Generation of human/rat xenograft animal model for the study of human donor stem cell behaviors in vivo

Table 1 Primer pair used for PCR detection of human gene

Gene	Primer name	Primer sequence	Annealing temperature	PCR product size (bp)
hAlu	hAlu-FP:	5'-CTGGGCGACAGAACGAGATTCTAT-3'	56°C	224
	hAlu-RP:	5'-CTCACTACTTGGTGACAGGTTCA-3'
Myo	Myo-FP:	5’-TTACGTCCATCGTGGACAGC-3’	56°C	245
	Myo-RP:	5’-TGGGCTGGGTGTTAGTCTTA-3’

Table 2 Engraftment of human CD45⁺ cells in PB of MNC-transplanted recipients

ExperimentNo.	AnimalNo.	Phenotype	Source	Cell dose	Chimeric/totalanimals
1	661	MNCs	hUCB	5 × 106	8/12
2	662	MNCs	hUCB	5 × 106	5/8
3	670	MNCs	hUCB	5 × 106	6/9

Three weeks after birth, PB was collected from MNC-transplanted rats, followed by measuring the expression of human CD45 maker using flow cytometry to screen human-rat chimeras. Detection of human cells by flow cytometry was defined by the presence of human CD45⁺ cells. No human marker-positive cells were detected in normal rats of the same age. hUCB: Human umbilical cord blood; MNCs: Low-density mononuclear cells. Other details are as in Figure 1.

Table 3 Percentage of human marker-positive cells detected in PB and BM from MNC-transplanted rats (mean ± SD)

Marker	PB			BM
	3 wk	2 mo	6 mo	2 mo	6 mo
CD45	0.2 ± 0.06	0.15 ± 0.04	0.09 ± 0.01	1.4 ± 0.36	0.1 ± 0.01
	(n = 19)	(n = 17)	(n = 4)	(n = 8)	(n = 4)

Human donation contribution in PB and BM were analyzed by two-color flow cytometry for human-specific marker, i.e. CD45. PB and BM were collected at 3 wk, 2 mo and 6 mo after birth, respectively. Values for human marker-positive cells represent the mean ± SD of human cells found in each of the animals analyzed at a given time point. Other details are as in Figure 1 and Table 2.