Builders and Blocks – Engineering Blood Vessels with Stem Cells

Back in 2001, when we first began studying how regenerative cells (stem cells or more mature progenitor cells) enhance blood vessel growth, our group as well as many of our colleagues focused on one specific type of blood vessel: arteries. Arteries are responsible for supplying oxygen to all organs and tissues of the body and arteries are more likely to develop gradual plaque build-up (atherosclerosis) than veins or networks of smaller blood vessels (capillaries). Once the amount of plaque in an artery reaches a critical threshold, the oxygenation of the supplied tissues and organs becomes compromised. In addition to this build-up of plaque and gradual decline of organ function, arterial plaques can rupture and cause  severe sudden damage such as a heart attack. The conventional approach to treating arterial blockages in the heart was to either perform an open-heart bypass surgery in which blocked arteries were manually bypassed or to place a tube-like “stent” in the blocked artery to restore the oxygen supply. The hope was that injections of regenerative cells would ultimately replace the invasive procedures because the stem cells would convert into blood vessel cells, form healthy new arteries and naturally bypass the blockages in the existing arteries.

 

Image of mouse red blood cells flowing through an engineered human blood vessel- Image from Paul and colleagues (2013), Creative Commons license.
Image of mouse red blood cells flowing through an engineered human blood vessel implanted in a mouse- Image from Paul and colleagues (2013), Creative Commons license.

As is often the case in biomedical research, this initial approach turned out to be fraught with difficulties. The early animal studies were quite promising and the injected cells appeared to stimulate the growth of blood vessels, but the first clinical trials were less successful. It was very difficult to retain the injected cells in the desired arteries or tissues, and even harder to track the fate of the cells. Which stem cells should be injected? Where should they be injected? How many? Can one obtain enough stem cells from an individual patient so that one could use his or her own cells for the cell therapy? How does one guide the injected cells to the correct location, and then guide the cells to form functional blood vessel structures? Would the stem cells of a patient with chronic diseases such as diabetes or high blood pressure be suitable for therapies, or would such a patient have to rely on stem cells from healthier individuals and thus risk the complication of immune rejection?

The complexity of blood-vessel generation became increasingly apparent, both when studying the biology of stem cells as well as when designing and conducting clinical trials. A large clinical study published in 2013 studied the impact of bone marrow cell injections in heart attack patients and concluded that these injections did not result in any sustained benefit for heart function. Other studies using injections of patients’ own stem cells into their hearts had led to mild improvements in heart function, but none of these clinical studies came close to fulfilling the expectations of cardiovascular patients, physicians and researchers. The upside to these failed expectations was that it forced the researchers in the field of cardiovascular regeneration to rethink their goals and approaches.

One major shift in my own field of interest – the generation of new blood vessels – was to reevaluate the validity of relying on injections of cells. How likely was it that millions of injected cells could organize themselves into functional blood vessels? Injections of cells were convenient for patients because they would not require the surgical implantation of blood vessels, but was this attempt to achieve a convenient therapy undermining its success? An increasing number of laboratories began studying the engineering of blood vessels in the lab by investigating the molecular cues which regulate the assembly of blood vessel networks, identifying molecular scaffolds which would retain stem cells and blood vessel cells and combining various regenerative cell types to build functional blood vessels. This second wave of regenerative vascular medicine is engineering blood vessels which will have to be surgically implanted into patients.  This means that it will be much harder to get approval to conduct such invasive implantations in patients than the straightforward injections which were conducted in the first wave of studies, but most of us who have now moved towards a blood vessel engineering approach feel that there is a greater likelihood of long-term success even if it may take a decade or longer till we obtain our first definitive clinical results.

The second conceptual shift which has occurred in this field is the realization that blood vessel engineering is not only important for treating patients with blockages in their arteries. In fact, blood vessel engineering is critical for all forms of tissue and organ engineering. In the US, more than 120,000 people are awaiting an organ transplant but only a quarter of them will receive an organ in any given year. The number of people in need of a transplant will continue to grow but the supply of organs is limited and many patients will unfortunately die while waiting for an organ which they desperately need. The advances in stem cell biology have made it possible to envision creating organs or organoids (functional smaller parts of an organ) which could help alleviate the need for organs. One thing that most organs and tissues need is a network of tiny blood vessels that permeate the whole tissue: small capillary networks. For example, a liver built out of liver cells could never function without a network of tiny blood vessels which supply the liver cells with metabolites and oxygen. From an organ engineering point of view, microvessel engineering is just as important as the building of functional arteries.

