Chan, Longaker and their colleagues had hoped to use what they learned from identifying the mouse skeletal stem cell to quickly isolate its human counterpart. But the quest turned out to be more difficult than they had anticipated. Most cell isolation efforts focus on using a technology called fluorescence activated cell sorting to separate cells based on the expression of proteins on their surface. Often, similar cell types from different species share some key cell surface markers.
But the human skeletal stem cell turned out to share few markers with its mouse counterpart. Instead, the researchers had to compare the gene expression profiles of the mouse skeletal stem cell with those of several human cell types found at the growing ends of developing human bone. Doing so, they were able to identify a cell population that made many of the same proteins as the mouse skeletal stem cell. They then worked backward to identify markers on the surface of the human cells that could be used to isolate and study them as a pure population.
“This was quite a bioinformatics challenge, and it required a big team of interdisciplinary researchers, but eventually Chuck and his colleagues were able to identify a series of markers that we felt had great potential,” Longaker said. “Then they had to prove two things: Can these cells self-renew, or make more of themselves indefinitely, and can they make the three main lineages that comprise the human skeleton?”
The researchers showed that the human skeletal stem cell they identified is both self-renewing and capable of making bone, cartilage and stroma progenitors. It is found at the end of developing bone, as well as in increased numbers near the site of healing fractures. Not only can it be isolated from fracture sites, it can also be generated by reprogramming human fat cells or induced pluripotent stem cells to assume a skeletal fate.
‘The perfect niche’
Intriguingly, the skeletal stem cell also provided a nurturing environment for the growth of human hematopoietic stem cells — or the cells in our bone marrow that give rise to our blood and immune system — without the need for additional growth factors found in serum.
“Blood-forming stem cells love the interior of spongy bone,” Chan said. “It’s the perfect niche for them. We found that the stromal population that arises from the skeletal stem cell can keep hematopoietic stem cells alive for two weeks without serum.”
By studying the differentiation potential of the human skeletal stem cell, the researchers were able to construct a family tree of stem cells to serve as a foundation for further studies into potential clinical applications. Understanding the similarities and differences between the mouse and human skeletal stem cell may also unravel mysteries about skeletal formation and intrinsic properties that differentiate mouse and human skeletons.
The skeletal stem cell we’ve identified possesses all of the hallmark qualities of true, multipotential, self-renewing, tissue-specific stem cells.
“Now we can begin to understand why human bone is denser than that of mice, or why human bones grow to be so much larger,” Longaker said.
In particular, the researchers found that the human skeletal stem cell expresses genes active in the Wnt signaling pathway known to modulate bone formation, whereas the mouse skeletal stem cell does not.
The ultimate goal of the researchers, however, is to find a way to use the human skeletal stem cell in the clinic. Longaker envisions a future in which arthroscopy — a minimally invasive procedure in which a tiny camera or surgical instruments, or both, are inserted into a joint to visualize and treat damaged cartilage — could include the injection of a skeletal stem cell specifically restricted to generate new cartilage, for example.
“I would hope that, within the next decade or so, this cell source will be a game-changer in the field of arthroscopic and regenerative medicine,” Longaker said. “The United States has a rapidly aging population that undergoes almost 2 million joint replacements each year. If we can use this stem cell for relatively noninvasive therapies, it could be a dream come true.”
Additional Stanford authors are CIRM Scholars Michael Lopez, Rachel Brewer, and Lauren Koepke, former graduate students Ava Carter, PhD, Ryan Ransom, graduate students Anoop Manjunath, and Stephanie Conley; former postdoctoral scholar Andreas Reinisch, MD, PhD; research assistant Taylor Wearda; clinical assistant professor of plastic and reconstructive surgery Matthew Murphy, MD; medical student Owen Marecic; former life sciences researcher Eun Young Seo; former research assistant Tripp Leavitt, MD; research assistants Allison Nguyen, Ankit Salhotra, Taylor Siebel, and Karen M Chan; instructor of stem cell biology and regenerative medicine Wan-Jin Lu, PhD; postdoctoral scholars Thomas Ambrosi, PhD, and Mimi Borrelli, MD; orthopaedic surgery resident Henry Goodnough, MD, PhD; assistant professor of orthopaedic surgery Julius Bishop, MD; professor of orthopaedic surgery Michael Gardner, MD; professor of medicine Ravindra Majeti, MD, PhD; associate professor of surgery Derrick Wan, MD; professor of surgery Stuart Goodman, MD, PhD; professor of pathology and of developmental biology Irving Weissman, MD; and professor of dermatology and of genetics Howard Chang, MD, PhD.
Researchers from the Medical University of Graz in Austria, RIKEN in Japan and the University of California-San Diego also participated in the study.
The study was supported by the National Institutes of Health (grants R01DE027323, R56DE025597, R01DE026730, R01DE021683, R21DE024230, U01HL099776, U24DE026914, R21DE019274, U01HL099999, R01CA86065, R01HL058770, NIAK99AG049958, P50HG007735, R01 R055650, R01AR06371 and S10 RR02933801), the California Institute for Regenerative Medicine, the Howard Hughes Medical Institute, the Oak Foundation, the Hagey Laboratory, the Pitch Johnson Fund, the Gunn/Oliver Research Fund, a Siebel Fellowship, a PCFYI Award, Stinehart/Reed, the Deutsche Forschungsgemeinschaft and the Ellenburg Chair.
The researchers have a pending patent for the isolation, derivation and use of human skeletal stem cells and their downstream progenitors.
Researchers from the Medical University of Graz in Austria, RIKEN in Japan and the University of California-San Diego also contributed to the study.
Stanford’s Department of Surgery also supported the work.