Granulocyte-Colony Stimulating Factor (G-CSF) is a glycoprotein (protein with sugars attached to it) that signals to the bone marrow to produce granulated white blood cells (specifically neutrophils), and to release stem cells and progenitor cells into the peripheral circulation.
This function of G-CSF makes it a candidate treatment for patients who have recently experienced a heart attack, since the release of stem cells from the bone marrow could, in theory, bring more stem cells to the damaged heart to heal it. Additionally, G-CSF is known to induce the proliferation and enhance the survival of heart muscle cells.
In several experiments with laboratory animals showed that G-CSF treatments after a heart attack significantly reduced mortality (Moazzami K, Roohi A, and Moazzimi B. Cochrane Database Systematic Reviews 2013; 5: CD008844. However, in a clinical trial known as the REVIVAL-2 trial, a double-blind, placebo-controlled study, G-CSG treatment failed to influence the performance of the heart six months after administration.
Now Birgit Steppich and others have published a seven-year follow-up of the subjects in the original REVIVAL-2 study to determine if G-CSF had long-term benefits that were not revealed in the short-term study. These results were published in the journal Thrombosis and Haemostasis (115.4/2016).
Of the initially enrolled 114 patients, 106 patients completed the seven-year follow-up. The results of this trial showed that G-CSF treatment for five days in successfully revascularized heart attack patients did not alter the incidence of death, recurrent heart attacks, stroke, or secondary adverse heart events during the seven-year follow-up.
These results are similar to those of the STEMMI trial, which treated patients with G-CSF for six days 10-65 hours after the reperfusion. In a five-year follow-up of 74 patients, there were no differences in the occurrence of major cardiovascular events between the G-CSF-treated group and the placebo group (Achili F, et al., Heart 2014; 100: 574-581).
Therefore, it appears that even though G-CSF worked in laboratory rodents that had suffered heart attacks, this treatment does not consistently benefit human heart attack patients. Although why it does not work will almost certainly require more insights than we presently possess.
The migration of several different types of stem cells is regulated by a receptor called “CXCR4” and the molecule that binds to this receptor, SDF-1. SDF-1 is a powerful summoner of white blood cells. During early development, SDF-1 mediates the migration of hematopoietic cells from fetal liver to bone marrow and plays a role in the formation of large blood vessels. During adult hood, SDF-1 plays an important role in making new blood vessels by recruiting endothelial progenitor cells (EPCs) from the bone marrow. Consequently, SDF-1 has a role in tumor metastasis where cancer cells that express the receptor CXCR4 are attracted to metastasis target tissues that release SDF-1. SDF-1 also attracts mesenchymal stem cells and helps them suppress the breakdown of bone.
Hopefully, I have convinced you that SDF-1 and its receptor CXC4 are important molecules. Can overexpression the CXCR4 receptor improve the retention of stem cells within an injured tissue?
Xiao-Tao Wu and Feng Wang from Zhongda Hospital in Nanjing, China and their colleagues have used this CXCR4 receptor/SDF-1 system to test this question in the damaged spinal cord. This work was published in the journal DNA and Cell Biology (doi:10.1089/dna.2015.3118).
Isolated MSCs were treated with genetically engineered viruses to so that would overexpress the CXCR4 receptor. In order to track these cells under medical imaging scans, the MSCs were also labeled with superparamagnetic iron oxide (SPIO). Next, rabbits that had suffered injuries to their intervertebral discs that lie between the vertebrae were given infusions of these labeled, genetically engineered MSCs. Images of the spine were taken at 0, 8, and 16 weeks after the surgery. The degeneration of the damaged intervertebral discs were also evaluated by disc height (damaged, degenerating intervertebral discs tend to shrink and lose height).
The SPIO-labeled CXCR4-MSC could be detected within the intervertebral discs by MRI 16 weeks post-transplantation. The MSCs that had been engineered to overexpress CXCR4 showed better retention within the discs, relative to implanted MSCs that had not been engineered to overexpress CXCR4.
Did the implanted MSCs affect the integrity of the intervertebral discs? Indeed they did. Compared to the control group, loss of disc height was slowed in the animals that received the CXCR4-overexpressing MSCs. Also, the genetically engineered MSCs seemed to make more cartilage-specific materials, like the giant molecule aggrecan and type II collagen. There is a caveat here, since there is no indication that measured protein directly; only mRNAs. Until the quantities of these molecules can be directly shown to increase in the disc, the increases in these cartilage-building molecules can be said to be presumptive, but not proven.
From these experiments, it seems reasonable to conclude that CXCR4 overexpression promoted MSC retention within the damaged intervertebral discs and the increased stem cell retention enhanced stem cell-based disc regeneration. Therefore this SDF-1/CXCR4 signaling pathway might be a way to drive stem cell migration and infiltration within degenerated intervertebral discs.
A growth factor called Glial cell line-derived neurotrophic factor (GDNF) has the remarkable ability to supports the growth and survival of dopamine-using neurons. Dopamine-using neurons are the cells that die off in Parkinson’s disease (PD). Providing GDNF to dopamine-using neurons can help them survive , but getting GDNF genes into the central nervous system relies on invasive intracerebral injections in order to pass through the blood-brain barrier.
Typically, genes are placed into the central nervous system by means of genetically engineered viruses. Viruses, however, are often recognized by the immune system and are destroyed before they can deliver their genetic payload. Therefore, non-viral gene delivery that can pass through the blood-brain barrier is an attractive alternative, since it is non-invasive. Unfortunately, such a high-yield technique is not yet available.
