HSC Daughter Cell Splitting

Isolation of HSC Daughter Cells or “Doublets Splitting”

Hematopoietic stem cells (HSC) sit on top of the hematopoietic hierarchy being in charge to establish and maintain a functioning blood system. HSC achieve this by being able to self-renew as well as to give rise to the multiple lineages of the hematopoietic blood system. Thereby, the balance of self-renewal and differentiation is of great importance for the healthy functioning of our blood system.

However, the dynamics leading to one or another stem cell fate are not fully understood. One possibility to study fate decision making is the CFC assay (see Hematopoietic Stem Cell Colonies). Though, the CFC assay studies colony formation from single stem or progenitor cells, but does not differentiate between single cells within these colonies. Therefore, another option for studying cell fate decisions is to study the “daughter” cells originating from one “mother” HSC on single cell level in molecular and functional assays like single cell sequencing or transplantation assays.

However, whatever method shall be applied, first, single “daughter” cells must be isolated (here also called “doublets splitting”). Especially for functional assays, but also for expression studies it is important that isolation is performed under conditions that minimally influence the cells molecular programs. Isolation of HSCs from bone marrow or blood samples is usually performed by FACS. However, because of low overall cell numbers (down to 2 daughter cells), this is not an option for doublets splitting. In the past, doublets splitting was realized by serial dilution, meaning pipetting the cells from one well to another until cells are singularized. However, this method is not very gentle and yields a high risk to damage or even loose the cells. Moreover, doublets tend to sit together decreasing the success of serial dilution.

Here, we present a better option for the gentle isolation of daughter cells enabling traceability of each single cell: with the CellCelector’s interactive picking mode, doublets can be split, while having full overview by a live-image of what is happening to the cells neglecting the risk of losing a cell. Moreover, picking by the CellCelector is very gentle to the cells minimizing the influence on the cells molecular programs. Finally, the picking process is tightly documented allowing full traceability.

A: Daughter cells originating from one HSC, the red arrow marks the cell to be isolated.

B: To split doublets, the capillary is placed at a position next to the cell that shall be picked (red arrow) and furthest from the other cells.

C:  Attracted by capillary forces, the cells now start moving into the direction of the capillary, with the cell sitting closest to the capillary moving fastest (red arrow).

D: As soon as the cell cannot escape any more, the capillary is moved away from the other cells.

E: Eventually, the distance between the attracted cell and the other daughter cells is big enough to enable safe single-cell picking.

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