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JoVE: Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells (Video Protocol)

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Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

Shannon McKinney-Freeman, George Daley
Children's Hospital, Harvard Stem Cell Institute, Harvard Medical School
0:06 Assembling handing drops differentiation culture
0:08 Collect undifferentiated ES cells
2:24 Prepare materials
2:57 Assemble hanging drop culture
4:34 Collecting differentiated ES cells form hanging drops
6:05 Starting EB culture
6:53 EB dissociation and infection
11:25 Preparation of ES cell-derived progenitors for FACS
15:18 Transplantation
16:19 Discussion
A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny. Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability. Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types. The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol. HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders. However, many patients that might benefit from HSC therapy lack access to suitable donors. ESC could provide an alternative source of HSC for these patients. The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC. In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation. EBs are then dissociated and infected with retroviral HoxB4. Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days. During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells. These cells can then be used to rescue and reconstitute lethally irradiated mice.

Differentiation of Embryonic Stem Cells

  1. Collect a near confluent flask of embryonic stem cells (ESC) via treatment with ESC trypsin.
  2. Resuspend cells in 5 mL of Differentiation media and transfer to a non-gelatin coated T25. Incubate at 37°C for 45 minutes. Retrieve supernatant and collect cells via centrifugation.

    Note: Mouse embryonic fibroblasts (MEFs) from the ESC cultures attach readily to the non-gelatin coated T25 and thus are depleted from the culture during this plating step. If you are not culturing your ESC on MEFs, you can skip the pre-plate.

  3. Resuspend MEF-depleted ESC in Differentiation media at 333,333 cells/50 mL. This concentration results in 100 ESC/15 mL.
  4. Using multi-channel pipettor, plate ESC at 100 cells/15 mL drop on 15 cm2 petri plates. With an 8-channel pipettor, you should be able to fit about 18-22 rows of drops per plate (about 5ml per plate).

    Note:For this protocol, 2-5 15 cm2 dishes of drops will be more than sufficient and should yield 2-5 x 106 day six EB-derived cells.