A new study by workers in the laboratories of Hao-Li Liu from Chang Gung University and Chih-Kuang Yeh from National Tsing Hua University, Taiwan has utilized a novel, non-viral gene delivery system to deliver genes into the central nervous system.
In this study, Lui and Yeh and their research teams used tiny bubbles made from positively-charged molecules to carry genes across the blood brain barrier. These bubbles formed stable complexes with GDNF genes, and when the skulls of laboratory animals were exposed to focused ultrasound, the bubble-gene complexes permeated the blood brain barrier and induced local GDNF expression.
In fact, this technique outperformed intracerebral injection in terms of targeted GDNF delivery. The amount of GDNF expressed in these laboratory animals that received the GDNF gene/microbubbles + ultrasound protocol was significantly higher than those animals that had genes directly injected into their brains. Furthermore, these higher levels of GDNF genes increased the levels of neuroprotection from PD. Animals that had a form of PD and had received nonviral GDNF gene therapy showed reduced disease progression and restored behavioral function.
This interesting study explores the potential of using ultrasound-induced passage through the blood brain barrier to bring genes into the central nervous system. This noninvasive technique successfully delivered genes into the brain to delay the effects of, and possibly treat, a neurodegenerative disease.
This study was published in Scientific Reports6, Article number: 19579 (2016), doi:10.1038/srep19579.
Retinal degenerations are the leading cause of blindness and fixing a defective retina is not an easy task.
Fortunately, a model system in nonhuman primates that has been used to test retinal replacement with stem cell-derived retinal cells has seen some success. In several experiment in small animals, retinal transplantations helped blind animals regain their sight. However, small laboratory rodents are not terribly good model systems for human eye problems.
To address the clinical relevancy of this transplantation system, Shirai and colleagues confirmed in rats and in macaques that transplantion of human embryonic stem cell (hESC)–derived retinas integrate into the already-existing retina and develop as fully mature retinal grafts.
In this paper, Shirai and others established the developmental stage at which embryonic stem cell-derived retinal cells could integrate into the retina and replace damaged cells. By transplanting cells into nude rats that do not have the ability to reject transplanted tissue, they refined their cell-based technique to heal damaged retinas. Then they took their refined technique into macques to treat two newly established monkey models of retinal degeneration.
In the first model system, Shirai et al. exposed one group monkeys to retina-damaging chemicals, and the other group had their retinas damaged by lasers. In both cases, the result was photoreceptor degeneration. Anywhere from 46 to 109 days after injury, the human embryonic stem cell-derived retinal sheets were implanted into the damaged retinas.
The retinal grafts integrated into the primate eyes and continued to differentiate into cone and rod cells, which are the two types of photoreceptor cells in the retina. Functional studies are still being conducted, but if vision can be improved, but these new macaque models confirm the clinical potential of stem cell–derived grafts for retinal blindness that results from photoreceptor degeneration.
See H. Shirai et al., Transplantation of human embryonic stem cell-derived retinal tissue in two primate models of retinal degeneration. Proc. Natl. Acad. Sci. U.S.A.113, E81–E90 (2015).
Jan Nolta and her colleagues at the Stem Cell Program and Institute for Regenerative Cures at UC Davis have published a remarkable paper in the journal Molecular Therapy regarding Huntington’s disease and a potential stem cell-based strategy to delay the ravages of this disease.
Huntington’s disease (HD) is an inherited neurodegenerative disease. It is inherited as an autosomal dominant disease, which means that someone need only inherit one copy of the disease-causing allele of the HTT gene to have this disease. HD is characterized by progressive cell death in the brain, particularly in a portion of the brain known as the striatum and by widespread brain atrophy.
The portion of the brain known as the striatum lies underneath the surface of the forebrain (subcortical) and it receives neural inputs from the cerebral cortex and is the primary source of neural inputs to the basal ganglia system. The basal ganglia system (BGS) is located underneath the surface of the brain but even deeper within the cerebral hemispheres. The BGS is part of the corpus striatum, it consists of the subthalamic nucleus and the substantia nigra. The BGS help with voluntary motor control, procedural learning relating to routine behaviors. otherwise known as “habits,” eye movements, and cognitive, and emotional functions. The ventral striatum is very important in addiction because it is the reward center on consists of the nucleus accumbens, olfactory tubercle, and islands of Calleja.
HD takes its largest toll on the striatum, which affects voluntary movement, routine behaviors, and personality. Disturbances of both involuntary and voluntary movements occur in individuals with HD. Chorea, an involuntary movement disorder consisting of nonrepetitive, non-periodic jerking of limbs, face, or trunk, is the major sign of the disease. Chorea is present in more than 90% of individuals, increasing during the first ten years. The choreic movements are continuously present during waking hours, cannot be suppressed voluntarily, and are worsened by stress. HD patients show impaired voluntary motor function early on and show a clumsiness in common daily activities.
With advancing disease duration, other involuntary movements such as slowness of movement (bradykinesia), rigidity, and involuntary muscle contractions that cause repetitive or twisting movements (dystonia) occur. Eye movement becomes progressively worse. So-called “gaze fixation” is observed in ~75% of symptomatic individuals. Unclear speech occurs early and Swallowing difficulties occur later.