In one of our recent projects, we engineered functional human blood vessels by combining bone marrow derived stem cells with endothelial cells (the cells which coat the inside of all blood vessels). It turns out that stem cells do not become endothelial cells but instead release a molecular signal – the protein SLIT3- which instructs the endothelial cells to assemble into networks. Using a high resolution microscope, we watched this process in real-time over a course of 72 hours in the laboratory and could observe how the endothelial cells began lining up into tube-like structures in the presence of the bone marrow stem cells. The human endothelial cells were like building blocks, the human bone marrow stem cells were the builders “overseeing” the construction. When we implanted the assembled blood vessel structures into mice, we could see that they were fully functional, allowing mouse blood to travel through them without leaking or causing any other major problems (see image, taken from reference 3).

I am sure that SLIT3 is just one of many molecular cues released by the stem cells to assemble functional networks and there are many additional mechanisms which still need to be discovered. We still need to learn much more about which “builders” and which “building blocks” are best suited for each type of blood vessel that we want to construct. The fact that human fat tissue can serve as an important resource for obtaining adult stem cells(“builders”) is quite encouraging, but we still know very little about the overall longevity of the engineered vessels, the best way to implant them into patients, and the key molecular and biomechanical mechanisms which will be required to engineer organs with functional blood vessels. It will be quite some time until the first fully engineered organs will be implanted in humans, but the dizzying rate of progress suggests that we can be quite optimistic.

(An earlier version of this article was first published on 3Quarksdaily.com).

 

References and links (all of them are open access, so you can read them for free, including the original paper we published):

 1. A recent longform overview article I wrote for “The Scientist” in which I describe the importance of blood vessel engineering for organ engineering:

J Rehman “Building Flesh and Blood“, The Scientist (2014), 28(5):48-53

 

2. An unusual and abundant source of adult stem cells which promote the formation of blood vessels: Fat tissue obtained from individuals who undergo a liposuction! 

J Rehman “The Power of Fat” Aeon Magazine (2014)

 

3. The study which describes how adult stem cells release a protein (SLIT3) which organizes blood vessel cells into functional networks (open access – can be read free of charge):

J.D. Paul et al., “SLIT3-ROBO4 activation promotes vascular network formation in human engineered tissue and angiogenesis in vivo” J Mol Cell Cardiol (2013), 64:124-31.

 

ResearchBlogging.org

Paul JD, Coulombe KL, Toth PT, Zhang Y, Marsboom G, Bindokas VP, Smith DW, Murry CE, & Rehman J (2013). SLIT3-ROBO4 activation promotes vascular network formation in human engineered tissue and angiogenesis in vivo. Journal of molecular and cellular cardiology, 64, 124-31 PMID: 24090675

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Does Human Fat Contain Stem Cells?

Aeon Magazine recently published my longform essay on our research with human liposuction samples and our attempts to use fat for regenerative and therapeutic purposes. Many research groups, including our own group, have been able to isolate stem cells from human fat. However, when it came to using this cells for treating cardiovascular disease, the cells behaved in a manner that we had not anticipated.

Undifferentiated mesenchymal stem cells (left) and their fat neighbors (right)
Undifferentiated mesenchymal stem cells (left) and their fat neighbors (right) – From our PLOS One paper

We were unable to convert them into heart muscle cells or blood vessel endothelial cells, but we found that they could help build large networks of blood vessels by releasing important growth factors. Within a few years of our initial publication, clinical trials with patients with blocked arteries or legs were already being planned, and are currently underway.

We decided to call the cells “adipose stromal cells” because we wanted to emphasize that they were acting as a “stroma” (i.e. supportive environment for blood vessels) and not necessarily as stem cells (i.e. cells that convert from an undifferentiated state into mature cell types). In other contexts, these same cells were indeed able to act like “stem cells”, because they could be converted into bone-forming or cartilage-forming cells, thus showing the enormous versatility and value of the cells that reside within our fat tissues.

The answer to the question “Does Human Fat Contain Stem Cells?” is Yes, but these cells cannot be converted into all desired tissues. Instead, they have important supportive functions that can be used to engineer new blood vessels, which is a critical step in organ engineering.

In addition to describing our scientific work, the essay also mentions the vagaries of research, the frustrations I had as a postdoctoral fellow when my results were not turning out as I had expected, and how some predatory private clinics are already marketing “fat-derived stem cell therapies” to paying customers, even though the clinical results are still rather preliminary.

