Quantifying levels of transplanted murine and human mesenchymal stem cells in vivo by real-time PCR.

BACKGROUND

Previously, we demonstrated that murine mesenchymal stem cells (MSCs) injected intracranially into mice expand throughout the central nervous system (CNS). This paper describes real-time PCR (RT-PCR) assays that enables accurate quantification of transplanted cells in vivo.

METHODS

RT-PCR assays that amplify sequences in the mouse Y chromosome or human Alu repeats were developed and used to quantify the number of male, murine, or human MSCs in the CNS at various times after intracranial injection into neonatal mice, or in various organs of adult mice after i.p. injection of cells into 3 day-old embryos.

RESULTS

In the CNS, levels of male mouse DNA in female transplant recipients increased on average 30-fold between 3 and 60 days post-injection but then was unchanged at 140 days post-transplant (P = 0.107). Male DNA accounted for up to 0.309% of the total DNA content of the brain, representing maximally 600000 donor cells. Human DNA was detected in the CNS up to 300 days post-transplant, but levels never exceeded 7.63 x 10 (-4) % of the total brain content. After in utero transplantation, human DNA levels ranged from 0.36 x 10(-5) % to 2.14 x 10 (-5) % of the total DNA content of liver, kidney and spleen. significantly higher levels were found in heart (P = 0.06), femur and brain (P = 0.0025).

DISCUSSION

RT-PCR assays were developed to quantify levels of male, murine and human cells in vivo following sex-mismatched or xeno-transplants. Due to their accuracy, precision, and sensitivity, these assays provide a versatile alternative to measuring stem-cell engraftment in vivo.