Animal models of HD used in the past have injected molecules into the brain that kill off striatal cells and mimic at least some of the characteristics of HD in laboratory animals. Unfortunately, such a model system is fat too clean, since implanted cells tend to survive perfectly well. However the brains of HD patients are like unto a toxic waste dumps and implanted cells are quickly killed off. Therefore, a better animal model system was required, and it came in the form of R6/2 and YAC128 mice. R6/2 mice have a part of the human HTT gene that has 150 CAG triplets, and show the characteristic cell death in the striatum and behavioral deficits. The only problem with this mouse strain is that the neurodegenerative decline is very rapid rather than slow and progressive. YAC128 mice have a full-length copy of the HTT gene and show a slower, more progressive neurological decline that more closely approximates the human clinical condition.
In this paper from the Nolta laboratory, they used a growth factor that is known to decrease precipitously in HD brains; a growth factor called Brain-Derived Neurotrophic Factor (BDNF). BDNF is known to mediate the survival and function of striatal neurons and the reduction of BDNF in the brains of HD patients correlates with the onset of symptoms and the greater the reduction in BDNF, the greater the severity of the disease (see Her LS & Goldstein LS, J Neurosci 2008; 28, 13662-13676).
However injecting BDNF into the brain is problematic, since the protein has a very short half-life. Delivering the growth factor by means of genetically engineered viruses shows promise, but most of the viral vectors used in such experiments are recognized by the immune system as foreign invaders. Therefore, Nolta and her colleagues decided to genetically engineer mesenchymal stem cells (MSCs) to overexpress BDNF and implant these cells into the brains of R6/2 and YAC128 mice.
MSCs have an added advantage over viral vectors: these cells migrate to damaged areas where they can exert their healing properties (see Olson SD et al., Mol Neurobiol 45; 2012: 87-98).
Nolta and her coworkers actually tested human MSCs in HD model mice. After completing all the necessary control experiments to ensure that their isolated and engineered MSCs were secreting BDNF, Nolta and others implanted them into the brains of R6/2 and YAC128 mice.
HD mice show greater anxiety, which is manifested in a so-called “open field assay” by not remaining the center of the field. The control HD mice did not stay long in the center of the open field, but the normal mice did. The MSC-BDNF-implanted mice spend far more time in the center of the field. Mind you, not as much as wild-type mice, but significantly more than their HD counterparts.
Next the volume of the striata of these mice were determined and compared to the normal mice. While all the HD mice showed shrinking of the striatum, the MSC-NDNF-implanted YAC128 mice show significantly less shrinking of the striatum. Then the degree of neurogenesis (formation of new neurons) was measured in normal, HD, HD + implanted MSCs, and HS + MSC-BDNF mice. This experiment measures the degree of healing that is occurring in the brain. The brain from HD + MSC and HD + MSC-BDNF mice showed significantly more new brain cell growth. This is probably the reason for the delayed onset of symptoms and the delayed shrinking of the striatum.
Finally, Nolta and others measured the lifespans of the R6/2 mice and compared them with R6/2 mice that had been implanted with MSCs-BDNF. Animals transplanted with the MSCs that made the most BDNF lived 15% longer than the nontreated R6/2 mice.
MSCs have been shown in several experiments to promote neuronal growth, decrease cell death and decrease inflammation through the secretion of trophic factors. MSCs can modify the toxic environment that is part of the brain of an HD patient and help damaged tissue out by inducing neural regeneration and protection (see Crigler L, et al., Experimental neurology, 198; 2–6, 54-64; Kassis I, et al., Archives of Neurology 65; 2008: 753-761).
The downside of using MSCs that they will only survive in the brain for a few months. However, several studies have shown that the benefits of modified MSC implantation persist after the MSCs are gone, since the neural reconstruction wrought by the secreted BDNF stay after the MSCs have died off (see Arregui L, et al., Cell Mol Neurobiol 31; 2011: 1229-1243 and many others).
At best this treatment would delay the ravages of HD, but delaying this disease might very well be the first step towards a cure. Hopefully, clinical trials will not be fat behind.
New papers in Science magazine and the journal Cell have addressed a long-standing question of how the descendants of hematopoietic stem cells in bone marrow make the various types of blood cells that course through our blood vessels and occupy our lymph nodes and lymphatic vessels.
Hematopoietic stem cells (HSCs) are partly dormant cells that self-renew and produce so-called “multipotent progenitors” or MPPs that have reduced ability to self-renew, but can differentiate into different blood cell lineages.
The classical model of how they do this goes like this: the MPPs lose their multipotency in a step-wise fashion, producing first, common myeloid progenitors (CMPs) that can form all the red and white blood cells except lymphocytes, or common lymphoid progenitors (CLPs) that can form lymphocytes (see the figure below as a reference). Once these MPPs form CMPs, for example, the CMP then forms either an MEP that can form either platelets or red blood cells, or a GMP. which can form either granulocytes or macrophages. The possibilities of the types of cells the CMP can form in whittled down in a step-by-step manner, until there is only one choice left. With each differentiation step, the cell loses its capacity to divide, until it becomes terminally differentiated and becomes platelet-forming megakarocyte, red blood cell, neutrophil, macrophage, dendritic cells, and so on.
These papers challenge this model by arguing that the CMP does not exist. Let me say that again – the CMP, a cell that has been identified several times in mouse and human bone marrow isolates, does not exist. When CMPs were identified from mouse and human none marrow extracts, they were isolated by means of flow cytometry, which is a very powerful technique, but relies on the assumption that the cell type you want to isolate is represented by the cell surface protein you have chosen to use for its isolation. Once the presumptive CMP was isolated, it could recapitulate the myeloid lineage when implanted into the bone marrow of laboratory animals and it could also produce all the myeloid cells in cell culture. Sounds convincing doesn’t it?
