang lab for helpful discussions. Support was provided by NIH (GM-83812; to F.W.), the Illinois Regenerative Medicine Institute (IDPH 2006-05516; to F.W.), NSF CARE E R award (0953267; to F.W.), the Beckman award from the University of Illinois and the National Natural Science Foundation of China (30728022; to F.W. and E.D.). Disclosure of Potential Conflicts of Interest The authors indicate no potential conflicts of interest.

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Fig. 1. Compound C suppresses mesoderm, endoderm and trophoectoderm differentiation in hESCs.

(A) Immunofluorescence studies of OCT-4, SOX2 and NANOG proteins (red) in H1 cell colonies cultured in complete growth medium (CM + bFGF), medium with KSR only, and medium with KSR and 1 P M compound C for 6 d. All three pluripotency markers were expressed in 92.5%±4.7% of the colonies (i.e., with CM and bFGF). Removal of CM and bFGF reduced the OCT-4-, SOX2- and NANOG-positive colonies to 8.1%±3.2%, and addition of compound C increased it to 88.4%±6.5%. Nuclei were stained with DAPI (blue). H9 cells showed similar response to compound C treatment. Dose-dependent experiments with compound C (100 nM, 200 nM, 500 nM, 1 P M and 2 P M) showed that 1 P M was necessary to give rise to the strong anti-differentiation effect. Scale bar, 200 μm. (B) mRNA levels of SOX2,POU5F1 and NANOG in H1 hE SCs cultured in aforementioned three conditions for 6 d, assessed by real-time PCR. All values were normalized to the level (=1) of mRNA in the cells treated with KSR alone. *, p<0.05; **, p<0.01. Cells treated with compound C were compared to control cells (i.e., with KSR only) treated with vehicle. ACTB (E-actin gene) was used as an internal control. (C) mRNA levels of differentiation markers in H1 hESCs cultured in medium with KSR only and medium with KSR and 1 P M compound C for 6 d, assessed by real-time PCR. All values were normalized to the level (=1) of mRNA in the cells treated with KSR alone. *, p<0.01; **, p<0.001. Cells treated with compound C were compared to control cells treated with vehicle. NE and TE denote neuroectoderm and trophoectoderm, respectively. (D) mRNA levels of control E Bs or compound C-treated EBs assessed by real-time PCR. Four separate experiments were conducted, and quantification of three replicates of a typical experiment is shown. E ach bar represents the mean ± SEM (error bars). All values were normalized to the mRNA level (=1) of in the control cells on day 0. *, p<0.05; **, p<0.01. EBs treated with compound C were compared to control EBs on day 4.

Fig. 2. Compound C induces high-efficiency neural conversion. (A) Immunofluorescence studies of PAX6 (left panel, red) and SOX1, (middle panel, green) and SOX2 (right panel, red) in differentiating H1 hE SCs. Cell nuclei were stained with DAPI (blue). In contrast to control cells, which exhibited minimal anti-PAX6 fluorescence, H1 cells treated with compound C alone (1 μM) or with compound C (1 P M) and SB431542 (50 nM) combined expressed high level of PAX6 after 7-d differentiation. SOX1 and SOX2 were also highly expressed in compound C or compound C and SB431542 treated cells. Bar, 200 P m. (B) Western blot analysis of PAX6, Nestin and SOX2 in H1 cells with three treatments. Cells were induced to differentiate for 7 d. A typical experiment from five separate experiments is shown. D-tubulin was a loading control. (C) The percentage of PAX6+ cells assessed by flow cytometry. H1 hE SCs treated with compound C alone or with compound C and SB431542 contained 88.7±2.5% and 80.3±10.3% (mean ± SEM) PAX6+ cells, respectively. In contrast, control cells and cells treated with SB431542 showed 16.9±1.9% and 17.7±0.4% PAX6+ cells. Treatment with Noggin (500 ng/mL) alone or combined with SB431542 gave rise to 36.9±1.6% and 84.7±3.9% PAX6+ cells, respectively. Similarly, compound C treatment of IMR-90-derived hiPSCs induced a high population of PAX6+ cells (70.3%±5.5%), in contrast to untreated cells (4.8%±2.1%). (D) Immunofluorescence studies of PAX6 (top panel, red) and SOX2 (bottom panel, red) in differentiating hiPSCs. Cell nuclei were stained with DAPI (blue). Cells were treated or not treated with 1 P M compound C for 7 d. scale bar, 50 μm. (E) Western blot analysis of PAX6 and SOX2 in hiPSCs treated or not treated with 1 P M compound C. Cells were induced to differentiate for 7 d. D-tubulin was a loading control. (F) Immunofluorescence studies of TUJ1 and NURR1 in terminally differentiated H1 hESCs. Anti-TUJ1 (?) and NURR1 (??) fluorescence (red) was observed in H1-derived differentiated cells after 2-w neural maturation. Nuclei were stained with DAPI (blue). Scale bar, 50 μm.

