Until now, scientists didn't know exactly where to find these extremely rare, elusive adult stem cells – the only cells capable of forming all the different types of blood and immune cells found in mammals. Previous research suggested, and most scientists believed, that hematopoietic stem cells were clustered together somewhere in bone marrow.
But a new study published this week in the Proceedings of the National Academy of Sciences online Early Edition provides compelling visual evidence that hematopoietic, or blood-forming, stem cells prefer a solitary life.
"We took time-lapse movies of sections from mouse leg bone as seen under a fluorescent microscope," says Douglas Engel, Ph.D., chair and professor of cell and developmental biology at the U-M Medical School, who is the corresponding author for the study. "They clearly show individual, isolated hematopoietic stem cells at the edge of the bone marrow."
According to Engel, the discovery will make it possible to study hematopoietic stem cells undisturbed and in their natural environment. That's important, he says, because when stem cells are removed from bone marrow, they either die or start differentiating – changing into different types of specialized blood cells.
"There's something about the physical location and cellular environment surrounding stem cells in their bone marrow niche that is at least partly responsible for their ability to maintain a primitive, pluripotent state," Engel says. "Now that we can visualize them in vivo, we are in a better position to find out how they do it."
In order to visualize hematopoietic stem cells or HSCs in living bone marrow, the researchers had to figure how to get the gene for green fluorescent protein, called GFP, to be expressed only in HSCs. When GFP is expressed inside a cell, it generates a vivid fluorescent green color that is easy to see under a microscope.
Norio Suzuki, Ph.D., the study's first author, and colleagues at the University of Tsukuba in Tsukuba, Japan solved the problem by splicing the GFP reporter gene into a gene called Gata2, which is known to regulate the activity of hematopoietic stem cells, and into a promoter called IS that specifically controls the expression level of the Gata2 gene in these stem cells. Suzuki and the research team then injected the two modified genes into laboratory mice to create two lines of mutant "knock-in" animals.
"Disrupting Gata2 is embryonic lethal, so mice with two copies of the modified Gata2 gene all died before birth and had few hematopoietic stem cells," Suzuki says. "But mice that inherited two copies of the modified Gata2 promoter were healthy with levels of hematopoietic stem cells similar to normal mice. By following the Gata2-directed expression of GFP in mouse bone marrow and another marker protein called Sca1, we could easily isolate the hematopoietic stem cells."
According to Engel, scientists currently identify a hematopoietic stem cell by looking for a unique pattern of protein markers found on the cell's surface. The process is complicated and the flow cytometry equipment used to sort the cells is expensive. Plus the sorting process removes stem cells from their natural bone marrow environment, which changes their properties in fundamental ways.
To verify that cells identified with their Gata2-GFP marker technique were true hematopoietic stem cells, Suzuki and his colleagues conducted a series of experiments to test the purity of their cell samples against cells selected using conventional hematopoietic stem cell markers.
"We validated our findings using existing techniques, and found that the hematopoietic stem cells identified in this new way have all the known properties of HSCs," Engel says.
Specifically, the researchers found that:
- GFP was seen only in immature hematopoietic progenitors that lacked the surface protein markers seen in more mature types of blood cells.
- Only those cells with GFP activity had the ability to reconstitute the bone marrow of mice whose hematopoietic stem cells were destroyed by high doses of radiation.
- HSCs with green fluorescent protein were immobile and found in contact with osteoblasts, or bone-forming cells, at the edge of mouse bone marrow.
"The association between HSCs and osteoblasts may be important to stem cell function," Engel says. "Something in the niche, or the surrounding micro environment, is key to maintaining the stem cell's pluripotency."
The research study was funded by JST-ERATO, the Japanese Ministry of Education, Science, Sports and Culture; PROBRAIN, Promotion of Basic Research Activities for Innovative Biosciences; and the National Institutes of Health.
Other collaborators from the University of Tsukuba include Osamu Ohneda, M.D., Ph.D.; Naoko Minegishi, M.D., Ph.D.; Satoru Takahashi, M.D., Ph.D.; and Masayuki Yamamoto, M.D., Ph.D., professor and chair of molecular and developmental biology. Additional collaborators Mitsuo Nishikawa and Takayuki Ohta are from the Kirin Brewery Pharmaceutical Research Laboratory in Takasaki, Japan.
Citation: Proceedings of the National Academy of Sciences: doi/10.1073/pnas.0508928103
Editors: Video clips showing glowing stem cells in bone marrow from adult mice are available on request
Proceedings of the National Academy of Sciences