Waste Not, Want Not—Making Human Neurons From Urine

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).

image

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

Comments

  1. This is an interesting paper, and certainly represents an advance in generating human neuronal progenitor cells (NPCs). The data are very convincing. It will be of interest to see if the oligodendrocytes are functional. Compared to previous attempts of generating human NPCs (Ring et al., 2012; Lujan et al., 2012), I see two attractive aspects in this paper: First is the use of human urine epithelial-like cells, which offers enormous advantages because this is probably the most non-invasive method of obtaining human cells; second is the integration-free system for generating neuroprogenitor cells, which contrasts with previous attempts that used viral-mediated gene transfer. The system seems to be very efficient, and it will offer an alternative method to generate human neurons and glial cells that will be useful for understanding the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease.

    References:

    Ring KL, Tong LM, Balestra ME, Javier R, Andrews-Zwilling Y, Li G, Walker D, Zhang WR, Kreitzer AC, Huang Y. Direct reprogramming of mouse and human fibroblasts into multipotent neural stem cells with a single factor. Cell Stem Cell. 2012 Jul 6;11(1):100-9. PubMed.

    Lujan E, Chanda S, Ahlenius H, Südhof TC, Wernig M. Direct conversion of mouse fibroblasts to self-renewing, tripotent neural precursor cells. Proc Natl Acad Sci U S A. 2012 Feb 14;109(7):2527-32. PubMed.

    View all comments by Zhiping Pang

  2. It is great that urine epithelium cells can be used to generate NPCs. This once again shows that solutions lie in simple things.

    It may have potential impact on Alzheimer's treatment.

    View all comments by A Alia

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References

News Citations

  1. Not So Fast: iPS Cells Have Potential Pitfalls
  2. Turning Human Fibroblasts Into Neurons; Making Safer Stem Cells
  3. Faster, Safer Ways to Cook Up Dopaminergic Neurons

Paper Citations

  1. Zhou T, Benda C, Dunzinger S, Huang Y, Ho JC, Yang J, Wang Y, Zhang Y, Zhuang Q, Li Y, Bao X, Tse HF, Grillari J, Grillari-Voglauer R, Pei D, Esteban MA. Generation of human induced pluripotent stem cells from urine samples. Nat Protoc. 2012 Nov 8;7(12):2080-9. PubMed.
  2. Pang ZP, Yang N, Vierbuchen T, Ostermeier A, Fuentes DR, Yang TQ, Citri A, Sebastiano V, Marro S, Südhof TC, Wernig M. Induction of human neuronal cells by defined transcription factors. Nature. 2011 Aug 11;476(7359):220-3. PubMed.

Other Citations

Further Reading

Papers

  1. Nguyen HN, Byers B, Cord B, Shcheglovitov A, Byrne J, Gujar P, Kee K, Schüle B, Dolmetsch RE, Langston W, Palmer TD, Pera RR. LRRK2 mutant iPSC-derived DA neurons demonstrate increased susceptibility to oxidative stress. Cell Stem Cell. 2011 Mar 4;8(3):267-80. PubMed.
  2. Israel MA, Yuan SH, Bardy C, Reyna SM, Mu Y, Herrera C, Hefferan MP, Van Gorp S, Nazor KL, Boscolo FS, Carson CT, Laurent LC, Marsala M, Gage FH, Remes AM, Koo EH, Goldstein LS. Probing sporadic and familial Alzheimer's disease using induced pluripotent stem cells. Nature. 2012 Jan 25;482(7384):216-20. PubMed.

Primary Papers

  1. 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; PubMed.

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