In a paper in Science magazine, Faiyaz Notta and colleagues from the University of Toronto beg to differ. By using a battery of antibodies to particular cell surface molecules, Notta and others identified 11 different cell types from umbilical cord blood, bone marrow, and human fetal liver that isolates that would have traditionally been called the CMP. It turns out that the original CMP isolate was a highly heterogeneous mixture of different cell types that were all descended from the HSC, but had different developmental potencies.
Notta and others used single-cell culture assays to determine what kinds of cells these different cell types would make. Almost 3000 single-cell cultures later, it was clear that the majority of the cultured cells were unipotent (could differentiate into only one cell type) rather than multipotent. In fact, the cell that makes platelets, the megakarocyte, seems to derive directly from the MPP, which jives with the identification of megakarocyte progenitors within the HSC compartment of bone marrow that make platelets “speedy quick” in response to stress (see R. Yamamoto et al., Cell 154, 1112 (2013); S. Haas, Cell Stem Cell 17, 422 (2015)).
Another paper in the journal Cellby Paul and others from the Weizmann Institute of Science, Rehovot, Israel examined over 2700 mouse CMPs and subjected these cells to gene expression analyses (so-called single-cell transriptome analysis). If the CMP is truly multipotent, then you would expect it to express genes associated with lots of different lineages, but that is not what Paul and others found. Instead, their examination of 3461 genes revealed 19 different progenitor subpopulations, and each of these was primed toward one of the seven myeloid cell fates. Once again, the presumptive CMPs looked very unipotent at the level of gene expression.
One particular subpopulation of cells had all the trappings of becoming a red blood cell and there was no indication that these cells expressed any of the megakarocyte-specific genes you would expect to find if MEPS truly existed. Once again, it looks as though unipotency is the main rule once the MPP commits to a particular cell lineage.
Thus, it looks as though either the CMP is a very short-lived state or that it does not exist in mouse and human bone marrow. Paul and others did show that cells that could differentiate into more than one cell type can appear when regulation is perturbed, which suggests that under pathological conditions, this system has a degree of plasticity that allows the body to compensate for losses of particular cell lineages.
Fetal HSCs, however, are a bird of a different feather, since they divide quickly and reside in fetal liver. Also, these HSCs seem to produce CMPs, which is more in line with the classical model. Does the environmental difference or fetal liver and bone marrow make the difference? In adult bone marrow, some HSCs nestle next to blood vessels where they encounter cells that hang around blood vessels known as “pericytes.” These pericytes sport a host of cell surface molecules that affect the proliferative status of HSCs (e.g., nestin, NG2). What about fetal liver? That’s not so clear – until now.
In the same issue of Science magazine, Khan and others from the Albert Einstein College of Medicine in the Bronx, New York, report that fetal liver also has pericytes that express the same cell surface molecules as the ones in bone marrow, and the removal of these cells reduces the numbers of and proliferative status of fetal liver HSCs.
Now we have a conundrum, because the same cells in bone marrow do not drive HSC proliferation, but instead drive HSC quiescence. What gives? Khan and others showed that the fetal liver pericytes are part of an expanding and constantly remodeling blood system in the liver and this growing, dynamic environment fosters a proliferative behavior in the fetal HSCs.
When umbilical inlet is closed at birth, the liver pericytes stop expressing Nestin and NG2, which drives the HSCs from the fetal liver to the other place were such molecules are found in abundance – the bone marrow.
These models give us a better view of the inner workings of HSC differentiation. Since HSC transplantation is one of the mainstays of leukemia and lymphoma treatment, understanding HSC biology more perfectly will certainly yield clinical pay dirt in the future.
Mesoblast Limited announced that the number of subjects treated in their ongoing Phase 3 clinical trial in chronic heart failure (CHF) that is testing their proprietary cell-based medicine MPC-150-IM will be substantially reduced.
CHF is characterized by an enlarged heart, coupled with insufficient blood supply to the organs and extremities of the body. Unfortunately, this is a progressing condition that tends to get worse with time. CHF is caused by many different factors such as chronic high blood pressure, faulty heart valves, infections, or congenital heart problems.
Mesoblast centers their company around the isolation and expansion of so-called mesenchymal precursor cells (MPCs) from bone marrow. Mesenchymal stem cells are found in many different tissues and organs throughout our bodies. They play vital roles in maintaining tissue health. However, relatively speaking, mesenchymal stem cells are rare cells. They are found around blood vessels and respond to signals associated with tissue damage. They secrete mediators and growth factors that promote tissue repair and control the immune response to prevent it from going out of control.
Mesoblast uses an array of monoclonal antibodies to isolate primitive mesenchymal stem cells that are actually precursors to mesenchymal stem cells or mesenchymal precursor cells (MPCs). These cells are then expanded in culture without being differentiated into any other cell type.
Initially, Mesoblast planned to test their product on 1,165 subjects, but have scaled that number back to approximately 600 patients.
Mesoblast’s development and commercial partner, Teva Pharmacueticals has communicated this reduction in the number of subjects to the US Food and Drug Administration (USFDA). “The reduction in the size of the Phase 3 trial may significantly shorten the time to trial completion,” said Mesoblast CEO Silviu Itescu.
The reduction in the number of patients was due to a proposed change in the primary endpoint of the trial. The revised primary endpoint is now a comparison of recurrent heart failure-related major adverse cardiovascular events (HF-MACE) between patients treated with Mesoblast’s MPC-150-IM cells and the control patients who were not treated with these cells.