Fig. 3. Compound C targets the TGF-E superfamily receptors and blocks Activin and BMP signaling.

(A) Some of the kinases inhibited by 1 μM compound C and the degree of inhibition, as revealed by the in vitro kinase assay. The complete list of kinases examined is shown in Supplementary Table S2.

(B) Relative luciferase activity of the pARE-GL3 reporter in H1 cells with or without Activin A (100 ng/mL, 24 h), treated with different concentrations of SB421542 or compound C. Results from four separate experiments are shown as mean±SE M. *, p<0.05 ; **, P<0.01; ***, p<0.001. Cells with various treatments were compared to control (with Activin stimulation). Hint amount of Rellina plasmid was co-transfected as an internal control. (C) Western blot analysis of phosphorylated Smad2/3 and total Smad2/3 in the nuclear fraction of H1 cells treated with various concentrations of compound C and stimulated with Activin-A (100 ng/mL) for 24 h. A typical experiment from four separate experiments is shown. Total Smad2/3 was a loading control. Compound C (1 P M) reduced the nuclear p-Smad2/3 levels by approximately 25%. (D) mRNA levels of endoderm markers SOX17, FOXA2 and GSC in H1 cells with various treatments, assessed by real-time PCR. Cells were stimulated with Activin A (100 ng/mL) for 5 d. Four separate experiments were conducted, and quantification of three replicates of a typical experiment is shown. E ach bar represents the mean ± SEM (error bars). All values were normalized to the mRNA level (=1) of in the control cells. Activin and compound C treatments vs Activin alone were compared (*, p<0.05; **, p<0.01). (E) Western blot analysis of Smad1/5/8 phosphorylation under various conditions. Similar to 1 P M compound C treatment, depletions of ALK2, ALK3 and ALK6 altogether (denoted as Triple KD) attenuated the phosphorylation of Smad1/5/8 induced by BMP-4 (20 ng/mL, 30 min). Further addition of 1 P M compound C to cells lacking the three receptors exerted no detectable effects. A typical blot is shown in the bottom panel, and quantification of blots from four separate experiments is shown in the top panel.D-tubulin was a loading control. Y-axis represents relative intensities (measured with Image J) with values normalized to the signal (=100%) without BMP-4 or compound C treatment. E ach bar represents mean ± SE M (error bars). Student t tests compared data between cells treated with compound C vs control cells after BMP-4 addition (**, p<0.01).

Fig. 4. MSX2 as a target gene of compound C and a key intermediate of the BMP pathway. (A) mRNA levels of ID1,ID2,ID3,MSX1 and MSX2 in H1 hESCs induced to differentiate by the removal of bFGF and CM, with or without the treatment of 1 P M compound C, assessed by real-time PCR. Four separate experiments were conducted, and quantification of four replicates of a typical experiment is shown. Bars represent mean ± SE M (error bars). All values were normalized to the mRNA level in the control cells at 0 h. Cells treated with compound C were compared to control cells treated with vehicle. **, p<0.01; ***, p<0.001. (B) The mRNA level of MSX2 in H1 hE SCs after BMP-4 treatment (20 ng/mL) for various time points assessed by real-time PCR. (C) Western blot analysis of MSX2, OCT-4 and SOX2 in H1 cells after BMP-4 treatment (20 ng/mL) for various time points. (D) Phase-contrast images of H1 cells with MSX2 depletion 4 d after BMP-4 treatment (20 ng/mL). Two separate shRNAs were used to verify the effects of MSX2 depletion, while scramble shRNA was used as a control. Scale bar, 50 μm. ( Western blot analysis of MSX2, OCT-4 and SOX2 in H1 cells with MSX2 depletion 4 d after BMP-4 treatment (20 ng/mL). Two separate shRNAs (sh4849 and sh4850) were used to verify the effects of MSX2 depletion. D-tubulin was used as loading controls for (C) and (E).

