14 December 2012. You may not like peeing in a cup at the doctor’s office, but what if it made stem cell creation a breeze? According to research published in the December 9 Nature Methods, a simple urine sample provided the human epithelial cells needed to create neural progenitor cells (NPCs). Researchers led by Duanqing Pei at the Guangzhou Institutes of Biomedicine and Health in China then transplanted these NPCs into rat brain, where the cells survived and became neurons. In the future, this method could provide NPCs to model disease or even to transplant back into donors. The researchers created the progenitor cells in a way that left the genome free of foreign DNA, which has become de rigueur for any cells that may be used therapeutically.
“The system seems to be very efficient,” Zhiping Pang, Child Health Institute of New Jersey in New Brunswick, told Alzforum in an e-mail (see full comment below). He was not involved in this work. “The use of human urine epithelial-like cells offers enormous advantages because this is probably the most non-invasive method of obtaining human cells,” he added.
Acquiring cells for reprogramming often requires invasive procedures, such as skin biopsies. However, researchers led by Pei and Miguel Esteban, also from the Guangzhou Institutes, reported last month that they could gather epithelial cells from urine—shed either from the kidney or bladder wall—and turn them into induced pluripotent stem cells (iPSCs) (see Zhou et al., 2012). Pei’s group wanted to improve on that process by creating iPSCs without a foreign DNA “footprint” left by viruses that integrate potentially disruptive reprogramming genes into cell DNA.
The scientists transfected urine epithelial cells with genetic reprogramming factors previously used to make induced pluripotent stem cells. These are Oct4, Sox2, SV40LT, Klf4, and microRNA cluster MIR302 367, all carried on episomal vectors that do not integrate into a cell’s DNA. Pei grew the cells on a medium containing FGF2 and a novel mix of small molecules known to promote reprogramming. After 12 days, 0.2 percent of the cells started to form compact colonies. When cultured on a special gel that mimics the extracellular environment, these cells formed little NPC-like rosette colonies that expressed typical NPC markers rather than those specific to iPSCs (see image below).
Neural progenitor cells derived directly from cells in urine stain for neural markers Pax6 (red) and nestin (green). Image courtesy of Lihui Wang, Guangjin Pan, and Duanqing Pei
“This was a pleasant surprise—we were trying to find a way to make naïve iPSCs, and instead the cells became neural progenitors, which may be better for therapeutic development,” Pei wrote to Alzforum. NPCs do not have the same cancer-forming tendency of iPSCs, nor do they require the same laborious, inefficient differentiation process required to coax out neural stem cells (see ARF related conference story).
The NPCs differentiated into several types of neural cells, including mature GABAergic, glutamatergic, and dopaminergic neurons, as well as astrocytes. About 85 percent were deemed potentially excitable, as evidenced by staining for synapsin, a protein that regulates neurotransmitter release. Tested neurons fired action potentials in response to stimuli. Four weeks after transplantation into the striatum of newborn rats, cells had survived, differentiated into both neurons and astrocytes, and formed no tumors.
“The generation of [DNA] integration-free neural progenitor cells directly from human cells has not been reported before,” Pei told Alzforum in an e-mail. Past direct induction methods have led to generic neural cells (see ARF related news story on Pang et al., 2011) and dopaminergic neurons (see ARF related news story), but used viral vectors to do so. Since cells in the current study contain no foreign DNA, they may be used for drug screening and possibly therapeutic transplants in the future, Pei wrote. He is unsure why his particular cocktail of reprogramming factors and chemical compounds turned epithelial cells directly into NPCs, but is pursuing that question now.
After perfecting the procedure, Pei plans to create NPCs from various donors, including those with neurodegenerative diseases, as well as to test the safety and efficacy of transplanting these cells into animals.—Gwyneth Dickey Zakaib.
Wang L, Wang L, Huang W, Su H, Xue Y, Su Z, Liao B, Wang H, Bao X, Qin D, He J, Wu W, So KF, Pan G, Pei D. Generation of integration-free neural progenitor cells from cells in human urine. Nat Methods. 2012 Dec 9. Abstract