Nanoscale Scaffolds and Stem Cells Show Promise For Cartilage Repair

Tissue engineers have designed scaffolds for stem cells made from nanotubes that induce them to form cartilage. These nanofibers are tiny, artificial fiber scaffolds that are thousands of times smaller than a human hair. Cartilage formation has succeeded in laboratory and animal model systems.

Much more work is necessary before these scaffolds can be used inside a human body; the results of this present study hold promise for designing new techniques to help millions who endure joint pain.

Jennifer Elisseeff, Ph.D., Jules Stein Professor of Ophthalmology and director of the Translational Tissue Engineering Center at the Johns Hopkins University School of Medicine, said: “Joint pain affects the quality of life of millions of people. Rather than just patching the problem with short-term fixes, like surgical procedures such as microfracture, we’re building a temporary template that mimics the cartilage cell’s natural environment, and taking advantage of nature’s signals to biologically repair cartilage damage,”

Unfortunately, cartilage does not repair itself when damaged. For the last decade, Elisseeff’s research team has been trying to better understand the development and growth of cartilage cells (chondrocytes). Part of these studies has involved building scaffolds that mimics the environment inside the body that produces new cartilage tissue. The cartilage-making environment consists of a three-dimensional mix of protein fibers and gel. This matrix provides support to connective tissue throughout the body, and physical and biological cues for cells to grow and differentiate.

In the laboratory, the Elisseeff team created a nanofiber-based network that utilized a process called electrospinning. Electrospinning shoots a polymer stream onto a charged platform, to which a compound called chondroitin sulfate is added. Chondroitin sulfate is commonly found in many joint supplements. After characterizing the manufactured fibers, they made several different scaffolds from spun polymer or spun polymer plus chondroitin sulfate. Elisseeff and her colleagues then seeded the scaffolds with bone marrow stem cells from goats in order to test how well the scaffolds supported the growth of the stem cells.

The results showed that the scaffold-supported stem cells formed more voluminous, cartilage-like tissues that those grown without the manufactured scaffolds.

“The nanofibers provided a platform where a larger volume of tissue could be produced,” said Elisseeff, adding that 3-dimensional nanofiber scaffolds were more useful than the more common nanofiber sheets for studying cartilage defects in humans.

Next, Elisseeff and her group tested the ability of these nanotube scaffolds and the cartilage they produce in an animal model. They implanted the nanofiber scaffolds into damaged cartilage in the knees of rats. They compared the quality of the knee repair to damaged cartilage in knees that were not treated with any stem cells. The nanofiber scaffolds improved tissue development and repair. The nanofiber-implanted knees produced far more cartilage as measured by the production of collagen, which is a component of cartilage. The nanofiber scaffolds induced the production of larger quantities of a more durable type of collagen, which is typically absent in surgically repaired cartilage tissue. In rats, the limbs with damaged cartilage treated with nanofiber scaffolds generated a higher percentage of the more durable collagen (type 2) than those damaged areas that were left untreated.

“Whereas scaffolds are generally pretty good at regenerating cartilage protein components in cartilage repair, there is often a lot of scar tissue-related type 1 collagen produced, which isn’t as strong,” said Elisseeff. “We found that our system generated more type 2 collagen, which ensures that cartilage lasts longer. Creating a nanofiber network that enables us to more equally distribute cells and more closely mirror the actual cartilage extracellular environment are important advances in our work and in the field. These results are very promising.”

Embryonic Stem Cell Cultures Fluctuate Between Pluripotence and Totipotence

In the journal Nature, a fascinating paper appeared from the laboratory of Samuel Pfaff at the Howard Hughes Medical Institute at the Salk Institute for Biological Studies in La Jolla, California near San Diego. In this paper, Todd Macfarlan and his colleagues show that embryonic stem cells cycle between a very primitive developmental stage and a later stage. This cycling is also due to gene expression that is linked to transposable DNA elements.

First, we need some background. The term “totipotent” means that a cell can form any structure in the embryonic or adult body. For example, when the egg undergoes fertilization, it becomes a zygote, which has the capacity to grow into the embryo and all the extraembryonic membranes (amnion, chorion, allantois, placenta, and so on). Another example is a sponge. When a small piece of the sponge is cut from it, the cells in that small piece can de-differentiate and grow into an entire new sponge. Sponge cells are, therefore, totipotent.

Secondly, there is a term “pluripotent,” which means that the cells can form all the adult cell types. Embryonic stem cells are generally thought to be pluripotent and not totipotent. Once the embryo forms the two-cell stage, these two blastomeres are totipotent. However, when the blastocyst stage forms, the inner cell mass cells become pluripotent and lose the ability to form the placenta.