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关于神经干细胞

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形态学观察方法：利用各种染色法可观察到凋亡细胞的各种形态学特征： （1）DAPI时常用的一种与DNA结合的荧光染料。借助于DAPI染色，可以观察细胞核的形态变化。 （2）Giemsa染色法可以观察到染色质固缩、趋边、凋亡小体形成等形态。 （3）吖啶橙(AO)染色，荧光显微镜观察，活细胞核呈黄绿色荧光，胞质呈红色荧光。凋亡细胞核染色质呈黄绿色浓聚在核膜侧，可见细胞膜呈泡状膨出及凋亡小体。 （4）吖啶橙(A())／溴化乙啶(EB)复染可以更可靠地确定凋亡细胞的变化，AO只进入活细胞，正常细胞及处于凋亡早期的细胞核呈现绿色；EB只进入死细胞，将死细胞及凋亡晚期的细胞的核染成橙红色。 （5）台盼蓝染色对反映细胞膜的完整性，区别坏死细胞有一定的帮助，如果细胞膜不完整、破裂，台盼蓝染料进入细胞，细胞变蓝，即为坏死。如果细胞膜完整，细胞不为台盼蓝染色，则为正常细胞或凋亡细胞。使用透射电镜观察，可见凋亡细胞表面微绒毛消失，核染色质固缩、边集，常呈新月形，核膜皱褶，胞质紧实，细胞器集中，胞膜起泡或出“芽”及凋亡小体和凋亡小体被临近巨噬细胞吞噬现象。 （6）木精-伊红（HE）染色是经典的显示细胞核、细胞质的染色方法，染色结果清晰。发生凋亡的细胞经HE染色后，其细胞大小的变化及特征性细胞核的变化：染色质凝集、呈新月形或块状靠近核膜边缘，晚期核裂解、细胞膜包裹着核碎片“出芽”凸出于细胞表面形成凋亡小体等均可明显显示出来。 DNA凝胶电泳：细胞发生凋亡或坏死，其细胞DNA均发生断裂，细胞小分子 质量DNA片段增加，高分子DNA减少，胞质出现DNA片段。但凋亡细胞DNA断裂点均有规律的发生在核小体之间，出现180～200 bp DNA片段，而坏死细胞的DNA断裂点为无特征的杂乱片段，利用此特征可以确定群体细胞的死亡，并可与坏死细胞区别。

神经干细胞的应用前景及研究进展

神经干细胞的应用前景及研究进展 生科1301班李桐 1330170031 神经干细胞( neuralstem cells, NSCs)是重要的干细胞类型之一，是神经系统发育过程中保留下来的具有自我更新和多向分化潜能的原始细胞,可分化为神经元、星形胶质细胞、少突胶质细胞等多种类型的神经细胞。具有很多的特性，如自我更新、多潜能分化、迁移和播散、低免疫原性、良好的组织相容性、可长期存活等。目前神经干细胞的分离与体外培养已取得可喜的进展，有关神经干细胞的研究已经成为国内外神经科学领域的热点。 一、神经干细胞的生物学特性 19世纪80年代提出了神经干细胞的概念，它是指一类多潜能的干细胞，能够长期自我更新与复制，并具有分化形成神经元、星形胶质细胞的能力。神经干细胞的主要特征：未分化、缺乏分化标记、能自我更新并具有多种分化潜能。它并不是指特定的单一类型的细胞，而是具有相类似性质的细胞群。Gage将神经干细胞的特性进一步描绘为以下三点，可生成神经组织或来源于神经系统，具有自我更新能力，可通过不对称法、分裂产生新细胞。神经干细胞经过不对称分裂产生一个祖细胞和另一个干细胞，祖细胞只有有限的自我更新能力，并自主分化产生神经元细胞和成胶质细胞。神经干细胞是具有自我更新和具有多种潜能的母系神经细胞，它能分化成各种神经组织细胞表型，如神经元、星形胶质细胞和少突胶质细胞.并能自我更新产生新的神经干细胞，在神经发育和神经损伤中发挥作用。神经干细胞移植、迁移及分化与局部环境密切相关，这种特性为移植及移植后的结构重建和功能恢复提供了依据，为移植治疗不同疾病提供了局可能。 二、神经干细胞的应用前景 1.细胞移植以往脑内移植或神经组织移植研究进展缓慢，主要受到胚胎脑组织的来源、数量以及社会法律和伦理等方面的限制。神经干细胞的存在、分离和培养成功，尤其是神经干细胞系的建立可以无限地提供神经元和胶质细胞，解决了胎脑移植数量不足的问题，同时避免了伦理学方面的争论，为损伤后进行替代治疗提供了充足的材料。研究表明，干细胞不仅有很强的增殖能力，而且尚有潜在的迁移能力，这一点为治疗脑内因代谢障碍而引起的广泛细胞受损提供了理论依据，借助于它们的迁移能力，可以避免多点移植带来的附加损伤。另外，神经干细胞移植也为研究神经系统发育及可塑性的实验研究提供了观察手段，前文提及细胞因子参与调控神经元增殖和分化，通过移植的手段对这些因素的具体作用形式和机制进行探索，为进一步临床应用提供了理论基础。 2.基因治疗目前诱导干细胞向具有合成某些特异性递质能力的神经元分化尚未找到成熟的方法，利用基因工程修饰体外培养的干细胞是这一领域的又一重大进展；另外已经发现许多细胞因子可以调节发育期甚至成熟神经系统的可塑性和结构的完整性，将编码这些递质或因子的基因导入干细胞，移植后可以在局部表达，同时达到细胞替代和基因治疗的作用。 3.自体干细胞分化诱导移植免疫至今为止仍是器官或组织移植的首要问题。前文提到已经证明成年动物或人脑内、脊髓内存在着具有多向分化潜能的干细胞，那么使人们很容易想到通过自体干细胞诱导来完成损伤的修复。中枢神经系统损伤后，首先反应的是胶质细胞，在某些因子的作用下快速分裂增殖，形成胶质瘢。其实在这个过程中也有干细胞的参与，可不幸的是大多数干细胞增殖后分化为胶

