Nurturing Stem Cells

The dream of regenerative medicine is to repair injury and disease by harnessing the versatility of human stem cells to form a wide range of healthy, specialized tissues. But the field faces many challenges: among them, getting stem cells and their progeny to grow and thrive in laboratory conditions.

HHMI investigator Shahin Rafii at Weill Cornell Medical College works on these problems in embryonic stem cells and the small populations of adult stem cells that are stored in various tissues in reserve, ready to be tapped for certain cell-replacement tasks.

“Stem cell biologists have been struggling for decades to expand these scarce populations [of adult stem cells], such as hematopoietic stem cells, and maintain them in vitro,” Rafii says. “Even in the presence of an elaborate array of cytokines, serum (a byproduct of clotted blood), and growth supplements, it has been impractical to keep adult stem cells alive or force them to self-renew for more than a few days in the laboratory.”

To extend the stem cells’ lives in the lab, Rafii’s research team has departed from the conventional methods of placing them in two-dimensional cultures with animal-based growth factors, serum, and oncogene-transformed, rapidly dividing immortalized “feeder” cells. Instead, novel three-dimensional (3-D) cultures mix mouse adult hematopoietic stem cells with unique primary endothelial cells, the building blocks of blood vessels. Rafii engineers them to maintain their biological properties in vitro, without added growth supplements.

Rafii had seen that after physiological stress, such as irradiation and chemotherapy, which deplete the bone marrow, activated endothelial cells help “wake up” adult blood stem cells in bone marrow and signal them to self-renew. In 3-D cultures, these activated endothelial cells produce factors that prompt stem cells to differentiate into specialized red and white and other mature blood cells. So Rafii dispensed with the commonly used growth factors and serum, which he concluded were a hindrance to stem cell long-term survival.

Employing this system to replicate the natural vascular niche of the bone marrow, Rafii and his colleague Jason Butler reported impressive results in a March 2010 publication in Cell Stem Cell. Mouse stem cells cocultured with endothelial cells thrived in the laboratory, retaining their self-renewal potential for more than 21 days and expanding their numbers hundreds of times.

The expanded in vitro stem cells also differentiated into large numbers of mature blood progenies. “This was a clinically relevant 3-D bone marrow microenvironment in which the endothelial cells could sustain the generation of all of the components of blood cells,” Rafii says.

Rafii is also evaluating the 3-D endothelial niche platform in expanding human organ-specific stem cells and their progeny. In an upcoming paper, he used his 3-D vascular niche platform to grow functional liver cells, which could ultimately be used to regenerate long-lasting vascularized liver tissue. Other preliminary results indicate that human endothelial cells could also sustain human blood and other organ-specific stem cells.

-- Richard Saltus
HHMI Bulletin, August 2010

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