Making Blood Cells in Culture – Done


 

One of the “Holy Grails” of stem cell biology has been growing blood cells in culture for use in clinical settings. Such a feat would provide large quantities of blood cells for post-surgical patients, or those with leukemia or other blood-based illness. The clinical applications are manifold and extensive.

Unfortunately, growing blood-making stem cells in the laboratory has proven to be a difficult task for even the most inventive and skilled stem cell laboratories. Nevertheless, several laboratories have been able to recapitulate the differentiation of pluripotent stem cells into cells that have the capacity to form T-cells and myeloid (non-lymphoid) cells (see Kennedy, M. et al. Cell Rep. 2, 1722–1735 (2012); Ditadi, A. et al. Nat. Cell Biol. 17, 580–591 (2015); and Elcheva, I. et al. Nat. Commun. 5, 4372 (2014)). Unfortunately, these experiments generated cells that were not able to engraft in the bone marrow of irradiated mice. Such an experiment is essential because radiation destroys the bone marrow of the mouse, and if a cultured cell is indeed and blood-cell-forming stem cell, then placing it into the bone marrow of irradiated mice should result in a functional restoration of the bone marrow. This, however, was not the case, which shows that whatever these pluripotent stem cells in these experiments differentiated into, they were not blood-cell-forming hematopoietic stem cells (HSCs).

Now, after a hiatus of almost 20 years, two different research groups have use two very different approaches to transform mature cells into primitive HSCs that are self-propagating and also form the cellular components of blood.

The first of these research teams was led by George Daley of Boston Children’s Hospital in Massachusetts. Daley’s group used induced pluripotent stem cell technology to reprogram adult human cells into cells that function as HSCs, even though they are not precisely like those found in the bone marrow in people. The second research team was led by Shahin Rafii of the Weill Cornell Medical College in New York City. Rafii and his coworkers used direct programming to differentiate mature cells from mice into fully functional HSCs.

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The Daley group isolated skin-based fibroblasts from adult donors and then reprogrammed then through a combination of genetic engineering and cell culture techniques. This technology is similar to that designed by Shinya Yamanaka and his colleagues at Kyoto University, for which Yamanaka won the Nobel Prize in Medicine in 2012.   Once these reprogrammed cells formed induced pluripotent stem cells (iPSCs), Daley and his group did something very creative. They inserted the genes that encode seven different transcription factors into the genomes of their iPSCs. Transcription factors are proteins that activate gene expression. Transcription factors do so either by binding specific sequences of DNA, or by tightly binding to other proteins involved in gene expression and activating them. The genes for these seven transcription factors (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1 and SPI1) are known to be sufficient to convert hemogenic endothelium (the cells from which HSCs develop) into HSCs.

After engineering their iPSCs with these seven genes, Daley’s group did yet another highly creative thing. Daley and his colleagues injected their modified human cells into developing mice. This provided the cells with the proper environment to differentiate into HSCs. Twelve weeks after injecting them into mouse embryos, the engineered iPSCs had differentiated into progenitor cells that could produce the full range of blood cells found in human blood. This included immune cells, platelets, and other types of red and white blood cells. These progenitor cells are, according to Daley, “tantalizingly close” to naturally occurring HSCs.

Rafii and his team made their mouse HSCs from mature mouse cells without going through an embryonic intermediate. The Rafii grouop isolated endothelial cells that line blood vessels from adult mice and genetically engineered them to overexpress four different genes (Fosb, Gfi1, Runx1, and Spi1). Upon culturing their genetically engineered cells in a culture system that mimicked blood vessels, these cells, over time, differentiated into HSCs.

For the next test, Rafii and others injected their cultures-derived HSCs into irradiated mice. These mice survived and showed a completely recapitulated bone marrow that produced immune cells, and all types of red and white blood cells, and lived than 1.5 years in the lab.

Rafii told Nature’s Amy Maxmen that his approach is like “a direct airplane flight, and Daley’s procedure to a flight that takes a detour to the Moon before reaching its final destination.” Rafii noted that how the cells are made matters when it comes to using them in the clinic. Every time genes are transformed into cultured cells, a significant percentage of the cells fail to incorporate one or all of these genes. Such cells must be removed from those cells that were successfully transformed. Genetically engineered cells also run the risk of having experienced mutations as a side effect of the genetic manipulation. If implanted into people, such cells might cause problems.

Daley, however, and other stem cell researchers remain sanguine about the possibility of making such cells in safer, more efficient and even cheaper ways that can be brought to the clinic. For example, Jeanne Loring from the Scripps Research Institute in La Jolla, CA has suggested that using techniques that cause transient rather than permanent expression of introduced genes might very well make such cells inherently safer. Loring also noted that the iPSCs had by Daley’s group are initially made from skin-based fibroblasts, which are easy to acquire and isolate, whereas Rafii’s method begins with endothelial cells, which are more difficult to gather and to keep alive in the lab.

“For many years, people have figured out parts of this recipe, but they’ve never quite gotten there,” says Mick Bhatia, a stem-cell researcher at McMaster University in Hamilton, Canada, who was not involved with either study. “This is the first time researchers have checked all the boxes and made blood stem cells.” Bhatia added: “A lot of people have become jaded, saying that these cells don’t exist in nature and you can’t just push them into becoming anything else. . . I hoped the critics were wrong, and now I know they were.”

So making blood cells and HSCs in the laboratory is possible.  Bring this into the clinic is going to be even tougher.

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mburatov

Professor of Biochemistry at Spring Arbor University (SAU) in Spring Arbor, MI. Have been at SAU since 1999. Author of The Stem Cell Epistles. Before that I was a postdoctoral research fellow at the University of Pennsylvania in Philadelphia, PA (1997-1999), and Sussex University, Falmer, UK (1994-1997). I studied Cell and Developmental Biology at UC Irvine (PhD 1994), and Microbiology at UC Davis (MA 1986, BS 1984).