There will be blood stem cells

Human cells are multiplied 20-fold in culture, offering promise for bone-marrow transplants

Patients with leukemia, certain autoimmune diseases and genetic defects such as sickle-cell anemia can be treated with blood stem cells either from a donor’s bone marrow or from cord blood—but the supply of effective stem cells often runs short.

Now, researchers in the lab of Whitehead Member Harvey Lodish have found a way to multiply in culture adult hematopoietic (blood-forming) stem cells from human cord blood 20-fold, a major milestone that offers promise for bone marrow transplants and perhaps even gene therapy. Cord blood can be easily collected and stored as a frozen product, making it readily available.

“Human cord blood is a rich source of stem cells, but offers too few of those cells to transplant into an adult,” says Lodish. “Previously we identified five growth factors that acting together in culture expanded mouse bone marrow hematopoietic stem cells 30-fold. Building on this research we’ve now identified five growth factors needed to stimulate human cord blood stem cells to divide in culture and make 20-fold as many stem cells.” The paper was published online in Blood in January.

Two novel growth factors (angiopoietin-like 5 and IGFBP2) work in combination with three previously identified growth hormones—stem cell factor (SCF), thrombopoietin (TPO) and Flt3 ligand—to stimulate the growth of these stem cells.

Hematopoietic stem cells give rise to oxygen-carrying red blood cells, white blood cells and all of the cells that comprise the immune system. Previous efforts to grow human hematopoietic stem cells in culture have proven extraordinarily difficult because they rapidly differentiate into mature blood or immune cells.

Down the road, advances in blood stem cell cultures may aid gene therapy by allowing tests of whether a repaired gene fits correctly into the genome, says Harvey Lodish. Images: John Soares

“Our finding builds on previous work studying hematopoietic cells in which we discovered a novel cell population that when cultured in a dish with stem cells enabled them to multiply,” says Chengcheng Zhang, first author of the paper, formerly a postdoctoral researcher in the Lodish lab. He is now an assistant professor of physiology and developmental biology at the University of Texas Southwestern Medical Center in Dallas.

“We searched for genes that were active in these and other stem-cell-supportive cells, and identified genes that encoded growth factors,” Zhang says. “We then added the growth factors to the isolated hematopoietic stem cells and increased the number of stem cells in culture.”

To make sure that these were still viable stem cells, the researchers transplanted them into immune-deficient mice and measured the resulting population of various sorts of human blood and immune system cells successfully growing in the mice.

The researchers note that this finding may also lead to advances in gene therapy, in which a genetic defect would be corrected by administering a healthy version of the gene into a patient.

During gene therapy, hematopoietic stem cells from a patient would be isolated and exposed to a virus that expresses a correct version of the mutated gene, and then the stem cells would be transferred back into the patient.

If we could first culture stem cells such that they divide and make more stem cells before they are reintroduced into the patient, assays could be used to determine if the virus had landed in any undesirable places, in order to ensure that the healthy version of the gene is administered to the patient,” says Lodish. “With a technique such as this, it may be possible to ensure that the gene is inserted into the genome in the correct place.”

How your red blood cells nuke their own nuclei

Unlike the rest of the cells in your body, your red blood cells lack nuclei. That quirk shows you’re a mammal—other vertebrates such as fish and birds have red cells that contain nuclei that are inactive. Losing the nucleus enables the red blood cell to hold more oxygen-carrying hemoglobin and thus transport more oxygen, boosting our metabolism.?

Scientists in the Lodish lab have modeled this complete process in vitro in mice, reporting their findings in Nature Cell Biology online in February. The first mechanistic study of how a red blood cell loses its nucleus, the research sheds light on one of the most essential steps in mammalian evolution.

As human red blood cells (here with several kinds of white blood cells) near maturity, a ring of actin filaments pinches off a segment of the cell that contains the nucleus. Images: National Cancer Institute

It was known that as a mammalian red blood cell nears maturity, a ring of actin filaments contracts and pinches off a segment of the cell that contains the nucleus, a type of cell division. The nucleus is then swallowed by macrophages (one of the immune system’s quick-response troops). The genes and signaling pathways that drive the pinching-off process, however, were a mystery.

“Using a cell-culture system, we actually were able to watch the cells divide, go through hemoglobin synthesis and then lose their nuclei,” says Lodish. “We discovered that the proteins Rac 1, Rac 2 and mDia2 are involved in building the ring of actin filaments.”

“Rac 1 and Rac 2 were involved in disposing the nuclei of red blood cells,” says Peng Ji, lead author and postdoctoral researcher in the Lodish lab. “These proteins are known for their role in creating actin fibers in many body cells and are a necessary component of many important cellular functions, including cell division, that support cell growth.”

His cell-culture system began with red blood cell precursors drawn from an embryonic mouse liver (in mammalian embryos, the liver is the main producer of such cells, rather than bone marrow as in adults).

The cultured cells, synchronized to develop together, divided four or five times before losing their nuclei and becoming immature red blood cells.? The researchers used fluorescence-based assays that enabled them to probe the changes in the red blood cells through the different stages leading up to the loss of the nucleus.

The researchers plan to further investigate the entire process of red blood cell formation, which may lead to insights about genetic alterations that underlie blood cell disorders.

“During normal cell division, each daughter cell receives half the DNA,” comments Lodish. “In this case, when the red blood cell divides, one daughter cell gets all the DNA. What’s fascinating is that in this case, that daughter cell gets eaten by macrophages. Until now, scientists were unable to study these cells because they were unable to see them.”.