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.

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.

Dosing Recent Heart Attack Patients with G-CSF Doesn’t Seem To Work


Granulocyte-Colony Stimulating Factor (G-CSF)is a small protein that stimulates the bone marrow to produce more of a particular class of white blood cells called granulocytes and release them into the bloodstream. A commercially available version of G-CSF called Filgrastim (Neupogen) is used to boost the immune system of cancer patients whose immune systems have taken a beating from chemotherapy.

Because several clinical trials have shown that implanting bone marrow mononuclear fractions into the hearts of heart attack patients can improve the heart health of some heart attack patients, clinicians have supposed that injecting heart attack patients with drugs like filgrastim, which moves many bone marrow-derived cells into the bloodstream might also provide some relief for heart attack patients.

Nice idea, but it does not seem to work. Two clinical trials, STEMMI and REVIVAL-2, have given G-CSF to heart attack patients at different times after their heart attacks. Unfortunately both studies have failed to show a difference from the placebo.

In the REVIVAL-2 study, 114 patients were enrolled, and 56 received 10 micrograms per kilogram body weight G-CSF for five days, and the remaining patients received a placebo treatment.  G-CSF and the placebo were administered to patients five days after the hearts were successfully reperfused by percutaneous coronary intervention (this is a fancy way of saying stenting).  This study was double-blinded, placebo-controlled and well designed.  Unfortunately, when patients were studied seven years after treatment, there were no statistically significant differences between the treatment and the placebo groups when it came to the number of deaths, heart attacks, and strokes.  Thus, the authors conclude that G-CSF administration did not improve clinical outcomes for patients who had a heart attack (see Birgit Steppich, et al, Atherosclerosis and Ischemic Disease 115.4, 2016).

A second clinical trial, the STEMMI trial, was a prospective trial in which G-CSF treatment was begun 10-65 hours after reperfusion.  Here again, there were no structural differences between the placebo group and the G-CSF-treated group six months after treatment and a five-year follow-up analysis of 74 patients revealed no differences in the occurrence of major cardiovascular incidents between the two treatment groups (R.S. Ripa, and others, Circulation 2006; 113: 1983-1992).

The STEM-AMI clinical trial also showed no differences in clinical outcomes after G-CSF treatment as compared to placebo in 60 patients after three years (F. Achilli, and others, Heart 2014, 100: 574-581).

Why does this technique fail?  It is possible that the white blood cells that are mobilized by G-CSF are low-quality and do not express particular genes.  A study in rats has shown that G-CSF infusion increases the number of progenitor cells in the bloodstream, but fails to increase the number of progenitor cells in the heart after a heart attack (D. Sato, and others, Experimental Clinical Cardiology, 2012; 17:83-88).  In order for cells to home to the infarcted heart, they must express particular proteins on their surfaces.  For example, the cell surface protein CXCR4 is known to play an integral role in progenitor cell homing, along with several other proteins (see Taghavi and George, American Journal of Translational Research 2013; 5:404-411; Shah and Shalia, Stem Cells International 2011;2011:536758; Zaruba and Franz, Expert Opinion in Biological Therapy 2010; 10:321-335).  Indeed, Stein and others have shown that progenitor cells mobilized with G-CSF in human patients lack CXCR4 and other cell adhesion proteins thought to play a role in homing to the infarcted heart (Thromb Haemost 2010;103:638-643).

Therefore, even though all of these studies have not uncovered a risk in G-CSF treatment, the consensus of the data seems to be there no clinical benefit is conferred by treating heart attack patients with G-CSF.

Hair Follicles Can Direct Wound-Based Cells to Induce Scar-Free Healing


News from the University of Pennsylvania reports a new method that involves the use of fat to help heal skin without the formation of scar tissue.  This work comes from the Perelman School of Medicine at the University of Pennsylvania, and it is the result of a large-scale, multi-year study that collaborated with the Plikus Laboratory for Developmental and Regenerative Biology at the University of California, Irvine.  Their findings were published online in the journal Science on January 5th, 2017.