牙髓干细胞向神经细胞方向的诱导分化实验

万方数据

万方数据

华西口腔医学杂志第篮卷第４期２００７年８月 ———— 一一里型！１１些地婴坐堂皇！型些！臣！！！堑盟型些ｇ：！婴！：塑！： 物经琼脂糖凝胶电泳分折，可见一条约３８０ｂｐ大小 的扩增产物条带，与ＧＦＡＰ预期片段大小基本一致。 内参对照ＧＡＰＤＨ也可见９００ｂｐ的清晰条带（图４）。 ２．２免疫细胞化学染色１＋Ｍａ妇２：Ｇ８Ａ９和诱导细胞，３：ｃＦＡ’耳口束诱导细胞４： 诱导细胞抗黑∑世氍ｅ鲞掣肌岬：、”冒：Ｉ鬻翟：詈：黧鬻：…。，。。呈 ３），对照组这两种抗体染色均呈阴性。一 阳性表选 囤２坤经诱导４ｄ盾牙髓干细胞抗ＧＦＡＰ的阳性表远ＡＢｃ×２００ ，培２ｈ椰ｖｅ８叩哪ｌ…ｍ曲ｔｌｂｏｄ）∞ｃＦＡＰ缸ｄｅⅡｋｌＦＬＩｌｐｓｔ…ｅⅡｓⅡｅａｔｅｄ诵ｔｈＩ）叫口ｌｊｎｄｕｃｂｖｅｒｒ?刚ｉｕｍＲⅡ４ｄＨＹｓ ＡＢＣＨ２００ 幽３砷经诱导４ｄ后，牙髓Ｔ细胞抗Ｎ虬的｜５Ｈ性表述ＡＢｃ×２００ Ｆ咱３Ｐｏｇｉｄ…砷…ｍ们【ｈａ埘ｂｏｄｙｔ０ＮｓＥ０ｆｄｅｎ词ｐＵ如“Ｌｅ…ｄｋｈ即Ｌ出ｗＪⅡＩＪ?ｅｍ“１ｎｄｌｍｄ…ｌ。出Ｉｌｒｔ＿ｈ４ｄａｖｓ ＡＢＣ×Ⅻ ２＿３１ｗ—Ｐｃｌ｛检测 咀未诱导细胞为对照，诱导细胞的ＲＴ—ＰｃＲ产３讨论 成体干细胞的横向分化潜能或称为可塑性是指其不仅能分化为来源组织的各种细胞类型，而且具有分化为其他胚层来源的成熟细胞的潜能Ｈ。自从１９９７年有关成体干细胞可塑性的系列研究＝ｌ挂道后，可塑性研究引起生命科学领域的极大关注，对干细胞的研究产生重要影响。而这其中研究最多、可塑性最强的是骨髓基质干细胞（ｂｏｎｃｍｃｓｅｎｃｈｙｍａｌｓｔｅｍｃｅｌｌｓ－ＢＭｓｃｓ），可以诱导分化为骨髂肌细胞、心肌细胞、内皮细胞、肝细胞、胆管上皮细胞、肺上皮细胞、肠上皮细胞、皮肤上皮细胞、神经细胞、脂肪细胞等，其中最引八注目的是其神经方向的分化潜能。 Ｅｄｉｔｉｓ等”研究表明小胶质细胞、星形胶质细胞是来源于骨髓的一种前体细胞。而Ｂｒａｚｅｌｔｏｎ等‘啦过绿色荧光蛋白基因转染示踪研究发现，在脑内微环境下，ＢＭｓｃｓ能分化为神经元样细胞、星形胶质样细胞，除表达相应神经细胞表型特点外，尚有神经元样功能。而在体外，削ＤＭｓＯ、ＢＨＡ、Ｂ—ＭＥ等作为主要诱导剂，可诱导成年大鼠和人的ＢＭＳＣｓ分化为神经元和神经胶质细胞，诱导后细胞在形态上出现类似于神经元样突起，表达ＮｓＥ、ｎｅｓｔｉｎ等神经细胞特异性标志㈣。此外，表皮生长｜盘｜子或脑源性神经生长因子与维甲酸或胎鼠的中脑、纹状体的悬液也可体外诱导ＢＭｓｃｓ分化为幼稚的神经兀和神经胶 质细胞口。可见体外不同培养条件下都可诱导ＢＭｓｃｓ　万方数据

