3203 – IDENTIFICATION OF DIFFERENTIATION ROOTS OF HEMATOPOIETIC STEM CELLS BY A PAIRED-DAUGHTER ASSAY COMBINED WITH MULTIPLE BARCODING

Abstract

Hematopoietic stem cells (HSCs) produce all blood cells with self-renewal potential. HSCs maintain their compartments by symmetric and asymmetric cell division. A paired daughter cell (PDC) assay combined with single-cell transplantation is a powerful method to compare lineage outputs of two HSC-daughter cells. However, it is difficult to analyze the large number of pairs at once. Here, we have developed a PDC assay combined with DNA barcoding to overcome the limitation. Briefly, we labeled HSCs by lentiviral DNA barcode in vitro, split them in half when most of the HSCs divide once, which can split HSC-daughter cells with fifty-fifty chance, transplanted each half to each lethally irradiated recipient mouse and analyzed barcodes detected as pairs later. This method enabled us to analyze the large number of paired-daughter cells of HSCs only from two recipient mice. The most common HSC-division pattern was balanced HSC (bHSC)-bHSC. We also detected the other following division patterns with frequencies in the following order: lymphoid-biased HSC (lyHSC)-lymphoid restricted progenitor (LyRP), myeloid-biased HSC (myHSC)-LyRP, short-term HSC (ST-HSC)-ST-HSC, myHSC-myHSC and myHSC-bHSC. Most of the HSCs except lyHSCs were generated by symmetric division. Differentiated progenitor outputs were often detected as a paired-daughter cell of lyHSC or myHSC, not bHSC, suggesting that differentiation of HSCs begins with lyHSCs and myHSCs and bHSCs primarily maintain the stem cell pool. Single-cell RNA-sequencing analysis and secondary transplantation confirmed the current HSC hierarchy in which myHSCs are at the apex, producing bHSCs and lyHSCs. Interestingly, a few myHSCs behaved as latent HSCs which don't produce any progenies. In summary, the combination of the PDC assay and DNA barcoding revealed the distinct division pattern and differentiation capacity in each HSC subtype. Hematopoietic stem cells (HSCs) produce all blood cells with self-renewal potential. HSCs maintain their compartments by symmetric and asymmetric cell division. A paired daughter cell (PDC) assay combined with single-cell transplantation is a powerful method to compare lineage outputs of two HSC-daughter cells. However, it is difficult to analyze the large number of pairs at once. Here, we have developed a PDC assay combined with DNA barcoding to overcome the limitation. Briefly, we labeled HSCs by lentiviral DNA barcode in vitro, split them in half when most of the HSCs divide once, which can split HSC-daughter cells with fifty-fifty chance, transplanted each half to each lethally irradiated recipient mouse and analyzed barcodes detected as pairs later. This method enabled us to analyze the large number of paired-daughter cells of HSCs only from two recipient mice. The most common HSC-division pattern was balanced HSC (bHSC)-bHSC. We also detected the other following division patterns with frequencies in the following order: lymphoid-biased HSC (lyHSC)-lymphoid restricted progenitor (LyRP), myeloid-biased HSC (myHSC)-LyRP, short-term HSC (ST-HSC)-ST-HSC, myHSC-myHSC and myHSC-bHSC. Most of the HSCs except lyHSCs were generated by symmetric division. Differentiated progenitor outputs were often detected as a paired-daughter cell of lyHSC or myHSC, not bHSC, suggesting that differentiation of HSCs begins with lyHSCs and myHSCs and bHSCs primarily maintain the stem cell pool. Single-cell RNA-sequencing analysis and secondary transplantation confirmed the current HSC hierarchy in which myHSCs are at the apex, producing bHSCs and lyHSCs. Interestingly, a few myHSCs behaved as latent HSCs which don't produce any progenies. In summary, the combination of the PDC assay and DNA barcoding revealed the distinct division pattern and differentiation capacity in each HSC subtype.