A fancy name for fat is “adipose tissue.”  Adipose tissue is actually a rather complicated pastiche of different cell types.  Specialized cells in adipose tissue that stores fat are called “adipocytes,” but they are more colloquially called fat cells.  Fat cells are normally found in the skin, but when wounds in the skin heal and form, those underlying population of fat cells are lost.  In skin tissue that is undergoing the process of healing, the most common cell types are known as “myofibroblasts.”  Myofibroblasts are large cells with ruffled membranes, that are kind of a cross between smooth muscle cells and fibroblasts.  They have the ability to contract like smooth muscle cells when exposed to molecules that induce smooth muscle to contract, such as angiotensin II or epinephrine.  Fibroblasts, which are numerous throughout the skin and other organs, can readily differentiate into myofibroblasts, as can stellate cells found in liver or the pancreas, some smooth muscle cells, progenitor cells in stromal tissue, epithelial cells, or circulating progenitor cells (see B. Hinz, et al, The myofibroblast: one function, multiple origins, Am J Pathol. 2007 Jun;170(6):1807-16).  Once it forms, scar tissue also does not properly form any hair follicles and this can give it a rather odd appearance relative to the rest of the skin. The Perelman researchers designed a new strategy to limit scar formation during healing by converting wound-based myofibroblasts into fat cells, which prevents the formation of scarring.

“Essentially, we can manipulate wound healing so that it leads to skin regeneration rather than scarring,” said George Cotsarelis, MD, the chair of the Department of Dermatology and the Milton Bixler Hartzell Professor of Dermatology at Penn, and the principal investigator of this project. “The secret is to regenerate hair follicles first. After that, the fat will regenerate in response to the signals from those follicles.”

Cotsarelis and his colleagues showed that the formation of fat in the skin and hair follicles are separate developmental events, but they are, nevertheless, linked.  Hair follicles form first, and the factors required to induce hair follicle formation that are produced by the regenerating hair follicle can also convert surrounding myofibroblasts into fat cells instead of a scar.  This underlying fat does not form without the formation of these new hair follicles.  These new fat cells are indistinguishable from pre-existing skin-based fat cells that give the healed wound a natural look instead of leaving a scar.  Cotsarelis and his gang discovered that a factor secreted by hair follicles called Bone Morphogenetic Protein (BMP) instructs the myofibroblasts to become fat.  This single finding represents a tectonic shift on our understanding of myofibroblasts.

“Typically, myofibroblasts were thought to be incapable of becoming a different type of cell,” Cotsarelis said. “But our work shows we have the ability to influence these cells, and that they can be efficiently and stably converted into adipocytes.” This was shown in both the mouse and in human keloid cells grown in culture.

“The findings show we have a window of opportunity after wounding to influence the tissue to regenerate rather than scar,” said the study’s lead author Maksim Plikus, PhD, an assistant professor of Developmental and Cell Biology at the University of California, Irvine. Plikus began this research as a postdoctoral fellow in the Cotsarelis Laboratory at Penn, and the two institutions have continued to collaborate.

These new findings might very well revolutionize dematological wound treatments.  These data might be useful for developing therapies that drive myofibroblasts to differentiate into adipocytes that can help wounds heal without scarring.

As Cotsarelis put it: “It’s highly desirable from a clinical standpoint, but right now it’s an unmet need.”

However, wound treatments are not the only use for this work.  Fat cell loss is a common complication of other clinical conditions.  HIV treatments, cancer, scleroderma, are just a few of the diseases that can cause wasting and drastic weight loss.  Also, because fat cells are also lost naturally because of the aging process, especially in the face, which leads to permanent, deep wrinkles, something that available anti-aging treatments cannot satisfactorily address.

“Our findings can potentially move us toward a new strategy to regenerate adipocytes in wrinkled skin, which could lead us to brand new anti-aging treatments,” Cotsarelis said.

The Cotsarelis Lab is now examining how hair follicle regeneration can promote skin regeneration.  The Plikus Laboratory would like to know more about the role of BMP in wound healing and are conducting further studies with using human cells and human scar tissue.

Stem Cell-Based Skin Graft for Severe Burns


Severe wounds are typically treated with full thickness skin grafts. Some new work by researchers from Michigan Tech and the First Affiliated Hospital of Sun Yat Sen University in Guangzhou, China might provide a way to use a patient’s own stem cells to make split thickness skin grafts (STSG). If this technique pans out, it would eliminate the needs for donors and could work well for large or complicated injury sites.

This work made new engineered tissues were able to capitalize on the body’s natural healing power. Dr. Feng Zhao at Michigan Tech and her Chinese colleagues used specially engineered skin that was “prevascularized, which is to say that Zhao and other designed it so that it could grow its own veins, capillaries and lymphatic channels.

This innovation is a very important one because on of the main reasons grafted tissues or implanted fabricated tissues fail to integrate into the recipient’s body is that the grafted tissue lacks proper vascular support. This leads to a condition called graft ischemia. Therefore, getting the skin to form its own vasculature is vital for the success of STSG.