神经干细胞体外诱导分化为胆碱能神经元的研究进展.

神经干细胞体外诱导分化为胆碱能神经元的研究进展 硕士研究生孙晓静导师孙莉 传统的观点认为，“中枢神经细胞不可再生”，然而自1992年Reynolds等从成年鼠脑纹状体和海马中首次分离出了神经干细胞（NSC）,且它与多能干细胞一样具有自我更新、分裂增殖、多向分化的潜能，此后人们开始深入了解神经干细胞。Erikson等于1998年也证实了人脑中同样存在神经干细胞，随后的研究发现成年脑中神经干细胞主要存在于侧脑室外侧壁脑室下区和海马齿状回中。随着近年来对中枢神经系统的损伤或病变部位实行细胞替代或移植治疗的研究深入，为中枢神经系统损伤、神经系统变性疾病或神经退行性疾病等的治疗提供了新的方向。 阿尔茨海默病（AD）是老年人常见的一种慢性进行性神经系统变性疾病。长期以来认为前脑胆碱能投射系统（尤其是Meynert基底核）胆碱能神经元变性、细胞数量减少等导致AD认知功能不可逆的减退。目前就其病因有多种学说：如基因学说（APP基因[ 1 ]、早老蛋白基因[ 2 ] 、ApoE基因[ 3 ] ）、胆碱能假说、雌激素水平下降学说[4] 、氧化应激学说[ 5 ] 、铝中毒学说、炎症学说[6 ] 、神经营养因子学说[7 ] 等。此外，AD还可能与年龄、种族、社会心理因素及各种疾病史等多种因素有关。 综上所述, 多因素参与AD 发病, 但目前为止确切的发病机制尚不清楚。其治疗方法包括对于轻度和中度AD 予以乙酰胆碱酯酶(AChE)抑制剂以提高认知功能, 以及N-甲基-D-天冬氨酸(NMDA)拮