Why the change in the primary endpoint? The reason lies in the success that MPC-150-IM cells had their Phase 2 clinical trial. In this trial, a single injection of MPC-150-IM cells successfully prevented HF-MACE over three years. This second, confirmatory study will be conducted in parallel with a patient population that has an identical clinical profile; approximately 600 of them using the same primary endpoint.
In the completed Phase 2 trial, patients treated with MPC-150-IM had no HF-MACE over 36 months of follow-up, compared with 11 HF-MACE in the control group. From this same clinical trial, of those patients who suffered from advanced heart failure (defined by baseline Left Ventricular Systolic Volume being greater than 100 milliliters), 71 percent of the controls (who received no cells) had at least on HF-MACE versus none of those who received a single injection of MPC-150-IM cells. As it turns out, this Phase 2 patient population closely resemble the patients being recruited in the Phase 3 trial.
“Patients with advanced heart failure continue to represent among the largest unmet medical needs, where existing therapies are inadequate and the economic burden is the greatest. The current Phase 3 trial targets this patient population, continues to recruit well across North America, and is now expanding to Europe,” said Itescu.
Amnion-derived stem cells show a dizzying set of regenerative properties, at least in the laboratory (see Miki T, Stem Cell Res Ther 2011, 2:25; Miki T, Grubbs B, J Obstet Gynaecol Res 2014, 40:360-8). Bringing these cells into the clinic will definitely take a lot more work, but a new paper examines a culture system that does not use any animal products. Such culture systems (known as “xeno-free) are vitally important if stem cells are going to be used in human trials.
When stem cells are grown in culture, typically serum from animal blood is used to provide the cells with the growth factors and things they need to kick the cells into the growth phase. However, occasionally, some animal-based products have animal viruses that can infect human cells with unpredictable consequences (see Karlsson JOM, Toner M, Biomaterials 1996, 17:243-56). Also, cells grown in animal-based culture media can have animal proteins around them that are very hard to get rid of. Implanting such cells into a human patient would cause their immune system to react against the animal proteins. Such immune responses might result in something as innocuous as itching at the site of injection or as dangerous as anaphylactic shock. Therefore, growing stem cells under xeno-free conditions is important for many stem cell applications. Expanding and preserving stem cells under so-called Good Manufacturing Pracctices (GMP) is essential for their use in human patients.
However, now we have just opened another can of worms. If all the characterizations of stem cells have been under culture conditions that utilize animal-based culture media, will the cells have the same capabilities if grown under xeno-free conditions?
Herein is the reason why this paper by Toshio Miki from the Keck School of Medicine at the University of Southern California and his colleagues is so important. In this paper, which was published in the journal Stem Cell Research and Therapy (Stem Cell Research & Therapy 2016, 7:8 doi:10.1186/s13287-015-0258-z), Miki and others examined the characteristics of amniotic stem cells grown in xeno-free media (see Mitry RR, Lehec SC, Hughes RD, Methods Mol Biol 2010, 640:107-13; Polchow B, et al. J Transl Med 2012, 10:98; Stacy GN, Masters JR, Nat Protoc 2008, 3:1981-89; Miki T, et al. Curr Protoc Stem Cell Biol 2010, Chapter 1:Unit 1E.3) and compared them to stem cells grown in standard culture media. This is a very important exercise, since amniotic stem cells are easily accessible as source of material for regenerative treatments that can be banked and immunotype matched for clinical applications.
Miki and others isolated human amniotic epithelial cells from newborn babies with the consent of their parents and then stored the cells at −160 °C in one of five commercially available culture media. Miki and his group used cells frozen in standard media containing fetal bovine serum as controls to which all the other cells were compared. Then they thawed the cells, and tested their viability, mitochondrial integrity, and senescence status (tendency to fall asleep in culture and stop growing). They also examined gene expression profiles in these cells by using quantitative real-time PCR. Flow cytometry was used to identify the stem cell surface markers.
The results were encouraging and interesting. There were no significant differences in viability and growth in cells grown and preserved in xeno-free media versus standard cryopreservation medium. Additionally, comparisons of the cells grown in the different cryopreservation media did not reveal significant differences in the senescence status, mitochondria, or overall morphology of the cells. The upshot is that the cells preserved in standard or xeno-free media looked and grew the same after being thawed. There were some differences in the expression of stem cell marker genes (e.g., OCT4, SOX2, and NANOG) and a particular cell surface marker (TRA1-60) following cryopreservation in different xeno-free media. However, it turns out these differences were slight and, overall, not statistically significant. Again, the upshot is that there were small differences in gene expression, but these differences did not amount to a hill of beans.
Miki and his colleagues have nicely shown that cryopreserving amnion-derived stem cells in xeno-free media is feasible and does not affect the characteristics of the cells. This paper also suggests that such xeno-free media can be used to establish a bio-bank of human amnion-derived stem cells for future clinical application.
Well that’s one hurdle vaulted. Now Miki and others need to figure out which of the xeno-free media is the best and optimize that medium to for improved preservation of stem cell-like characteristics in these cells.
Within the tiny alveolar sacs of our lungs is an immune cell that surveys and directs the immune response within the lung. This immune cell is called an “alveolar macrophage,” and this cell is an actively phagocytic cell. It gobbles up invading bacteria and foreign material in order to keep the lungs clean. When these cells work normally, they help our lungs function properly. When, however, they go rogue, they can fill the lungs with cells that clog the lungs and prevent you from breathing.
