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Methods for generating functional hematopoietic stem cells

A technology of hematopoietic stem cells and cells, applied in the field of generating functional hematopoietic stem cells, which can solve problems such as high mortality and affecting patient survival

Active Publication Date: 2018-09-28
THE BRIGHAM & WOMEN S HOSPITAL INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Even if someone finds a suitable match, immune complications such as graft-versus-host disease (GVHD), donor rejection, and high treatment-related mortality can affect patient survival

Method used

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  • Methods for generating functional hematopoietic stem cells
  • Methods for generating functional hematopoietic stem cells
  • Methods for generating functional hematopoietic stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Cellular Physiological Analysis of the Effect of Circumferential Stretching on Endothelial Emergence of HSCs.

[0050] introduction

[0051] During embryonic development, blood flow generates shear stress on the abdominal wall lining the aortic endothelium and stimulates endothelial transition to HSCs (12). Shear stress further activates the endothelial NOS pathway, which triggers the endothelial emergence of HSCs (13). Having recently demonstrated that functional HSCs emerge, develop, engraft, and reconstitute multilineage adult blood in the absence of active blood circulation (14), this example therefore investigates novel cell-extrinsic mechanisms that stimulate endothelial emergence of HSCs . Using 3D Doppler ultrasonography, microangiography, echocardiography, and confocal imaging, we elucidate the periodic stretching of vessels by pulsatile pressure (Figs. 1-3). We developed an AGM-on-a-chip to reproduce the circumferential stretch condition ex vivo ...

Embodiment 2

[0072] Example 2: Examination of the molecular mechanism of Trpv4 signaling during HSC formation.

[0073] introduction

[0074] The effect of circumferential stretching is conveyed by one or more members of the K1 selectivity family, the Trp family, the ENaC / DEG family, and / or the Piezo family (36-39). Our gene expression analysis revealed that Trpv4 is expressed in both endothelial and hematopoietic cells. We therefore investigated the role of Trpv4 in the endothelial to HSC transition. We demonstrated that nonspecific inhibition of stretch-activated ion channels (40,41) and Trpv family channels (41,42), as well as targeted inhibition of Trpv4 (42) reduced expression of HSPC markers and HSPC quantity( Figure 7 ,9). We also established a functional connection between trpv4 signaling and HSC generation by demonstrating that activation of trpv4 channels (29) increases HSC formation in zebrafish and mice ( Figure 7 ,8A-B,9), while rescue of hematopoietic defects in sih(tn...

Embodiment 2A

[0084] Example 2A: Study of the functional utility of HSCs generated after Trpv4 activation.

[0085] Rationale, strategy and analysis plan: Circumferential stretch activates TRPV4 on endothelial cells (45). Here we demonstrate that circumferential stretch-mediated TRPV4 activation stimulates endothelial-to-HSC transition. To show that HSCs generated following Trpv4 activation are functional, we incubated zebrafish embryos with a Trpv4 agonist, followed by embryo-embryo HSC transfer and ex vivo incubation of E10.5 mouse embryos with a Trpv4 agonist, AGM transplantation was performed subsequently.

[0086] Zebrafish Functional Analysis and Embryo-Embryo HSC Transfer:

[0087] We utilized the zebrafish transgenic line cd41:eGFP for HSCs and flk1:mCherry for endothelial cell expression analysis (14). We injected trpv4 morpholino rings (morpholino) in zebrafish embryos between developing 19 and 42hpf and / or incubated cd41:eGFP::flk1:mCherry transgenic embryos with trpv4 agonist...

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Abstract

Methods for preparing populations of hematopoietic stem cells (HSCs), e.g., autologous and / or allogenic HSCs, using mechanical stretching or transient receptor potential cation channel-subfamily vanilloid member 4 (Trpv4) agonists, and methods of use of the HSCs in transplantation are provided. Specifically, the methods comprising providing population cells comprising hemogenic endothelial (HE) cells, contacting the HE cells with an amount of an agonist of Trpv4; and / or subjecting the cells to cyclic 2-dimensional stretching, for a time and under conditions sufficient to stimulating endothelial-to-HSC transition. Further disclosed are methods of treating malignant or non-malignant hematologic, metabolic, or immune disorders comprising administering to a subject a therapeutically effectiveamount of HSCs obtained by the methods.

Description

[0001] priority statement [0002] This application claims the benefit of US Provisional Application Serial No. 62 / 262,464, filed December 3, 2015, and US Provisional Application Serial No. 62 / 332,853, filed May 6, 2016. The entirety of the foregoing is hereby incorporated by reference. [0003] Federally funded research or development [0004] This invention was made with government support under grant numbers 5K01DK085217, 2R03DK100672, and 5R01HL131645 awarded by the National Institutes of Health. The government has certain rights in this invention. technical field [0005] Described herein are methods of using mechanical stretching or Trpv4 agonists to prepare populations of hematopoietic stem cells (HSCs), such as autologous and / or allogeneic HSCs, and methods for the use of HSCs in transplantation. Background technique [0006] HSC transplantation (HSCT) is widely used to treat patients with blood, bone marrow, metabolic and immune diseases (1-5). Despite improveme...

Claims

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Application Information

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IPC IPC(8): A01N63/00C12N5/071C12N5/02
CPCA61K35/28A01K67/0271C12N2501/999C12N2506/28C12N2533/52A01K2207/12C12N5/0647A61P25/00A61P3/00A61P35/00A61P35/02A61P37/02A61P37/06A61P43/00A61P7/00C12N2506/45C12N2506/00
Inventor D·I·莎G·Q·戴利
Owner THE BRIGHAM & WOMEN S HOSPITAL INC
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