间充质干细胞研究

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It has also been proposed that the function of MSCs are to provide replacement cells for those that expire in the differentiated tissues.
How can we culture them?
What is their phenotype (表型)?
>95% of population must express:
§ § § CD105 CD73 CD90
Cells must not express (<2% express)
§ § § § § CD45 CD34 CD14 or CD11b CD79α CD19 HLA-DR (class II)
CFU-F Assay
- to quantify the number of stem/progenitors in the sample
Expansion
- to obtain more cells
Frequency of CFU-F
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In human bone marrow, the frequency is 1:100,000 but is negatively correlated with age (Clarke & McCann, 1989) Frequency is reduced following chemotherapy (化疗) or irradiation (辐射) (Galotto et al, 1999) Frequency remains decreased after bone marrow transplantation 骨髓移植 (Galotto et al, 1999)
• • • • Inhibit apoptosis (抑制凋亡) and limit injury Inhibit fibrosis (抑制纤维化) /scarring Stimulate angiogenesis (刺激血管新生) Stimulate mitosis (刺激细胞有丝分裂) of tissue-specific progenitors
What is their function?
BM-MSCs (most studied): • regulate the proliferation, differentiation and survival of hematopoietic stem cells. 调控造血干细胞的增殖,分化和存活
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Cell Expansion
BM Sample A B C D Fold Expansion 3732x 6966x 1770x 230x Days in Culture 40 58 41 28
Note variability in expansion observed with different bone marrow samples.
al., 2006)
Also termed:
§ § § § Multipotent mesenchymal stromal cells (ISCT – International Society of Cell Therapy - favored name) Mesenchymal progenitor cells Non-hematopoietic stem cells Multipotent marrow stromal cells
Medium typically used for mesenchymal progenitor cell culture: • basal medium + 10% FBS (homemade formulation) Problems: • variability (差异) due to serum performance MesenCult® Advantages: 1. medium is standardized and performance-tested to ensure increased reproducibility/consistency (增加 重复性和一致性) 2. formulation has been optimized for efficient cell expansion
What are mesenchymal stem cells?
The term mesenchymal stem cell was initially used because the identified cell type was capable of differentiating into cells of mesenchymal tissues (bone, adipose, cartilage, muscle). • Cells also showed self-renewal and proliferation properties.
Don’t need to use RosetteSep Enrichment kit with MesenCult-ACF Medium b/c there is little hematopoietic cell contamination
Culture and quantitate: MesenCult® Proliferation Kit (Human)
Isolate: Human MSC Cell Separation Products
Our cell separation products: • RosetteSep® enrichment cocktail (mature cells removed with lineage-specific markers) advantage: enriched cells are antibody-free • CD271 positive selection kit
(似神经细胞) (脂肪细胞)
Differentiate
Chondrocytes
(关节软骨细胞)
osteogenic cells
(成骨细胞)
Where do we find them?
1. 2. 3. 4. Bone Marrow (骨髓) Peripheral blood (外周血) Adipose tissue (脂肪组织) Cord blood/Placenta/HUPVC - human umbilical cord perivascular cells (脐带 血/胎盘/脐血血管周细胞) 5. Fetal liver and fetal lung (胎肝和胎肺) 6. Amniotic fluid (羊水) 7. Compact bone (密质骨) 8. Skeletal muscle (骨骼肌) 9. Synovium (滑膜) 10.Menstrual blood (经血)
Mesenchyme is embryonic connective tissue (结缔组织) that is derived from the mesoderm (中胚层) and that differentiates into hematopoietic ( 造血) and connective tissue.
What are mesenchymal stem cells?
How can they be manipulated?
Whole Bone Marrow
bmMSC (precursor)
Frequency =1/100,000
Quantitate (CFU-F)
Expand ceMSC (ce=culture expanded) Adipocytes neural-like cells
• • help to promote successful engraftment of hematopoietic progenitors (造 血祖细胞的植入) MSCs secrete bioactive molecules with immunomodulatory effects 免疫 调节作用 (turn off T cell surveillance and chronic inflammatory processes) MSCs secrete bioactive molecules that:
Reference: Dominici et al., Cytotherapy 8: 315-317, 2006
Why do we want to study them?
Substantial interest in investigating therapeutic applications of MSCs. Current clinical trials (临床试验) : Osteogenesis imperfecta (OI) 成骨不全症 Graft-Versus-Host Disease (GVHD) 移植物抗宿主病 Myocardial Infarct (MI) 心肌梗死 Crohn’s Disease 克罗恩氏病, 节段性回肠炎 Stroke 脑中风 Pulmonary Disease 肺疾病 Diabetes 糖尿病 Enhance engraftment of HSC 提高造血干细胞植入
http://clinicaltrials.gov/
Animal Models
It is important to have animal models to further investigate the therapeutic potential of MSCs.
1. Marrow stromal cells as a source of progenitor cells for non-hematopoietic tissue in transgenic mice with phenotype of osteogenesis imperfecta (Periera et al., 1998) 2. ceMSC (rat) can regenerate a critical sized segmental bone defect (Kadiyala et al., 1997) 3. Non-hematopoietic mesenchymal stem cells can be mobilized and differentiate into cardiomyocytes after MI 心肌梗死 (Kawada et al., 2004) 4. Diabetes, myocardial infarct, stroke, melanoma, EAE (实验性自身免疫性脑脊髓 炎), lung injury, renal failure, hepatic failure (Uccelli et al., 2008)
间充质干细胞研究
What are mesenchymal stem cells (间充质干 细百度文库)?
The term mesenchymal stem cell (MSC) is often used to describe a phenotypically (表型上)and functionally (功能上) heterogeneous ( 异质型) cell population. In culture they are defined as plastic-adherent, fibroblast-like cells which are able to self-renew (自我更新)and differentiate (分化) into bone (骨), adipose (脂肪) and cartilage (软骨) tissue (Dominici et