Certain diseases like chronic obstructive pulmonary disease, asthma, and lung fibrosis, have abnormal alveolar macrophages and no specific treatments can appropriately compensate for these abnormalities.
Since alveolar macrophages (AMs) can be made from pluripotent stem cells, perhaps transplanting exogenous AMs derived from pluripotent stem cells can clean up messy lungs.
Martin Post, from the University of Toronto, in Ontario, Canada, and his colleagues tested this very hypothesis in mice. Post and his coworkers differentiated mouse embryonic stem cells by using factor-defined media in order to generate embryonic macrophages that could be grown in culture. Then they conditioned their cells into an alveolar-like phenotype by treating them with the cytokine GM-CSF. The cells were surprisingly like normal AMs, at least in culture.
To test these cells in mice, Post and his group created mice that lacked the ADA (adenine deaminase) gene and these mice lacked proper AM activity and suffered chronic lung damage.
Next, Post’s team transplanted their embryonic stem cell-derived alveolar-like macrophages into the tracheas (windpipes) of these injured animals in order to view their therapeutic potential.
What Post and others saw truly amazed them. Not only was their differentiation protocol wonderfully efficient and adaptable to human pluripotent stem cells, but their PSC-derived macrophages essentially “walked and talked” like regular, normal AMs. These cells made all the right cell surface proteins to be identified as AMs and they engulfed bacteria and dying cells. In fact, they were better phagocytes than bone marrow-derived macrophages.
The implanted macrophages stayed in the airways of the recipient mice for at least 4 weeks, and were able to gobble up other types of rogue white blood cells (i.e., neutrophils) during acute lung injury. Thus, the implanted cells were able to protect the lung from further damage under conditions of lung injury. Additionally, the implanted AMs enhanced tissue repair in the lungs and promoted survival of these mice. Interestingly, the mice did not develop abnormal pathology or teratomas as a result of the implanted macrophages.
Thus, this work from Post and his colleagues shows that pluripotent stem cells are a viable source of therapeutically effective alveolar-like macrophages that can be implanted into the lungs and treat airway diseases. Further experiments in larger animals should prepare this strategy for clinical trials.
This study was published in the American Journal of Respiratory and Critical Care Medicine. published online 05 Jan 2016 as DOI: 10.1164/rccm.201509-1838OC.
The biotechnology company BrainStorm Cell Therapeutics Inc. has developed an autologous stem cell therapy for several neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS, also known as Lou Gehrig’s disease), Multiple Sclerosis (MS) and Parkinson’s Disease (PD). BrainStorm has designed a proprietary product called NurOwn™ that is made from the patient’s own bone marrow mesenchymal stem cells (BM-MSCs). Essentially, the patient’s BM-MSCs are isolated, purified, and cultured in a specialized culture system that drives the BM-MSCs to differentiate into nerve-like cells that Neurotrophic Factors (NTF). These NTFs have the capacity to keep nerve cells alive and prevent moribund cells from dying.
By transplanting NurOwn cells back into the patient at or near the site of neural damage, in the spine and/or muscles, it could potentially delay or even roll back damage from neurodegeneration. NurOwn cells have proven their efficacy in animal experiments (e.g., STEM CELLS 2008;26:2542–2551), and in a few small clinical trials.
In one case, a 75-year-old man who suffered from ALS and myasthenia gravis (the immune system attacks your own receptors for acetylcholine at the neuromuscular junction, which prevents the muscle contraction), was treated with NurOwn cells, and experienced the following improvement 1 month later.
This is only a case study and involves only one patient, which is the absolute lowest-quality evidence you can have in medicine. Therefore, this study is suggestive that NurOwn cells can help ALS patients improve.
Now BrainStorm Cell Therapeutics has entered into a collaborative agreement with Hadassah Medical Center in Jerusalem, Israel, to conduct a Phase 2 clinical trial to test the ability of NurOwn cells to treatment patients with Amyotrophic Lateral Sclerosis (ALS).
This clinical trial is not BrainStorm’s first rodeo, since they have conducted two other clinical trials in collaboration with Hadassah Medical Center. BrainStorm hopes that the results of this clinical trial will provide guidance in preparing a Phase 3 clinical trial that will test their NurOwn® stem cell based therapy in patients suffering from ALS.
In this trial, BrainStorm plans to enroll up to 24 ALS patients, all of whom will receive three consecutive stem cell transplantations of their own BM-MSCs that have been genetically engineered to secrete NFTs. The goal of this trial is to establish the safety and efficacy of a treatment regimen that includes multiple doses of stem cells. Because this trial includes human subjects, it must be approved by Hadassah’s Helsinki Committee and the Israeli Ministry of Health before the study can commence.
Professor Dimitrios Karussis, MD, PhD, Head of the Unit of Neuroimmunology and Cell Therapies at Hadassah’s Department of Neurology, who served as Principal Investigator in Brainstorm’s prior ALS studies, will serve as the Principal Investigator for this trial.
“NurOwn has generated promising clinical data in ALS and has the potential to offer a new approach for the treatment of patients afflicted with this disease,” stated Professor Karussis. “We are excited to be collaborating with BrainStorm to advance this product to the next phase of development and the application of stem cell therapies in similar neurological diseases in general.”
“Evaluating multiple doses with NurOwn is an important next step in our efforts to understand the treatment effect of this investigative medicine,” stated Chaim Lebovits, CEO of Brainstorm. “We are pleased to continue our partnership with Hadassah Medical Center, which has long maintained a reputation for excellence in the treatment of neurological disorders.”
