Adjustable Gels Used to Determine Those Molecules That Drive Stem Cell Differentiation

Scientists have been very interested in the details of stem cell differentiation. To that end, several laboratories have designed hydrogels that mimic the stiffness of biological tissue in order to grow stem cells and study their differentiation.

In one enterprising laboratory, led by Rein Ulijn of the City University of New York and the University of Strathclyde, scientists have used a novel culture-based gel system to study mesenchymal stem cell differentiation and identify those metabolites used by stem cells when they select bone and cartilage cell fates. When these molecules are provided to standard stem cell cultures, these molecules can guide stem cells to generate desired cell types. This new study illustrates how new biomaterials can provide an exacting model system that can help scientists precisely determine those identifying factors that drive stem cell differentiation.

Stem-cell scientists have known that the rigidity of a hydrogel surface can instruct stem cells to differentiate. A rigid surface, as it turns out, can result in bone cell formation, whereas soft surfaces induce the differentiation of cells into neuron-like cells. With this information, Ulijn and others developed a protocol that generates gels by combining small building-block molecules that spontaneously form a network of nanosized fibers. Furthermore, by varying the concentration of these building blocks, the stiffness of these gels can be adjusted. By mimicking the stiffness of bone (40 kilopascal) or cartilage (15 kilopascal), the gel stimulates stem cells applied to its surface to differentiate accordingly.

“This paper is a great example of how chemistry can help make step changes in biology,” said Matthew Dalby of the University of Glasgow and co-senior author. “As a biologist, I needed simple yet tunable cell-culture gels that would give me a defined system to study metabolites in the laboratory. Rein had developed the chemistry to allow this to happen.”

The available gels for growing stem cells are typically derived from animal products. Unfortunately, this can affect the reproducibility of results, since different preparations of particular animal products can have rather different properties. Synthetic components usually require coatings or coupling of cell-adhesive ligands. However, the gel developed by Ulijn’s group is composed of two simple synthetic peptide derivatives. One component binds to copies of itself with high directional preference, which results in the spontaneous formation of nanoscale fibers when the molecules are dissolved in water. The second components consists of a surfactant-like molecule that binds to the fiber surface and presents simple, cell-compatible chemical groups to any cells.


The components are held together by relatively weak and reversible interactions, e.g., hydrogen bonding and aromatic stacking. Interestingly, variants of these gels are commercially available through a spinoff company called Biogelx, Ltd., where Ulijn serves as chief scientific officer.

“We wanted a platform that provides nanofiber morphology and as-simple-as-possible chemistry and tunable stiffness to serve as a blank-slate background so that we could focus on changes in stem cell metabolism,” said Ulijn. “Matt and his team performed metabolomics analysis to find out how the key metabolites within a stem cell are used up during the differentiation process.”

Particular transcription factors are often the ingredients scientists use to induce stem cell fate in the case of induced pluripotent stem cells. However, Dalby and Ulijn think that certain metabolites might drive those pathways that cause the different intracellular concentrations of transcription factors that drive the various differentiation pathways.

One metabolite featured in the study is cholesterol sulfate. Cholesterol sulfate is used up during osteogenesis on a rigid matrix and can also be used to convert stem cells into bone-like cells in cell culture.

In their paper, Ulijn and his coworkers showed how small molecules, like cholesterol sulfate, can put into motion those cell-signaling pathways that culminate in the activation of the transcription factors that drive the transcription of major bone-related genes. The expression of these bone-specific genes drives bone formation, and this demonstrates a connection between the metabolites and the activation of transcription factors.

It must be noted that this gel does not precisely recapitulate the microenvironment inside the body. Therefore, it is unclear if the stem cells grown on it behave differently on the designed gel surfaces than they would in the body.

Although the full list of metabolites derived from the analysis is preliminary, “it could certainly point researchers in the right direction,” Ulijn said. “Our ambition is to simplify drug discovery by using the cell’s own metabolites as drug candidates,” Dalby said.

This paper was published here: Alakpa et al., “Tunable Supramolecular Hydrogels for Selection of Lineage Guiding Metabolites in Stem Cell Cultures,” Chem, 2016 DOI:10.1016/j.chempr.2016.07.001.


Hematopoietic Stem Cells Use a Simple Heirarchy

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 Cell by 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.

A model of the changes in human My-Er-Mk differentiation that occur across developmental time points. Graphical depiction of My-Er-Mk cell differentiation that encompasses the predominant lineage potential of progenitor subsets; the standard model is shown for comparison. The redefined model proposes a developmental shift in the progenitor cell architecture from the fetus, where many stem and progenitor cell types are multipotent, to the adult, where the stem cell compartment is multipotent but the progenitors are unipotent. The grayed planes represent theoretical tiers of differentiation.
A model of the changes in human My-Er-Mk differentiation that occur across developmental time points.
Graphical depiction of My-Er-Mk cell differentiation that encompasses the predominant lineage potential of progenitor subsets; the standard model is shown for comparison. The redefined model proposes a developmental shift in the progenitor cell architecture from the fetus, where many stem and progenitor cell types are multipotent, to the adult, where the stem cell compartment is multipotent but the progenitors are unipotent. The grayed planes represent theoretical tiers of differentiation.

