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Method for inducing pluripotency in a hematopoietic cell

A technology of hematopoietic cells and pluripotency, applied in the direction of cell culture active agents, blood/immune system cells, animal cells, etc., can solve the problem of low reconstruction efficiency

Inactive Publication Date: 2017-03-01
AGENCY FOR SCI TECH & RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Reconstitution efficiency was often low in these studies, forcing the addition of more factors (5 to 7 factors in some cases, including SV40LT, TP53shRNA) in the reconstitution protocol (Chou et al., 2011; Mack et al., 2011; Okita et al., 2013)

Method used

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  • Method for inducing pluripotency in a hematopoietic cell
  • Method for inducing pluripotency in a hematopoietic cell
  • Method for inducing pluripotency in a hematopoietic cell

Examples

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Embodiment 1

[0101] Efficient derivation of transgene-free hiPSCs from human finger-prick blood is described. A single drop volume of finger prick samples was found to be sufficient in parallel for cell reconstruction, DNA sequencing, and blood serotyping.

[0102] Materials and methods

[0103] Finger prick and venous blood sample. In a sterile laboratory setting, collect 10 µL of blood in a fingertip capillary. Samples were lysed in 2 mL of 1× RBC Lysis Buffer (00-4300-54, eBioscience) for 10 minutes, followed by spinning at 250 g for 5 minutes. Lysis buffer was aspirated immediately after centrifugation. Purified cells were resuspended in 500 μL of cell expansion medium and seeded into one well of a 24-well tissue culture plate (3536, Corning). For DIY experiments, donors were asked to perform their own finger pricks and collect blood into Microtainer tubes containing anticoagulant ((422) 365974, Becton Dickinson). The tube can be pre-sterilized by flame or under UV irradiation. D...

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Abstract

There is provided a method of inducing pluripotency in a hematopoietic cell. The method comprises providing a blood sample that has been collected from a subject in the absence of any polysaccharide preparation used for separating blood components. The sample is depleted of red blood cells by treating the sample with a hypotonic erythrocyte lysis buffer, in the absence of any polysaccharide preparation used for separating blood components, thus obtaining a remaining cell population. The remaining cell population in the sample is expanded in a hematopoietic expansion medium to obtain an expanded cell population that contains CD71+ cells, and the expanded cell population containing the CD71+ cells is then cultured in the presence of Oct4, Sox2, and Klf4, and optionally c-MYC, in human embryonic stem cell medium to induce pluripotency.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of and priority to US Provisional Application No. 61 / 951,051, filed March 11, 2014, the contents of which are hereby incorporated herein by reference. technical field [0003] The present invention relates to a method of inducing pluripotent stem cells from hematopoietic cells obtained from a blood sample. Background technique [0004] Induced pluripotent stem cells (iPSCs) are an important source of stem cells to replace embryonic stem cells for the treatment of diseases, the study of developmental stages, and the detection of disease mechanisms. This is especially important when studying human cells and disease mechanisms and treatments due to the ethical issues involved in the use of human embryos. [0005] Human iPSCs (hiPSCs) were initially generated by overexpressing several transcription factors in dermal fibroblasts (Takahashi et al., 2007; Yu et al., 2007, Park et al., 2008...

Claims

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Application Information

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IPC IPC(8): C12N5/074C12N5/10C12N5/0789C12N5/02
CPCC12N5/0696C12N2501/602C12N2501/603C12N2501/604C12N2501/606C12N2506/11C12N2510/00
Inventor Y·H·卢H·K·陈
Owner AGENCY FOR SCI TECH & RES
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