Loïc Reppel and his colleagues at CNRS-Université de Lorraine in France have found that mesenchymal stem cells from human umbilical cord can not only be induced to make cartilage, but that these remarkable cells can make cartilage without the use of exogenous growth factors.
Mesenchymal stromal/stem cells from bone marrow (BM-MSC) have, for some time, been the “all stars” for cartilage regeneration. In fact, a very innovative clinic near Denver, CO has pioneered the use of BM-MSCs for patients with cartilage injuries. Chris Centeno, the mover and shaker, of this clinic has carefully documented the restoration of articular cartilage in many patients in peer-reviewed articles.
However, there is another “kid’ on the cartilage-regeneration block; mesenchymal stromal/stem cells from Wharton’s jelly (WJ-MSC). The advantages of these cells are their low immunogenicity and large cartilage-making potential. In this paper, which was published in Stem Cell Research and Therapy, Reppel and others evaluated the ability of WJ-MSCs to make cartilage in three-dimensional culture systems.
Reppell and his coworkers embedded WJ-MSCs isolated from the umbilical cords of new-born babies in alginate/hyaluronic acid hydrogel and grew them for over 28 days. These hydrogels were constructed by the spraying method. The hydrogel solution (for those who are interested, it was 1.5 % (m/v) alginate and hyaluronic acid (ratio 4:1) dissolved in 0.9 % NaCl) was sprayed an airbrush connected to a compressor. The solution was seeded with WJ-MSCs and then sprayed on a sterile glass plate. The hydrogel was made solid (gelation) in a CaCl 2 bath (102 mM for 10 minutes). Then small cylinders were cut (5 mm diameter and 2 mm thickness) with a biopsy punch. Then Reppel and others compared the chondrogenic differentiation of WJ-MSC in these three-dimensional scaffolds, without adding growth factors with BM-MSC.
After 3 days in culture, WJ-MSCs seemed nicely adapted to their new three-dimensional culture system without any detectable damage. From day 14 – 28, the proportion of WJ-MSC cells that expressed all kinds of cell surface proteins characteristic of MSCs (i.e., CD73, CD90, CD105, and CD166) decreased significantly. This suggests that these cells were differentiating into some other cell type.
After 28 days in this scaffold culture, both WJ-MSCs and BM-MSCs showed strong upregulation of cartilage-specific genes. However, WJ-MSCs exhibited greater type II collagen synthesis than BM-MSCs, and these differences were evident at the RNA and protein levels. Collagen II is a very important molecule when it comes to cartilage synthesis because chondrogenesis, otherwise known as cartilage production, occurs when MSCs differentiate into cartilage-making cells known as chondroblasts that begins secreting aggrecan and collagen type II that form the extracellular matrix that forms cartilage. Unfortunately, in order to complete the run to mature cartilage formation, the chrondrocytes must enlarge (hypertrophy), and express the transcription factor Runx2 and secrete collagen X. Unfortunately, WJ-MSCs expressed Runx2 and type X collagen at lower levels than BM-MSCs in this culture system.
These experiments only examined cells in culture, which is not the same as placing cells in a living animal, but it is a start. Thus, when they are seeded in the hydrogel scaffold, WJ-MSCs and BM-MSCs, after 4 weeks, were able to adapt to their environment and express specific cartilage-related genes and matrix proteins in the absence of growth factors. In order to properly make cartilage in clinical applications, WJ-MSCs must go the full way and express high levels of Runx2 and collagen X. However, these experiments show that WJ-MSCs, which in the past were medical waste, are a potential alternative source of stem cells for cartilage tissue engineering.
Reppel and his colleagues note in their paper that to improve cartilage production from WJ-MSCs, it might be important to mimic the physiological environment in which chondrocytes normally find themselves. For example, they could apply mechanical stress or even a low-oxygen culture system. Additionally, Reppel and others could apply stratified cartilage tissue engineering. Reppel thinks that they could adapt their spraying method to design new stratified engineered tissues by applying progressive cells and spraying hydrogel layers one at a time.
All in all, cartilage repair based with WJ-MSC embedded in Alginate/Hyaluronic Acid hydrogel will hopefully be tested in laboratory animals and then, perhaps, if all goes well, in clinical trials.
The thyroid gland sits over the main cartilage of the larynx and produces thyroid hormone (thyroxine); a hormone that regulates the basal metabolic rate. When the thyroid slows down and fails to make sufficient amounts of thyroid hormone, the result is a condition called hypothyroidism. The symptoms of hypothyroidism are fatigue, weakness, weight gain or increased difficulty losing weight, coarse, dry hair, dry, rough pale skin, hair loss, cold intolerance, muscle cramps and frequent muscle aches, constipation, depression, irritability, memory loss, abnormal menstrual cycles and decreased libido.
If someone has any evidence of a thyroid tumor, then the thyroid is removed, and the patient must take oral thyroid hormone. Because getting the dose right can be difficult, we might ask, “Can we replace the thyroid with stem cell treatments?”
Human pluripotent stem cells can differentiate into balls of cells that are mini-organs called “organoids.” Unfortunately, if left to themselves, the formation of these organoids is rather haphazard and the cells tend to differentiate into a whole host of different cell types. This is not fatal, however, since the differentiation of these stem cells can be orchestrated by using growth factors or certain culture conditions. Can we use such innovations to make a minithyroid?
