Table 4. In vivo engraftment of... | American Society of Hematology

Table 4.

In vivo engraftment of transfected and 4-week–expanded CB CD34⁺ cells

In vitro expansion			In vivo engraftment
Cell counts	T₀	+4 Weeks	Engraftment	Primary mice, n = 4, mean ± SD	Secondary mice, n = 4, mean ± SD	Tertiary mice, n = 4, mean ± SD
Cells, × 10⁶	0.06	3.24 ± 0.6	% CD45⁺	22.7 ± 5.2^4-150	11.5 ± 1.4^4-150	6.3 ± 1.4^4-150
GFP⁺ cells, × 10⁶	0	0.84 ± 0.15	% CD45⁺GFP⁺	12 ± 3.5^4-151	15.4 ± 2.6^4-151	11 ± 4.5^4-151
CD34⁺ cells, × 10⁶	0.06	0.7 ± 0.02	%CD34⁺	ND	ND	ND
GFP⁺ CD34⁺cells	0	0.21 ± 0.02	%CD34 GFP⁺	ND	ND	ND

In vitro expansion			In vivo engraftment
Cell counts	T₀	+4 Weeks	Engraftment	Primary mice, n = 4, mean ± SD	Secondary mice, n = 4, mean ± SD	Tertiary mice, n = 4, mean ± SD
Cells, × 10⁶	0.06	3.24 ± 0.6	% CD45⁺	22.7 ± 5.2^4-150	11.5 ± 1.4^4-150	6.3 ± 1.4^4-150
GFP⁺ cells, × 10⁶	0	0.84 ± 0.15	% CD45⁺GFP⁺	12 ± 3.5^4-151	15.4 ± 2.6^4-151	11 ± 4.5^4-151
CD34⁺ cells, × 10⁶	0.06	0.7 ± 0.02	%CD34⁺	ND	ND	ND
GFP⁺ CD34⁺cells	0	0.21 ± 0.02	%CD34 GFP⁺	ND	ND	ND

Three hundred thousand CB CD34⁺ cells (at 5 × 10⁴/mL) were preincubated for 96 hours with FL, SCF, TPO, and IL6. Cells were then washed, counted, and incubated for 24 hours with lentiviral particles at an MOI of 50. All cells were then washed, counted and seeded in duplicate T₂₅ flasks at 5 × 10⁴/mL with the same growth factors for a 4-week expansion. Expansion, as explained in “Materials and methods,” was carried out by addition of growth factors twice a week and by weekly addition of new media so that the cells were kept at a concentration of less than 5 × 10⁵/mL. At the end of the 4 weeks all cells were harvested, pooled, counted, and washed. The expansion equivalents of 6 × 10⁴ initial CD34⁺ cells (second column) were injected in each of 4 mice, which were killed 6 to 8 weeks after transplantation.

F4-150

Human engraftment levels were assessed by FACS analysis as percentage of CD45⁺ cells in total BM cells (reported is the mean ± SD of the 4 primary, the 4 secondary, and the 4 tertiary mice).

F4-151

The percentage of EGFP-expressing CD45⁺ cells (mean ± SD of 4 mice per transplant).

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