抗剂对重度AD 的治疗[8],其他药物如脑循环改善药物剂、AChE 抑制剂、M1 胆碱受体激动剂、乙酰胆碱释放促进剂、神经生长因子、抗氧化药物和抗β-淀粉样药物等。这些治疗手段虽能减轻AD 的症状, 但无法补充大脑皮层和海马大量丢失的神经细胞,因而对中、晚期AD 患者的疗效有限。神经干细胞(NSCs)的广泛研究及重大进展为AD 的治疗提供了一个更有前景的治疗策略，NSC移植治疗AD等退行性疾病成为近年来的研究热点。然而不管是诱导、促进内源性NSCs增殖分化，产生相应的神经细胞代替缺损变性的细胞，还是体外培养需要的NSCs移植入体内，如何诱导NSCs定向分化为胆碱能神经元都是一个至关重要的问题。 神经干细胞增殖、分化相关因素 神经干细胞的增殖、迁移、分化与多种因素相关。目前的研究认为决定神经干细胞定向分化的机制有两种：一种是细胞自身基因调控；另一种是外来因素调控，主要为各类细胞因子家族，如神经营养因子、生长因子、细胞黏附因子等均可影响神经细胞的分化，其影响机制各不相同。不同的细胞因子诱导分化出的神经元不同。研究最多有生长因子如：表皮生长因子（EGF）家族、碱性成纤维细胞生长因子（FGF）家族、β-转导生长因子（β-TGF）超家族、神经营养因子、脑源性生长因子及化学诱导剂及药物等。 一、生长因子 碱性成纤维生长因子(FGF)在神经干细胞增殖的早期阶段发挥促有丝分裂的作用, 使神经干组胞获得对另一作用更强的促有丝分裂

神经母细胞瘤

中文名：神经母细胞瘤 英文名：neuroblastoma 别名：成神经细胞瘤 目录 1概述 2流行病学 3病因 4实验室检查 5其它辅助检查 6临床表现 展开 目录 1概述 2流行病学 3病因 4实验室检查 5其它辅助检查 6临床表现 7并发症 8诊断 9治疗 10预后 概述 神经母细胞瘤(neuroblastomaNB)从原始神经嵴细胞演化而来，交感神经链、肾上腺髓质是最常见的原发部位不同年龄、肿瘤发生部位及不同的组织分化程度使其生物特性及临床表现有很大差异部分可自然消退或转化 成良性肿瘤，但另一部分病人却又十分难治，预后不良鶒。在过去的30 年中，婴儿型或早期NB预后有了明显的改善，但大年龄晚期病人预后仍 然十分恶劣在NB中有许多因素可影响预后，年龄和分期仍然是最重要的 因素健康搜索。 流行病学 NB是儿童最常见的颅外实体瘤，占所有儿童肿瘤的8%～10%，一些高发 地区如法国、以色列瑞士、新西兰等的年发病率达11/100万(0～15岁)，美国为25/100万，中国和印度的报道低于5/100万。

病因 属胚胎性肿瘤，多位于大脑半球。 发病机制： NB来自起源于神经嵴的原始多能交感神经细胞，形态为蓝色小圆细胞。从神经嵴移行后细胞的分化程度、类型及移行部位形成不同的交感神经系统正常组织，包括脊髓交感神经节、肾上腺嗜铬细胞健康搜索。 NB组织学亚型与交感神经系统的正常分化模型相一致。经典的病理分类 将NB分成3型即神经母细胞瘤神经节母细胞瘤、神经节细胞瘤，这3个 类型反应了NB的分化、成熟过程。典型的NB由一致的小细胞组成，约 15%～50%的病例，母细胞周围有嗜酸性神经纤维网。 另一种完全分化的、良性NB为神经节细胞瘤，由成熟的节细胞神经纤维 网及Schwann细胞组成神经节母细胞瘤介于前两者之间含有神经母细胞 和节细胞混杂成分。 Shimada分类结合年龄将病理分成4个亚型，临床分成2组。4个亚型即 包括NB(Schwannin少基质型)；GNB混合型(基质丰富型)；GN成熟型和(3NB 结节型(包括少基质型和基质丰富型)。前3型代表了NB的成熟过程，而 最后一型则为多克隆性。对NB而言，细胞分化分为3级，包括未分化、 分化不良分化型；细胞的有丝分裂指数(MKI)也分为低、中、高3级。 Shimada分类综合肿瘤细胞的分化程度、有丝分裂指数和年龄将NB分为 临床预后良好组(FH)和预后不良组(UFH)： 1.FH包括以下各类 1)NB，MKI为低中度，年龄＜1.5岁 (2)分化型NB，MKI为低度，年龄1.5～5岁。 (3)GNB混合型。 (4)GN。 2.UFH包括 (1)NBMKI高级。 (2)NBMKI为中级年龄1.5～5岁 (3)未分化或分化健康搜索不良型NB，年龄1.5～5岁。 (4)所有＞5岁的NB。 (5)GNB结节型。在病理上除HE染色外，可进一步做免疫组化电镜检查来 与其他小圆细胞肿瘤相鉴别，NB时神经特异性酯酶(NSE)阳性电镜下可见

牙髓干细胞向神经细胞方向的诱导分化实验

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