Many years ago, Beddington and Robertson, (1989) implanted mouse embryonic stem cells into the outer layer of cells (trophoblast) to determine if the inner cell mass cells could form the placenta (Development 105, 733–737). The embryonic stem cells were incorporated into the placenta at a very low rate. These data led to an intriguing question: Was the low incorporation due to contamination with trophoblast cells, or could a small proportion of the embryonic stem cells actually become placenta? When gene expression studies examined embryonic stem cells, gene expression was stable in the majority of the cells, but unstable in a small minority of cells (a condition called metastable). It was not surprising that embryonic stem cells were a mixed bag of different cells, but some cells expressed genes that were normally found at earlier developmental stages or were normally expressed in cells that make placenta:
1. Niakan, K. K. et al. Sox17 promotes differentiation in mouse embryonic stem cells by directly regulating extraembryonic gene expression and indirectly antagonizing self-renewal. Genes Dev. 24, 312–326 (2010).
2. Hayashi, K., Lopes, S. M., Tang, F. & Surani, M. A. Dynamic equilibrium and heterogeneity of mouse pluripotent stem cells with distinct functional and epigenetic states. Cell Stem Cell 3, 391–401 (2008).
3. Singh, A. M., Hamazaki, T., Hankowski, K. E. & Terada, N. A heterogeneous expression pattern for Nanog in embryonic stem cells. Stem Cells 25, 2534–2542 (2007).
4. Chambers, I. et al. Nanog safeguards pluripotency and mediates germline development. Nature 450, 1230–1234 (2007)
5. Zalzman, M. et al. Zscan4 regulates telomere elongation and genomic stability in ES cells. Nature 464, 858–863 (2010).

Weird, huh?

Into the fray comes Macfarlan and company to save (or explain) the day. It turns out that our genomes are loaded with DNA from transposable elements. These DNA elements either have or had at one time, the ability to jump from one location in the genome to another. There are large numbers of these transposable elements in our genomes and almost 50% of the human genome is composed of the remains of these elements.  Current transposable elements include Long INterspersed Elements (LINEs), Short INterspersed Elements (SINEs) and SVA (SINE/VNTR/Alu) elements.  Others include elements such as Mariner, MIR, HERV-K, and others.  The significance of all this is that during development, when the embryo gets to the two-cell stages, in the mouse, particular transposable elements are expressed at very high levels (they produce 3% of the transcribed messenger RNAs, see Peaston, A. E. et al., Dev. Cell 7, 597–606 (2004); Evsikov, A. V. et al., Cytogenet. Genome Res. 105, 240–250 (2004); Kigami, D., et al., Biol. Reprod. 68, 651–654 (2003)), and after two-cell stage, the expression of these transposable elements is silenced (Svoboda, P. et al. Dev. Biol. 269, 276–285 (2004); Ribet, D. et al. J. Virol. 82, 1622–1625 (2008)).

Since these transposons are characteristic of gene expression at the two-cell stage, they can be used as a marker for embryonic stem cells that have reverted back to the two-cell stage.  MacFarlan and his co-workers made a reporter gene and placed it into embryonic stem cells that was controlled by the same sequences as the transposons that are activated at the two-cell stage.  After growing these engineered embryonic stem cells in culture, they discovered that a small minority of cells expressed this reporter gene.

Did these reporter-expressing cells have characteristics like unto those of the two-cell stage embryos?  Yes they did.  When Macfarlan and his buddies examined the genes expressed in the cells that expressed the reporter, they found that the traditional genes that are so characteristic of inner cell mass cells (Oct4, Nanog, Sox2, etc.) were not expressed and other genes normally expressed in two-cell stage embryos, such as Zscan4, were expressed.  Other features that are found in two-cell-stage embryos were also found in these cells that expressed the reporter gene. (methylation of histone 3 lysine 4 (H3K4) and acetylation of H3 and H4 for those who are interested).

Finally, the reporter-expressing cells were able to contribute to the formation of the placenta when transplanted into a mouse embryo.  This shows that these cells not only express the genes of the totipotent stage of development, but they also are totipotent.

These experiments show that most, maybe all embryonic stem cells pass through a short-lived state during which they display features that are characteristic of the totipotent two-cell stage: unlike the vast majority of the ES cells in the culture.  During this transition, they lack expression of the pluripotency-promoting proteins Oct4, Sox2 and Nanog, and have the ability to form cells of both the placenta and the fetus.

There is also a moral implication of these experiments.  In his book Challenging Nature and on the book’s web site, Lee Silver argues that embryonic stem cells are essentially embryos, and if we don’t object to using and discarding embryonic stem cells, then we should not have any problem with using and discarding embryos.  His reasons for asserting that embryonic stem cells are embryos is that in the mouse, embryonic stem cells can be inserted into the inner cell mass of an embryo that has four copies of each chromosome.  The tetraploid embryos can form the placenta, but they cannot form the embryo that is attached to the embryo.  Inserting embryonic stem cells into the inner cell mass of these embryos will rescue them from dying because the embryonic stem cells with make the embryo and the tetraploid embryos will form the placenta.  This experiment is called “tetraploid rescue” and Silver uses it to argue that embryonic cells are essentially embryos.

I find this argument unconvincing for several reasons.  First of all, these embryonic stem cells are being manipulated by being inserted into an embryo.  Granted this embryo is abnormal, but it is an embryo all the same and it provides a vital function that the embryonic stem cells cannot supply – the making of the placenta.  This manipulation helps the embryonic stem cells make the embryo, but not everything else.  In this case the embryonic stem cells are only doing part of the job and they are also receiving the structure and inductive signals from the tetraploid embryo to form the embryo proper.  This is something that embryonic stem cells do not do in culture.