STSG is a rather versatile procedure that can be used under unfavorable conditions, as in the case of patients who have a wound that has been infected, or in cases where the graft site possess less vasculature, where the chances of a full thickness skin graft successfully integrating would be rather low. Unfortunately, STSGs are more fragile than full thickness skin grafts and can contract significantly during the healing process.

In order to solve the problem of graft contraction and poor vascularization, Zhao and others grew sheets of human mesenchymal stem cells (MSCs) and mixed in with those MSCs, human umbilical cord vascular endothelial cells or HUVECs. HUVECs readily form blood vessels when induced, and growing mesenchymal stem cells tend to synthesize the right cocktail of factors to induce HUVECs to form blood vessels. Therefore this type of skin is truly poised to form its own vasculature and is rightly designated as “prevascularized” tissue.

Zhao and others tested their MSC/HUVEC sheets on the tails of mice that had lost some of their skin because of burns. The prevascularized MSC/HUVEC sheets significantly outperformed MSC-only sheets when it came to repairing the skin of these laboratory mice.

When implanted, the MSC/HUVEC sheets produced less contracted and puckered skin, lower amounts of inflammation, a thinner outer skin (epidermal) thickness along with more robust blood microcirculation in the skin tissue. And if that wasn’t enough, the MSC/HUVEC sheets also preserved skin-specific features like hair follicles and oil glands.

The success of the mixed MSC/HUVEC cell sheets was almost certainly due to the elevated levels of growth factors and small, signaling proteins called cytokines in the prevascularized stem cell sheets that stimulated significant healing in surrounding tissue. The greatest challenge regarding this method is that both STSG and the stem cell sheets are fragile and difficult to harvest.

An important next step in this research is to improve the mechanical properties of the cell sheets and devise new techniques to harvest these cells more easily.

According to Dr. Zhao: “The engineered stem cell sheet will overcome the limitation of current treatments for extensive and severe wounds, such as for acute burn injuries, and significantly improve the quality of life for patients suffering from burns.”

This paper can be found here: Lei Chen et al., “Pre-vascularization Enhances Therapeutic Effects of Human Mesenchymal Stem Cell Sheets in Full Thickness Skin Wound Re-pair,” Theranostics, October 2016 DOI: 10.7150/ thno.17031.

Activation of the Proteasome Enhances Stem Cell Function and Lifespan


As we age, the capacity of our stem cells to heal and replace damaged cells and tissues decline. This age-associated decrease in adult stem cell function seems to be a major contributor to the physiological decline during aging. A new paper, by Efstathios Gonos and his colleagues at the National Hellenic Research Foundation in Athens, Greece gives one possible technique that might improve the function of stem cells in an aging body.

Cells contain a multiprotein complex called the “proteasome” that degrades unneeded or defective proteins. The proteasome controls protein half-lives, function, and the protein composition of the cell. Functional failure of the proteasome has been linked to various biological phenomena including senescence and aging. The role of the proteasome in stem cells aging, however has received little attention to date.

Proteasome figure

Gonos and his coworkers used mesenchymal stem cells from umbilical cord Wharton’s Jelly and human fat. Because they were able to compare the proteasome activity in very young and aged stem cells, Gonos and others discovered a significant age-related decline in proteasome content and activity between these two types of stem cells. The proteasome from Warton’s Jelly mesenchymal stem cells were consistently more active and displayed more normal function and activity than those from human fat.  In fact, not only were the protease activities of the proteasomes from the aging stem cells decreased, but they also displayed structural alterations.

These differences in proteasomal activity were not only reproducible, but when the proteasome of young stem cells were compromised, the “stemness,” or capacity of the stem cells to act as undifferentiated cells, was negatively affected.

Even more surprisingly, once after mesenchymal stem cells from human donors lost their ability to proliferate and act as stem cells (their stemness, that is) their decline could be counteracted by artificially activating their proteasomes. Activating the proteasome seems to help the cell “clean house,” get rid of junk proteins, and rejuvenate themselves.

proteasomes-and-stem-cells

Gonos and his team found that the stem cell-specific protein, Oct4, binds to the promoter region of the genes that encode the β2 and β5 proteasome subunits. Oct4 might very well regulate the expression of these proteasome-specific genes.

From this paper, it seems that a better understanding the mechanisms regulating protein turnover in stem cells might bring forth a way to stem cell-based interventions that can improve health during old age and lifespan.

This paper was published in Free Radical Biology and Medicine, Volume 103, February 2017, Pages 226–235.