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.


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.

Repeated Administrations of Stem Cells are More Effective than Single Administration

After a heart attack, the heart can undergo several structural and functional changes. Even after oxygen delivery to the heart muscle has been restored, a temporary loss of contractile function can persist for several hours or even days. This phenomenon is called myocardial stunning and it can also occur in people who have undergone cardiovascular procedures or central nervous system trauma. Myocardial stunning seems to result from the release of toxic molecules by the dying heart muscle cells, and an imbalance in ions required for heart muscle contraction, such as calcium ions.

If the heart muscle remains deprived of oxygen for some time, then the heart muscle adapts to low-oxygen conditions and “hibernates.” Hibernating myocardium contracts very little, and has “battened down the hatches,” metabolically speaking, in order to survive. However, hibernating myocardium takes even more heart muscle cells off-line and further reduces the performance of the heart.

Reduced heart performance leads to the production of molecules by the blood-starved kidneys that enlarge the heart and further compromise its efficiency. This leads to congestive heart failure and death. The term for such post-heart attack heart disease is ischemic cardiomyopathy” and it refers to a heart after a heart attack that is deprived of oxygen in many places and has heart walls filled with dead tissue that struggles to properly supply to body with blood, and often deteriorates as a result of this struggle.

Several different cell types have been used in many different studies to treat ischemic (oxygen-deprived) heart disease. The results, though positive in many cases, only show modest improvements in most cases. Furthermore, clinical studies and pro-clinical studies in laboratory animals tend to produce inconsistent results in which some patients or animals significantly improve while others either fail to improve at all. However, all of these studies have one feature in common: the subject is treated with only one infusion of stem cells. What if only one treatment is not enough to properly heal the damaged heart after a heart attack?

Workers from Roberto Bolli’s laboratory at the University of Louisville, Kentucky have treated laboratory rodents with heart attacks with three doses of cardiac progenitor cells. These treatments were given 35 days apart and were compared with single treatments and placebo treatments.

In their paper, which was published in Circulation Research (DOI:10.1161/CIRCRESAHA.116.308937), Bolli and his group gave heart attack to 85 Fischer 344 rats, but 13 died one week after the procedure. The remaining 72 rats were split into three groups: vehicle only, single-treatment, and triple-treatment. The vehicle-only rats received injections of saline into their heart muscle 30 days after the heart attack, and then again 35 days later and then again 35 days after that. The single-treatment rats received an injection of 12 million cardiac progenitor cells into their heart muscle 30 days after their heart attacks, but then received injections of saline 35 days later and 35 days after that. The triple-treatment rats received an injection of 12 million cardiac progenitor cells into their heart muscle 30 days after their heart attack, and then another injection 35 days later and a third inject 35 days after that. Of the 72 rats that survived the laboratory-induced heart attacks, 9 died as a result of injections into the heart. Thus, 63 rats completed the protocol.

Cardiac progenitor cells (CPCs) are resident stem cells in the heart that can be isolated with small heart tissue biopsies (see Tang XL, et al., Circ Res 2016;118:1091-1105). They can differentiate into heart muscle, blood vessels, or other heart-specific cell types (See Parmacek and Epstein, Cell 2005;120;295-298). Bolli and his co-workers have shown that the infusion of CPCs into the hearts of laboratory rodents after a heart attack can improve heart function, but the CPCs do not engraft into the heart at a terribly high rate. Furthermore, the functional improvements in the heart of these rodent elicited by CPC implantation are long-term (see Circ Res 2016;118:1091-1105).

When heart function of the laboratory rats were assessed prior to the procedure, no significant differences were observed between any of the rats in the three groups. However, after the procedure, the hearts of those rats that were injected with saline continued to deteriorate throughout the duration of the experiment. This deterioration was functional in nature and structural.

Animals that had received only one injection of 12 million CPCs into the left ventricles of their hearts showed significant improvements over the saline-injected animals. These animals showed less dilation of the heart (lower end-systolic volume), their hearts pumped more blood per beat (stroke volume), better thickness in the damaged heart wall, and improved ejection fraction (average percentage of blood pumped from the left ventricle during each beat). These hearts also had smaller heart scars, great elasticity, and greater amounts of viable muscle.

While all that sounds great, the triply-injected hearts that received three injections of CPCs, showed even more robust and significant improvements in heart structure and function. Whatever the single injection of CPCs did, the triple injections did even better. However, it must be noted that even with three injections of CPCs, the level of engraftment of the injected cells remained poor.

To summarize these results observed in this paper, the 3 doses of CPCs 35 days apart resulted in increases in local and global heart function that were, roughly, triple that produced by a single dose. The multiple CPC administrations were associated with more viable tissue, less scar tissue, less collagen, and greater heart muscle cell density in the infarcted region. Still, the level of engraftment and differentiation of the injected cells accounted for <1% of total heart muscle cells.

Bolli and his coworkers believe that their work suggests that all clinical and preclinical trials should at least try multiple stem cell treatments in order to maximize the clinical benefit of the injected stem cells. Furthermore, Bolli and others suggest that the use of single injection protocols are the reason so many stem cell-based clinical trials have resulted in inconsistent and inconclusive results.

This study is a large preclinical trial that used large numbers of animals. The data are solid and the results are believable. My problem with the clinical implications of this study are as follows: A heart attack causes a good deal of inflammation in the heart that culminates in wound healing. Within 24 hours of the heart attack, white blood cells infiltrate the damaged area of the heart. Protein-degrading enzymes from scavenger neutrophils (a type of white blood cell) degrade dead tissue. The damaged cells degenerate, and collagen-making fibroblasts divide and lay down scar tissue. The initially-deposited collagen deposited in the wall of the heart is weak, mushy, and vulnerable to re-injury. Unfortunately, this is precisely the period of time (10-14 days after the heart attack) that patients feel better and want to increase their activity levels. However, this greater activity level can stress the heart and cause rupture of the heart wall. After 6 weeks, the dead (necrotic) area is completely replaced by scar tissue, which is strong but incapable of contracting or relaxing.

In light of this timeline, multiple injections of stem cells into the heart after a heart attack might very increase the risk of rupture of the heart wall. This is particularly the case if implanted cells secrete tissue proteases that degrade surrounding tissue. Thus, timing and dose will be extremely important in such multiple treatments. Too many cells can rupture the heart, treatments too early when the healing heart walls are sift and weak will prove inimical to the heart and treatments given too late might very well be too late for the cells to do any good. Therefore, while this paper seems to move the ball down the field of regenerative medicine, it creates a fair number of questions that will need to be answered before such a strategy can come to the clinic.