 

For the readers who want to dig a bit deeper, here are some references and links:

 

1. The original paper by Patricia Zuk and colleagues which described the presence of stem cells in human liposuction fat:

Zuk, P et al (2001) “Multilineage Cells from Human Adipose Tissue: Implications for Cell-Based Therapies

 

2. Our work on how the cells can help grow blood vessels by releasing proteins:

Rehman, J et al (2004) “Secretion of Angiogenic and Antiapoptotic Factors by Human Adipose Stromal Cells

 

3. Preliminary findings from ongoing clinical studies in which heart attack patients receive infusions of fat derived cells into their hearts to improve heart function and blood flow to the heart:

Houtgraf, J et al (2012) “First Experience in Humans Using Adipose Tissue–Derived Regenerative Cells in the Treatment of Patients With ST-Segment Elevation Myocardial Infarction

 

4. Preliminary results from an ongoing trial using the fat-derived cells in patients with severe blockages of leg arteries:

Bura, A et al (2014) “Phase I trial: the use of autologous cultured adipose-derived stroma/stem cells to treat patients with non-revascularizable critical limb ischemia

 

5. Example of how “cell therapies” (in this case bone marrow cells) are sometimes marketed as “stem cells” but hardly contain any stem cells:

The Largest Cell Therapy Trial in Heart Attack Patients Uses Hardly Any Stem Cells

 

6. The major scientific society devoted to studying the science of fat and its cells as novel therapies is called International Federation for Adipose Therapeutics and Science (IFATS).

I am not kidding, it is I-FATS!

Explore their website if you want to learn about all the exciting new research with fat derived cells.

 

7. Some of our newer work on how bone marrow mesenchymal stem cells turn into fat cells and what role their metabolism plays during this process:

Zhang, Y et al (2013) “Mitochondrial Respiration Regulates Adipogenic Differentiation of Human Mesenchymal Stem Cells

 

ResearchBlogging.org

Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, & Hedrick MH (2001). Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue engineering, 7 (2), 211-28 PMID: 11304456

 

 

 

ResearchBlogging.org
Rehman J, Traktuev D, Li J, Merfeld-Clauss S, Temm-Grove CJ, Bovenkerk JE, Pell CL, Johnstone BH, Considine RV, & March KL (2004). Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells. Circulation, 109 (10), 1292-8 PMID: 14993122

Synthetic Biology: Engineering Life To Examine It

Two scientific papers that were published in the journal Nature in the year 2000 marked the beginning of engineering biological circuits in cells. The paper “Construction of a genetic toggle switch in Escherichia coli” by Timothy Gardner, Charles Cantor and James Collins created a genetic toggle switch by simultaneously introducing an artificial DNA plasmid into a bacterial cell. This DNA plasmid contained two promoters (DNA sequences which regulate the expression of genes) and two repressors (genes that encode for proteins which suppress the expression of genes) as well as a gene encoding for green fluorescent protein that served as a read-out for the system. The repressors used were sensitive to either selected chemicals or temperature. In one of the experiments, the system was turned ON by adding the chemical IPTG (a modified sugar) and nearly all the cells became green fluorescent within five to six hours. Upon raising the temperature to activate the temperature-sensitive repressor, the cells began losing their green fluorescence within an hour and returned to the OFF state. Many labs had used chemical or temperature switches to turn gene expression on in the past, but this paper was the first to assemble multiple genes together and construct a switch which allowed switching cells back and forth between stable ON and OFF states.

dna-163466_640

The same issue of Nature contained a second land-mark paper which also described the engineering of gene circuits. The researchers Michael Elowitz and Stanislas Leibler describe the generation of an engineered gene oscillator in their article “A synthetic oscillatory network of transcriptional regulators“. By introducing three repressor genes which constituted a negative feedback loop and a green fluorescent protein as a marker of the oscillation, the researchers created a molecular clock in bacteria with an oscillation period of roughly 150 minutes. The genes and proteins encoded by the genes were not part of any natural biological clock and none of them would have oscillated if they had been introduced into the bacteria on their own. The beauty of the design lay in the combination of three serially repressing genes and the periodicity of this engineered clock reflected the half-life of the protein encoded by each gene as well as the time it took for the protein to act on the subsequent member of the gene loop.

Both papers described the introduction of plasmids encoding for multiple genes into bacteria but this itself was not novel. In fact, this has been a routine practice since the 1970s for many molecular biology laboratories. The panache of the work lay in the construction of functional biological modules consisting of multiple genes which interacted with each other in a controlled and predictable manner. Since the publication of these two articles, hundreds of scientific papers have been published which describe even more intricate engineered gene circuits. These newer studies take advantage of the large number of molecular tools that have become available to query the genome as well as newer DNA plasmids which encode for novel biosensors and regulators.

