Seeking the true nature of "stem cells" that support the continual
sperm production
National Institute for Basic Biology, National Institutes of Natural Sciences
Professor Shosei Yoshida[About my research]
The average human male's testes produce 100 million sperm per day for more than 60 years.
"Sperm stem cells” propagate to produce the numerous sperm, but some of them remain as stem
cells to prevent their loss (Fig. 1). Some calculations predict that human sperm stem cells may
divide more than 1,000 times during their lifetime. It is not at all an easy task to maintain aconstant number of stem cells (called self-renewal) while continuing to produce sperm (calleddifferentiation) for such a long period. We can say that the testis is an excellent factory thatcombines productivity, sustainability, and accuracy.
How is the balance between sperm stem cell differentiation and self-renewal maintained?
This question has attracted many researchers.

However, finding and characterizing sperm stem

cells is not easy, as the stem cells are very rare. Based on the microscopic observations of tissue
preparations conducted in the 1950s, new techniques including gene and protein expression
analysis and gene knock-out using mice flourished from the end of the last century to the presentcentury.

Transplanting the sperm stem cells into the testis of other mice to retore

spermatogenesis was achieved in 1994; culturing the sperm stem cells was made in 2003.

It hasbeen generally believed to be true that stem cells are tethered to a fixed place, called a "niche", andthat when a stem cell divides one stem cell and one differentiated cell are always produced (Fig.
2). Indeed, it is a simple, beautiful, and reasonable concept.
Fig. 1

Fig. 2
Stem cells

Stem cells

Stem cellniche

Self-renewal

Differentiation
into sperm

Differentiation
into sperm

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However, these studies did not directly examine the stem cell behavior in the testis.
Fixation (freezing time) and staining were inevitable, if you wanted to look into the testis with
its normal architecture. On the other hand, to explore the functions of living stem cells, eachcell had to be separated individually. This was a big dilemma in studying time-livingorganisms, especially multicellular ones. I thought it was essential to track the behavior of
every stem cell in the testis over time, so I conducted two experiments. One was "intra-vital liveimaging” – using the green fluorescent protein (GFP), for which Osamu Shimomura et al. won
the Nobel Prize (2008), I was able to visualized the testicular stem cells in live. Furthermore, I
succeeded in continuously observing and filming fluorescent stem cells in the live mouse testis.
The other is the "pulse labeling” experiments – by marking stem cells at a certain time point, I
could successfully trace the fate of their progeny.
These experiments revealed the behavior of sperm stem cells over time, which wassurprisingly unrestrained. They did not stay in the same place but actively moved throughout
the testis (Fig. 3).

The division pattern was also inconsistent, giving rise to two stem cells, twodifferentiated cells, or one stem and one one differentiated cells. The fate of each stem cell wasdisparate and different from the authentic image of stem cells. However, as a population, thestem cells maintained a precise balance between self-renewal and differentiation (Fig. 4). In otherwords, while individual cells behaved randomly, collectively, they held a stable order without
descending into chaos.

Moreover, this system has also revealed that spermatogenesis can beflexibly regenerated when the testes are damaged.
Fig. 3Fig. 4
Stem cells

Selfrenewal

Differentiation
into sperm
Fig. 3: An example of an intravital live imaging of sperm stem cells. The 48-hour trajectory ofstem cells are Shown. The scale bar is 30 micrometers, reprinted from Cell Stem Cell (2014).
Organisms seem to be far ahead of the wisdom of researchers. Currently, I am trying toelucidate the mechanism by which "individual randomness" gives rise to "collective order andCopyright © Ohsumi Frontier Science Foundation. All Rights Reserved.

stability”. This is an essential question not only for sperm stem cells but also for understanding
the formation of multicellular organisms in general. Although I cannot intuitively understandthe meaning of the erratic behavior of stem cells, I am fortunate to have collaborators whoanalyze them using mathematical statistics and biophysics. The simple behavioral principles
that give rise to the complex behavior of stem cells and the molecular mechanisms responsible
for this behavior are actively investigated now. I am repeatedly excited every time I see an
unexpected aspect of stem cells.
Key publications: Cell Stem Cell 14, 658 (2014), Science 328, 62 (2010), Cell Stem Cell 7, 214(2010), Science 317, 1722 (2007), Dev Cell 12, 195 (2007)[My basic science approach]I have continued my research according to my curiosity.

I think that there are two types ofresearchers: those who "want to be able to do what cannot be done" and those who "want to knowwhat is not known," and I am the latter.

Living organisms are full of things that humans do notknow (we know much less than what we don’t, indeed), which are fascinating subjects for basicresearchers. As many people say, the motivation for basic research comes down to the individual
researcher's curiosity. “Basic research" is often contrasted with "applied research”. However, I
don't think that basic research is conducted to form the foundation for successful applied research.
Rather, basic research means nothing other than "the research that genuinely pursues themechanism of nature (or life).” This is philosophy, the “Ph” of Ph.D. meaning a doctor. In thissense, the term "basic research," which reminds us of building foundations, may not the best term.
Although I cannot tell the best words, "academic research" or "pure research" are words with thisin mind.
I have to confess that, when I started my research, I thought that basic research was sublimeand superior than applied research. However, my thinking changed 180 degrees when I met a
top-notch applied researcher who is also a respected friend of mine. He said, "If it is not useful,what is the point?"

He is willing to do whatever is necessary to achieve his goal.

Impressively,

the results that he obtains are not only beneficial but also exceptional as basic research, and they
perfectly capture the essence of living organisms. I now believe that really beneficial appliedresearch can be made, only if you capture the essence of living organisms. Visa versa, only basic
research that captures the essence of living organisms can lead to truly useful applications.
Whether it is basic research or applied research, the essence of living organisms can only bediscovered when a researcher is motivated to confront living organisms thoroughly.

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There are many good things about being a basic researcher, but I will mention only one
here. I have learned that there are always some people (though not many) in the world who
find the discoveries that one person has worked so hard to make and found truly interesting tobe just as interesting from the bottom of their hearts. With these people, you quickly become
like longtime friends. It doesn't matter what country you are from or sometimes even what fieldof research you are in. It is a great pleasure to talk with such casual friends on various
occasions, such as joint research projects and conferences, and I learn many things outside ofresearch. I feel that science is a common language that connects us all.

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