Okayama University Medical Research Updates （OU-MRU）
Source: Okayama University (JAPAN), Center for Public Information
For immediate release: 30 November 2014
Okayama University research: Organ regeneration research leaps forward(Okayama, 30 November 2014) Researchers at Okayama University Graduate
School of Medicine and Kyorin University School of Medicine have successfully
generated a kidney-like structure from just a single cell.
“It has been predicted that the kidney will be among the last organs successfully
regenerated in vitro due to its complex structure and multiple functions,” state Shinji
Kitamura, Hiroyuki Sakurai and Hirofumi Makino at the beginning of their latest
report, before continuing to describe results suggesting a far more positive prognosis
for the pace of kidney regeneration research. Despite the anatomical challenges
posed by the kidney anatomy and the complexities understood from embryonickidney development processes, the researchers have demonstrated that kidney-likestructures can be generated from just a single adult kidney stem cell.
In embryos, kidney development requires two types of ‘primordial’ cells – cells at theearliest stage of development. However by generating kidney-like structures from asingle type of kidney stem cell the researchers provide evidence for differences in
the organ development in adults and embryos.
Kitamura, Sakurai and Makino – researchers from Okayama and Kyorin Universities took kidney stem cells from the different kidney components of microdissected adultrats and grew them in culture. A method for growing three-dimensional cell clusters
showed that kidney-like structures could form so long as the initial cell cluster waslarge enough.
The minimum cluster size required might suggest that not all the kidney stem cells
have stem cell characteristics. Therefore the researchers cloned kidney stem cells
and confirmed that kidney-like structures still formed from the clusters of clone cells
after a few weeks.
The researchers add, “Although the physiological roles of such cells are currently
unclear, analogous cells in the adult human kidney would be a valuable resource forthe regeneration of kidneys in vitro.”

OKAYAMA UNIVERSITY
1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530 JAPAN© 2014 OKAYAMA UNIV.

Background
Kidney structure
There are more than a dozen distinct types of cell in the kidneys. The basic structuralunit of the kidney is the nephron, which filters the blood to regulate theconcentration of water and soluble substances such as sodium salts. Each nephroncomprises several well-defined segments: the glomerulus, the proximal tubule, theloop of Henle, the distal tube and the collecting duct.
In embryo kidney organogenesis two primordial cell types are required todifferentiate into all the different cell types in the kidney: metanephric mesenchymal
cells and uteric bud cells. Kitamura, Sakurai and Makino produced kidney cells that
could differentiate into a kidney-like structure without these primordial cell types,
suggesting these are adult kidney stem cells.
Obtaining kidney stem cells
The researchers microdissected adult rat kidneys into segments from the glomeruli,
proximal convoluted tubule (S1/PCT), proximal straight tubule (S2, S3), medullary
thick ascending limb of Henle’s loop and the collecting duct. They then grew the cells
on mouse mesenchymal cells. While there is no known single biomarker for adultkidney stem cells, immunohistochemical anaylysis identified a number of markers in
the kidney stem cells- that are found in embryonic or adult kidneys.
Three-dimensional culture and morphogenesis
The cells were initially grown on type IV collagen and in two-dimensional growth
conditions there were no signs of kidney structure development. Instead theresearchers used a ‘hanging drop’ method for making cell cluster to generate 3dimensional structure. Cell clusters were placed in a half MatrigelTM - a gelatinous
protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells. This
protein gel provides a complex extracellular environment similar to that found inmany tissues.
The cell clusters cultured in this way reproducibly formed kidney-like structures
depending on the size of the initial cell cluster. When less than 6250 cells were used
only cystic structures developed. When the initial cluster contained up to 50,000
cells distinct tubular structures were observed and when the cluster size exceeded
100,000 cells full kidney-like structures containing a glomerulus, as well as proximaland distal tubules developed.

OKAYAMA UNIVERSITY
1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530 JAPAN© 2014 OKAYAMA UNIV.

The kidney-like structures formed did not show the formation of blood vessels or
make urine. Further research is required to understand these aspects of kidney
regeneration.
Acknowledgments
A portion of this study was supported by the Health and Labor Sciences Research
Grants for Research on Tissue Engineering from the Ministry of Health Labor andWelfare of Japan, the programme for the promotion of fundamental studies in
Health Sciences of the National Institute of Biomedical Innovation (VIBIO), a Grantin-Aid for Young Scientists (B) and a grant from the Okayama Medical Foundation,
the Ministry of Economy, Trade and Industry (METI), Japan, and joint research with
Organ Technologies, Inc. We appreciate the technical contributions of Dr. Takeshi
Sugaya (Cimic, Co., Ltd., Japan) and Dr. Naoshi Shinozaki (Cornea Center & Eye Bank,Tokyo Dental College, Ichikawa General Hospital) and the kind advice of Dr. Kazuhiro
Sakurada (Sony Computer Science Laboratories, Inc., Japan) and Dr. Naoya Kobayashi(Okayama Saidaiji Hospital).
Reference
Shinji Kitamura, Hiroyuki Sakurai, Hirofumi Makino, “Single adult kidney
stem/progenitor cells reconstitute three-dimensional nephron structures in vitro” ,Stem Cells, x, (x), pp.yy-zz, (2014) in press.
Abstract URL: http://onlinelibrary.wiley.com/doi/10.1002/stem.1891/abstract
DOI: 10.1002/stem.1891

OKAYAMA UNIVERSITY
1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530 JAPAN© 2014 OKAYAMA UNIV.

Figure caption:
The relationship between cell number in the cluster and the ability to reconstitute akidney-like structure. Red bar: cyst formation, Blue bar: long distinct tubules
formation, Green bar: tubules with ball-like structures at the tip. The horizontal axis
represents kidney stem/progenitor cell (KS cell) number (6.25–200 × 103cells)/cluster. The KS cell clusters were cultured for 3 weeks. Representative
photomicrographs were from three independent experiments. Scale bar = 100 μm.

OKAYAMA UNIVERSITY
1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530 JAPAN© 2014 OKAYAMA UNIV.

Figure caption:
A KS cell cluster formed a kidney-like structure (KLS) after 4weeks of incubation using
DMEM/F12 plus 10% FCS, 250ng/ml GDNF, 250ng/ml b-FGF, 250ng/ml HGF,
250ng/dl BMP-7 and 500 ng/ml EGF. Glomerulus-like structures were formed at thetips of the tubular structures, proximal like tubules, distal like tubules, collecting
duct-like tubules and renal pelvis-like structures. G, glomerulus-like structure; Pr,proximal tubule-like structure; H, loop of Henle-like structure; D/C, distal tubulelike structure or collecting duct-like structure; Pe, renal pelvis-like structure.

Correspondence toSenior Assistant Professor Shinji Kitamura, M.D., Ph.D.
Department of Medicine and Clinical Sciences, Okayama
University Graduate School of Medicine, Dentistry andPharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku,
Okayama 700-8558, Japan
E-mail: s-kita@md.okayama-u.ac.jp

OKAYAMA UNIVERSITY
1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530 JAPAN© 2014 OKAYAMA UNIV.

Further information
Okayama University
1-1-1 Tsushima-naka , Kita-ku , Okayama 700-8530, Japan
Planning and Public Information Division, Okayama University
E-mail: www-adm@adm.okayama-u.ac.jp
Website: http://www.okayama-u.ac.jp/index e.html

Okayama University Medical Research Updates （OU-MRU）
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cells from blood samples for genetic testing
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