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.


Fat-Derived Stem Cells Form Muscle in Muscular Dystrophy Mice

Stem cell therapy for Duchenne muscular dystrophy (DMD) has been plagued by poor cell engraftment into diseased muscles. Additionally, there are no reports to date describing the efficient generation of muscle progenitors from fat-derived stem cells (ADSCs) that can contribute to muscle regeneration.

A study by Cheng Zhang and others from Sun Yat-sen University in Guangzhou, China, Guangdong Province has examined the ability of progenitor cells differentiated from ADSCs using forskolin, basic fibroblast growth factor, the glycogen synthase kinase 3β inhibitor 6-bromoindirubin-3′-oxime as well as the supernatant of ADSC cultures to form workable muscle cells.

When these fat-derived stem cells were treated as described above, they formed a proliferative population of muscle progenitors from ADSCs that had characteristics similar to muscle satellite cells. Furthermore, in culture, these cells were capable of terminal differentiation into multinucleated myotubes.

When these fat-derived stem cells were transplanted into mice that had an inherited type of DMD, the progenitor cells successfully engrafted in skeletal muscle for up to 12 weeks, and generated new muscle fibers, restored dystrophin expression, and contributed to the satellite cell compartment.

These findings highlight the potential application of ADSCs for the treatment of muscular dystrophy. They also illustrate the ability of ADSCs to differentiate into functional skeletal muscle cells when treated properly in culture. These same cells might serve as a treatment for DMD patients.

This article was published in Hum. Mol. Genet. (2015) doi: 10.1093/hmg/ddv316.

Cartilage-Making Stem Cells from Joints

Chiharo Akazawa from the Tokyo Medical and Dental University and his colleagues have tested two types of mesenchymal stem cells from human patients for their ability to make bone, cartilage, or fat. Their tests illustrated what has been shown several time before; mesenchymal stem cells tend to differentiate into the tissues that most closely resemble their tissue of origin.

Akazawa and his colleagues previously discovered a way to effectively isolated mesenchymal stem cells from bone marrow, which is no small feat because mesenchymal stem cells (MSCs) are a minority of the cells in bone marrow (Mabuchi and others (2013), Stem Cell Reports 1: 152-165). In a recent paper in the journal PLoS ONE, Akazawa and others used this technique to isolate MSCs from bone marrow and from synovial membrane – the fluid-filled sac that encases joints. In large joints, this synovium is large and called a “bursa.”.

In culture, the bone marrow-derived MSCs from several different human donors showed a marked tendency to form bone, but they did not make good cartilage or fat. The synovial MSCs, on the other hand, did not do so well at making bone, but made very good fat and cartilage. These differentiation trends were observed in MSCs culture for several different human donors. All cells were collected during arthroscopic surgery.

Since the synovial membrane of patients suffering from osteoarthritis undergoes, increased cell division, it is possible that the number of stem cells also increases. Alternatively, using MSCs from healthy donors who do not have arthritis may be even more preferable. Nevertheless, MSCs from synovial membrane show excellent cartilage-making potential and they may be a suitable source of cell for cartilage regeneration.

Toxic Gas Prompts Mesenchymal Stem Cells to Become Bone Cells

Hydrogen sulfide smells like rotten eggs and is toxic to human life at moderate concentrations. Therefore, imagine the surprise of researchers when they discovered that low concentrations of this poisonous gas actually stimulate mesenchymal stem cells from bone marrow to differentiate into bone-making cells.

In a paper published in the journal Cell Stem Cell, Yi Liu from the Ostrow School of Dentistry at the University of Southern California and colleagues have discovered that hydrogen sulfide (H2S), acts as a “gaseous signaling molecule” that mesenchymal stem cells actually produce at sub-lethal concentrations.

H2S acts as a “gasotransmitter” that regulates multiple signaling pathways. To determine the extent of these pathways, Liu and his colleagues made mice that were unable to synthesize any H2S. The H2S-deficient mice showed distinct abnormalities in bone marrow mesenchymal stem cells. Namely, mesenchymal stem cells (MSCs) from H2S-deficient mice were unable to properly self=-renew or differentiate into bone-making cells (osteoblasts).

When Liu and others dug a little deeper, they found that H2S deficiency results in aberrant influx of intracellular Ca2+. Problems with calcium handling arose because calcium channels have amino acids that actually react with the H2S. This reaction between the calcium channels and H2S opens the channels and allows entry of calcium into the cell. Now cells contain a host of enzymes that need calcium to operate properly.  Without the reaction of the calcium channels with H2S, calcium does not influx into the cell and the differentiation of mesenchymal stem cells into bone-making cells stops.

schematic diagram R1.ppt

Why is this important? Consider some of the diseases of bone, such as osteoporosis, in which the bones thin and become fragile. Restoring mesenchymal stem cell function in osteoporotic patients with treatments of H2S levels at nontoxic levels may provide treatments for diseases such as osteoporosis that might arise from H2S deficiencies.