Synthetic biology is an area of science devoted to engineering novel biological circuits, devices, systems, genomes or even whole organisms. This rather broad description of what “synthetic biology” encompasses reflects the multidisciplinary nature of this field which integrates ideas derived from biology, engineering, chemistry and mathematical modeling as well as a vast arsenal of experimental tools developed in each of these disciplines. Specific examples of “synthetic biology” include the engineering of microbial organisms that are able to mass produce fuels or other valuable raw materials, synthesizing large chunks of DNA to replace whole chromosomes or even the complete genome in certain cells, assembling synthetic cells or introducing groups of genes into cells so that these genes can form functional circuits by interacting with each other. Synthesis in the context of synthetic biology can signify the engineering of artificial genes or biological systems that do not exist in nature (i.e. synthetic = artificial or unnatural), but synthesis can also stand for integration and composition, a meaning which is closer to the Greek origin of the word.  It is this latter aspect of synthetic biology which makes it an attractive area for basic scientists who are trying to understand the complexity of biological organisms. Instead of the traditional molecular biology focus on studying just one single gene and its function, synthetic biology is engineering biological composites that consist of multiple genes and regulatory elements of each gene. This enables scientists to interrogate the interactions of these genes, their regulatory elements and the proteins encoded by the genes with each other. Synthesis serves as a path to analysis.

One goal of synthetic biologists is to create complex circuits in cells to facilitate biocomputing, building biological computers that are as powerful or even more powerful that traditional computers. While such gene circuits and cells that have been engineered have some degree of memory and computing power, they are no match for the comparatively gigantic computing power of even small digital computers. Nevertheless, we have to keep in mind that the field is very young and advances are progressing at a rapid pace.

One of the major recent advances in synthetic biology occurred in 2013 when an MIT research team led by Rahul Sarpeshkar and Timothy Lu at MIT created analog computing circuits in cells. Most synthetic biology groups that engineer gene circuits in cells to create biological computers have taken their cues from contemporary computer technology. Nearly all of the computers we use are digital computers, which process data using discrete values such as 0’s and 1’s. Analog data processing on the other hand uses a continuous range of values instead of 0’s and 1’s. Digital computers have supplanted analog computing in nearly all areas of life because they are easy to program, highly efficient and process analog signals by converting them into digital data. Nature, on the other hand, processes data and information using both analog and digital approaches. Some biological states are indeed discrete, such as heart cells which are electrically depolarized and then repolarized in periodical intervals in order to keep the heart beating. Such discrete states of cells (polarized / depolarized) can be modeled using the ON and OFF states in the biological circuit described earlier. However, many biological processes, such as inflammation, occur on a continuous scale. Cells do not just exist in uninflamed and inflamed states; instead there is a continuum of inflammation from minimal inflammatory activation of cells to massive inflammation. Environmental signals that are critical for cell behavior such as temperature, tension or shear stress occur on a continuous scale and there is little evidence to indicate that cells convert these analog signals into digital data.

Most of the attempts to create synthetic gene circuits and study information processing in cells have been based on a digital computing paradigm. Sarpeshkar and Lu instead wondered whether one could construct analog computation circuits and take advantage of the analog information processing systems that may be intrinsic to cells. The researchers created an analog synthetic gene circuit using only three proteins that regulate gene expression and the fluorescent protein mCherry as a read-out. This synthetic circuit was able to perform additions or ratiometric calculations in which the cumulative fluorescence of the mCherry was either the sum or the ratio of selected chemical input concentrations. Constructing a digital circuit with similar computational power would have required a much larger number of components.

The design of analog gene circuits represents a major turning point in synthetic biology and will likely spark a wave of new research which combines analog and digital computing when trying to engineer biological computers. In our day-to-day lives, analog computers have become more-or-less obsolete. However, the recent call for unconventional computing research by the US Defense Advanced Research Projects Agency (DARPA) is seen by some as one indicator of a possible paradigm shift towards re-examining the value of analog computing. If other synthetic biology groups can replicate the work of Sarpeshkar and Lu and construct even more powerful analog or analog-digital hybrid circuits, then the renaissance of analog computing could be driven by biology.  It is difficult to make any predictions regarding the construction of biological computing machines which rival or surpass the computing power of contemporary digital computers. What we can say is that synthetic biology is becoming one of the most exciting areas of research that will provide amazing insights into the complexity of biological systems and may provide a path to revolutionize biotechnology.

ResearchBlogging.orgDaniel R, Rubens JR, Sarpeshkar R, & Lu TK (2013). Synthetic analog computation in living cells. Nature, 497 (7451), 619-23 PMID: 23676681

 

 

 

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An earlier version of this article was first published here on the 3Quarksdaily blog.