In order to make thyroid cells from embryonic stem cells (ESCs), Kotton and his group had to make endodermal progenitors from them first. Fortunately, a study from Kotton’s own laboratory that was published in 2012 employed a technique used in several other papers that grew ESCs in a serum-free medium with a growth factor called activin. Christodoulou, C., and others (J. Clin. Invest. 121, 2313–2325) showed that over 80 percent of the ESCs grown under these conditions differentiated into endodermal progenitors. When Kotton and his colleagues cultured these endodermal progenitors in BMP4 and FGF2, some of them differentiated into thyroid progenitor cells. Interestingly, this mechanism by which thyroid-specific cell fates are specified is conserved in creatures as disparate as frogs and mice.
To make mature thyroid cells from these progenitors, a three-dimensional culture system was used in combination with thyroid stimulation hormone and dexamethasone. Under these conditions, the cells formed spherical thyroid follicles that secreted thyroid hormone. To perfect their protocols, Kotton’s group used mouse ESCs, but they additionally showed that this same strategy can make mature thyroid cells from human induced pluripotent stem cells (iPSCs).
The appearance of cells in a culture system that look like mature thyroid follicles and express many of the same iodine-metabolizing enzymes as mature thyroid cells is exciting, but can such cells stand in for thyroid tissue in an animal that lacks sufficient thyroid tissue?
Kotton’s laboratory took this to the next step by transplanting their cultured thyroid follicles into laboratory mice that lacked a functional thyroid. These transplants were not inserted into the neck of the animal, but instead were place underneath the kidney, which is area rich in blood vessels. Interestingly, the implanted thyroid “organoids” or little organs did not fall apart upon transplantation. Instead they retained their characteristic structure. More interestingly, these organoids kept expressing iodine-metabolizing enzymes and made thyroid hormone. The synthesis and release of thyroid hormone was also regulated by the hypothalamic hormone thyroid stimulating hormone (TSH). TSH is made and released in response to insufficient thyroid hormone levels. The thyroid responds to TSH by making a releasing more thyroid hormone, which causes a feed-back inhibition of the release of TSH. The fact that these implanted organoids were properly regulated by TSH bespeaks of the maturity of these cells. Also, significantly, none of the laboratory animals showed any signs that the implanted cells had formed any tumors.
Kotton and his coworkers were also able to used human ESCs and human iPSCs to make thyroid organoids. Human iPSCs-derived thyroid organoids were made from human patients with normal thyroid function and from hypothyroid children who carry a loss-of-function mutation in the NKX2-1 gene. This show that Kotton’s system can be used as a model to study inherited thyroid deficiencies. However, there is even more excitement that this system or something similar to it might be useful to safely treat thyroid loss in patients who have lost their thyroid as a result of cancer, or injury.
A new study that was published in the journal Cancer Cell, has introduced a way to treat colon, which is not only a leading cause of cancer deaths worldwide, but is notoriously resistant to treatment.
This collaboration between Swiss and Japanese scientists has identified an anticancer mechanism that includes vitamin A that can be tapped to inhibit colon cancer.
Colon cancer patients are typically treated with chemotherapy, which kills off most of the cancer cells, but leaves a few resistant cells that then aggressively grow back to form another deadlier tumor that can readily spread throughout the body. Chemotherapy ultimately fails because there are a core of rouge stem cells that divide uncontrollably called cancer stem cells that drives the growth of the cancer. These cancer stem cells are what is driving the growth of the tumor and if these cancer stem cells are not eliminated, the tumor will simply come back after chemotherapy. When a colon cancer patient receives treatment such as chemotherapy, most of the cancer cells die off.
Joerg Huelsken, Ph.D., of Ecole Polytechnique Federale de Lausanne (EPFL) led his research team to understand how stem cells populations in the colon give rise to new colon cells to replace dead, dying or cells that have been sloughed off. In mice and in tissue samples from human patients, a protein called HOXA5 kept asserting itself. HOXA5 turns out to be an integral part of the machinery of the cell that ensures that the cells of the colon properly differentiate after they are born from colon stem cells.
Huelsken’s team showed that in the colon, HOXA5 helps restrict the number of stem cells. Cancerous stem cells, however, block the expression of HOXA5 and prevent it from restricting stem cell numbers. HOXA5 is part of a signaling pathway that activates aspects of the cellular machinery that negatively controls cell growth. By blocking expression of the HOXA5 gene, these cancerous stem cells in the colon can grow uncontrollably and spread, thereby causing relapses and metastasis or spread of the colon cancer.
Huelsken and his team, in collaboration with Japanese researchers from Kyoto University investigated ways to unblock the expression of HOXA5 in colon cancer stem cells. The answer came from an unexpected corner – vitamin A. Vitamin A is a member of the retinoid family of molecules and has been known for some time to be able to induce differentiation of skin-based stem cells. Huelsken’s group showed that retinoids like vitamin A can upregulate HOXA5 and antagonize the mechanism in colon cancer stem cells that staunch its expression.
In a mouse colon cancer model system, treatment with retinoids not only blocked progression of the tumors, but normalized the tissue. The activation of the expression of the HOXA5 gene eliminated cancer stem cells and prevented metastasis in live animals. These results were then faithfully recapitulated in samples from actual patients.
From this study, it seems that screening tumors for the absence of HOXA5 expression is a relatively easy way to determine if a patient’s colon cancer will respond to treatment with vitamin A. Treatment with vitamin A or other retinoids might not only prove effective against colon cancer, but also as a preventive measure in high-risk patients.