Cold spring harbor Asia

STEM CELLS & DEVELOPMENTAL MECHANISMS

From 2012-12-3 to 2012-12-7

123/4/20

Presented by ShenBin

One of the hotspots in stem cells

• – Stem cell niche is the microenvironment and in which

stem cells are found and which interacts with stem cells to regulate stem cell fate.

– The word 'niche' can be in reference to the in vivo or in vitro stem cell microenvironment.

– "Stem-cell populations are established in 'niches' — specific anatomic (组织学上的 )locations that regulate how they participate in tissue generation, maintenance and repair

David T. Scadden, The stem-cell niche as an entity of action, Nature, 441 (7097), 1075-1079 (29 June 2006)223/4/20

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N-cadherin marked reserve HSCsSupport long-term hematopoiesis

and longer life spanLinheng Li

Stowers Institute For Medical Research

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Multiple niche components were

proposed

CAR: chemokine (C-X-C motif) ligand 12 (CXCL12)-abundant reticular (CAR) cells

Sugiyama et al., 2006 523/4/20

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HSCs in the bone marrow vascular niche.

Nature Medicine 18,1613–1614(2012) 1023/4/20

Nonmyelinating Schwann cells maintain hematopoietic stem cell hibernation in the bone marrow niche

Satoshi Yamazaki et al. Cell Vol. 147, Issue 5, pp. 1146-1158 1123/4/20

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• Glial fibrillary acidic protein (GFAP)-expressing cells (‘‘glial cells’’) were unexpectedly identified as the cells that principally process latent TGF-b into active TGF-b in BM

• These GFAP-positive cells were nonmyelinating Schwann cells

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• TGF-b negatively regulates HSC and hematopoietic progenitor cell proliferation in vitro

• TGF-bs are secreted as part of the large latent complex (LLC) consisting of TGF-b, latency-associated protein (LAP), and latent TGF-b binding protein-1 (LTBP-1)

• Under most conditions TGF-b in the LLC is inactive and is thus called latent TGF-b

(Larsson and Karlsson, 2005)

(Annes et al., 2003)

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• TGF-bs are multipotent cytokines that modulate cell growth, inflammation, matrix synthesis, and apoptosis

• Tgfbr1 null mice develop a lethal inflammatory disorder (LID) affecting multiple organs that is caused by uncontrollable overreaction of lymphocytes. Similarly, immune cells from Tgfbr2 knockout mice should induce LID upon transplantation.

(Waite and Eng, 2003; Schmierer and Hill, 2007),

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• The recombination activating genes (RAGs) encode enzymes that play an important role in the rearrangement and recombination of the genes of immunoglobulin and T cell receptor molecules during the process of VDJ recombination. There are two recombination activating gene products known as RAG-1 and RAG-2, whose cellular expression is restricted to lymphocytes during their developmental stages. RAG-1 and RAG-2 are essential to the generation of mature B and T lymphocytes, two cell types that are crucial components of the adaptive immune system.

Jones, J.M.; Gellert, Martin (Aug 2004). The taming of a transposon: V(D)J recombination and the immune system. Immunological Reviews 200: 233–248 1723/4/20

Mx-1-Cre:Tgfbr2flox/-:Rag2-/- mice

Mx-1-Cre:Tgfbr2+/-:Rag2-/- mice

B6-Ly5.1 x Ly5.2 F1 mice

lethally irradiated recipients (B6-Ly5.2) 1823/4/20

Phosphorylation status of Smad2 and Smad3 in wild-type BM as detected by immunostaining of frozen BM sections with DAPI (blue) and anti-p-Smad2/3(green).

1923/4/20

Loss of Tgfbr2 was associated with reduced p-Smad2/3, increased cycling, and reduced long-term repopulating activity in HSCs.

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• These results demonstrated for the first time that TGF-b/Smad signaling is active in BM HSCs and maintains their dormancy.

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Detection of specificity of anti-active TGF-b antibody by ELISA assays

Citric acid treatment can release TGF-b from the latent TGF-b complex

Lyons et al., 1990

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in vitro colony formation by CD34KSL HSCs is inhibited by active TGF-b, but not by latent TGF-b. This colony formation inhibited by active TGF-b was completely rescued by addition of anti-TGF-b antibody, but not of anti-LAP antibody

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Representative megakaryocytes are indicated by arrows

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integrin-b8 (Itgb8) was specifically expressed by the long, spindled-cell structure that marked abundantly for active TGF-b 2623/4/20

• The aforementioned findings led us to identify BM cell types that express Itgb8.

B220: leukocyte common antigen

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• Myelinating Schwann cells, which provide the neural myelin sheath, express myelin basic protein (MBP), but not GFAP. Myelination occurs for large axons 1–25 um in diameter

• nonmyelinating Schwann cells, which ensheath several small axons, express GFAP, but not MBP

• Focus on nonmyelinating Schwann cells

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S100b, a marker antigen for glial cells

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• To confirm that these GFAP-positive cells were nonmyelinating Schwann cells– confocal laser microscope under

high magnification

Neurofilament (NF)-positive axons神经纤毛

tyrosine hydroxylase (TH)-positive axons酪氨酸羟化酶

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• These results strongly suggested that the cells that play a role in HSC hibernation were nonmyelinating Schwann cells.

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costaining with anti-VE-cadherin and anti-GFAP or anti-Itgb8 antibodies revealed that the VE-cadherin positive endothelial cells and GFAP- and Itgb8-positive cells of interest were distinct and lay in parallel in close proximity

Distributions of GFAP- and Integrin-b8-Expressing Cells in BM

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• HSCs Exists in Contact with Glial Cells？？

Positional relationship of HSCs with niche cells

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Frozen wild-type BM sections were stained with antibodies against CD150 (red), against CD48, CD41, and lineage markers (green), and against VE-cadherin or osteocalcin (blue). 3523/4/20

Expression of HSC Niche Factor Genes by Glial Cells

housekeeping gene Gapdh：磷酸甘油醛脱氢酶（ reduced glyceraldehyde-phosphate dehydrogenase）

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Discussion • The present work implicates BM glial cells of the autonomic

nervous system in direct regulation of HSCs. By regulating the activation process of TGF-b, these glial cells appear to control hibernation of HSCs in BM.

• How these two components of the BM niche are related in the maintenance of homeostasis(内稳态 ) of HSCs under the influence of the autonomic nervous system remains an intriguing issue.

• Identification of glial cells as BM HSC niche components opens up a new area of research that links the neural and hematopoietic systems.

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Thinking: contradiction between proliferation and maintenance in vivo. Break the balance in viro

The law of life ???

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Thanks for your attention

Other hotspot such reprogramming, regulation ….

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