Thus by understanding stem cell biology better, we can potentially treat a disease like osteoporosis with small amounts of a stinky gas. Incredible, isn’t it?

Skin Tissue Grown From Human Stem Cells

A research team from King’s College, London, in collaboration with the San Francisco Veteran Affairs Medical Center has succeeded in growing the epidermal layer of skin in culture, this cultured skin has many of the mechanical and biological properties of actual human skin.

The outermost layer of the skin, known as the epidermis forms a protective barrier between the external environment and the body. It protects against water loss and prevents the entry of microorganisms.

Tissue engineers have been able to grow skin cells (keratinocytes) in culture, but getting them to organize into an organ that resembles biological skin has proven rather difficult. However, the ability to test drugs on cultured skin that greatly mimics human skin has been the goal of such research for several years.

For this present project, keratinocytes were made from induced pluripotent stem cells that were derived from skin cells obtained from biopsies. These keratinocytes made from induced pluripotent stem cells (iPSCs) were very similar to keratinocytes made from embryonic stem cells and primary keratinocytes isolated from skin biopsies.

To form a three-dimensional structure like skin, the keratinocytes were cultured in a high-to-low humidity environment and they assembled into a layer structure that looked like human skin. When this cultured skin was compared with skin made from embryonic stem cell-derived keratinocytes or from keratinocytes isolated from skin biopsies, there were no significant structural differences.

Scientists hope to use this cultured skin to study congenital skin diseases like ichthyosis (characterized by dry, flaky skin) or atopic dermatitis. Growing large quantities of skin in culture will also allow drugs and cosmetics to be effectively tested for safety without the use of expensive and sometimes highly variable animal models.

This technology would also allow different laboratories to grow skin from different ethnic groups that have distinct types of skin with variable biological properties.

Long-Term Survival of Transplanted Human Neural Stem Cells in Primate Brains

A Korean research consortium has transplanted human neural stem cells (hNSCs) into the brains of nonhuman primates and ascertained the fate of these cells after being inside the brains of these animals for 22 and 24 months. They discovered that the implanted hNSCs had not only survived, but differentiated into neurons and never caused any tumors.

This important study is slated to be published in the journal Cell Transplantation.

To properly label the hNSCs so that they were detectable inside the brains of the animals, Lee and others loaded them with magnetic nanoparticles to enable them to be followed by magnetic resonance imaging (MRI). Also, they did not use immunosuppressants when they transplanted their hNSCs into the animals. This study is the first to examine the long-term survival and differentiation of hNSCs without the need for immunosuppression.

“Stroke is the fourth major cause of death in the US behind heart failure, cancer, and lower respiratory disease,” said study co-author Dr. Seung U. Kim of University of British Columbia Hospital’s department of neurology in Canada. “While tissue plasminogen activator (tPA) treatment within three hours after a stroke has shown good outcomes, stem cell therapy has the potential to address the treatment needs of those stroke patients for whom tPA treatment was unavailable or did not help.”

Based on the ability of hNSCs to differentiate into a variety of types of nerves cells, Lee and his colleagues thought that these cells have remarkable potential to treat damaged brain tissue and replace what was lost after a stroke, head injury or other type of brain trauma. Cell regeneration therapy can potentially treat brain-specific diseases like Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), spinal cord injury and stroke.

Dr. Kim and colleagues in Korea grafted magnetic particle-labeled hNSCs into the brains of laboratory primates and evaluated their performance to assess their survival and differentiation over 24 months. Of particular interest was determining their ability to differentiate into neurons and to determine whether the cells caused tumors.

“We injected hNSCs into the frontal lobe and the putamen of the monkey brain because they are included in the middle cerebral artery (MCA) territory, which is the main target in the development of the ischemic lesion in animal stroke models,” commented Dr. Kim. “Thus, research on survival and differentiation of hNSCs in the MCA territory should provide more meaningful information to cell transplantation in the MCA occlusion stroke model.”

Lee’s team said that they chose NSCs for transplantation because the existence of multipotent NSCs “has been known in developing rodents and in the human brain with the properties of indefinite growth and multipotent potential to differentiate” into the three major CNS cell types – neurons, astrocytes and oligodendrocytes.

“The results of this study serve as a proof-of-principle and provide evidence that hNSCs transplanted into the non-human primate brain in the absence of immunosuppressants can survive and differentiate into neurons,” wrote the researchers. “The study also serves as a preliminary study in our planned preclinical studies of hNSC transplantation in non-human primate stroke models.”

“The absence of tumors and differentiation of the transplanted cells into neurons in the absence of immunosuppression after transplantation into non-human primates provides hope that such a therapy could be applicable for use in humans.” said Dr. Cesar V. Borlongan, Prof. of Neurosurgery and Director of the Center of Excellence for Aging & Brain Repair at the University of South Florida. “This is an encouraging study towards the use of NSCs to treat neurodegenerative disorders”.