Secondly, embryos undergo development, a process that we understand rather well.  This process of development has a goal toward which the embryo proceeds during development.   Embryonic stem cells are not in the process of development.  They can be induced to form particular cell types or even tissues, but this is part of the embryo or fetus and forming part of the fetus does not constitute embryonic development but only a small part of it.  Embryos do not go backwards during development.  Cells that do go backwards are usually cancer cells that grow uncontrollably and cannot move to a more differentiated state that puts the brakes on cell division.  The fact that embryonic stem cells do move developmentally backward is another indication that they are not embryos.  They do something that embryos do not do and this disqualifies them from being embryos.

Thus another argument against the humanity of the early embryo falls into the pit of very bad arguments.

Sox2 Marks Incisor Stem Cells

Finnish stem cell researchers have discovered a gene that serves as a marker for front teeth. Researchers in the group of Professor Irma Thesleff at the Institute of Biotechnology in Helsinki, Finland have developed a method to record the division, movement, and specification of these dental stem cells. Apparently, building a tooth requires a detailed recipe to instruct cells to differentiate towards proper lineages and form dental cells.

Building a tooth from stem cells is a very difficult talk. However the development of new bioengineering protocols might make this possible in a few years. There is definitely a demand for tissue engineered teeth, since tooth loss affects oral health, quality of life, and your appearance. To build a tooth, a detailed recipe to instruct cells to direct cells to differentiate towards proper cell lineages and form dental cells is needed. However, in order to study of stem cells, scientists need a specific protein that only those cells make (a marker), that allows the isolation of and purification of dental stem cells. Unfortunately, the lack of an identifiable marker has hindered such studies so far.

The mouse system is an excellent system for such studies, since mouse incisors grow continuously throughout life and this growth is fueled by stem cells located at the base of the tooth. In Professor Thesleff’s lab, her students traced the descendants of genetically labeled cells, and showed that a gene called Sox2 labels stem cells that give rise to enamel-forming ameloblasts as well as other cell lineages of the tooth.

Even though human teeth don’t grow continuously, the mechanisms that control and regulate their growth are similar to those in mouse teeth. Therefore, the discovery of Sox2 as a marker for dental stem cells is an important step toward developing a complete bioengineered tooth.

In the future, it may be possible to grow new teeth from stem cells to replace lost ones, said researcher Emma Juuri, a co-author of the study.

Letter from a Nurse With MS – FDA’s Cells = Drugs Hurts People

At the Regenexx Blog site is a letter from a Registered Nurse who has Multiple Sclerosis. The drugs for MS do little to stop the progression of the disease and they have remarkably bad side effect. On top of that they are very expensive. Despite some exceedingly robust results with animals models with a form of MS and stem cell treatments, and human clinical trials with a patient’s own mesenchymal stem cells, the FDA has yet to budge because according to your FDA cells = drugs and they get to regulate them into the ground.

This letter from the nurse really nails it on the head and shows how the FDA’s policy is a) crap, and b) actively hurting sick people. Read about it here, and then write your Congressperson.

Getting Genes into Stem Cells Without Viruses

Genetic engineering of cells and, in particular, of stem cells has the ability to adjust the functional capacities of cells. Unfortunately, genetically engineering cells requires the use of viruses that introduce genes into cells and, by doing so, produce mutations in cells.

However, there are new ways to put genes into cells without the use of viruses. By surrounding DNA that encodes the genes you want to put into cells with positively-charged lipids, you have made a structure called a liposome. Liposomes can fuse with the membranes of cells and deliver the genes to cells without viruses that can cause mutations.

A paper that has appeared in the journal Stem Cells and Development examined the use of liposomes to introduce genes into blood cell-making stem cells (HSCs). They used commercially-available systems to transfer genes into these stem cells, but they found that their own lab-designed system did a better job than the commercially-available systems.

The lead author of this paper is Hilal Gul-Uludag and the senior author is Jie Chen from the University of Alberta in Edmonton, Alberta, Canada. In this paper, Chen’s research group isolated blood cell-making stem cells from umbilical cord blood. Then they used liposomes to insert the CXCR4 gene. The CXCR4 gene encodes a receptor for “stromal cell-derived factor-1alpha” (SDF-1alpha). When cells bind to SDF-1alpha, they move towards the source of SDF-1alpha.

Interestingly, one of the best sources of SDF-1alpha is the bone marrow. If HSCs could be engineered to make CXCR4, then they would readily move into the bone marrow. This means that implanted HSCs would only need to be introduced into the peripheral blood and not into the bone. This would increase the efficiency of bone marrow or umbilical cord transplants.

Chen’s group showed the feasibility of such experiments, and that these treatments are not toxic in any way to the HSCs. Thus, such a strategy could potentially increase the efficiency of bone marrow and umbilical cord blood transplantation.

Neurons Derived from Cord Blood Cells

A research group at the Salk Institute in San Diego has discovered a new protocol for converting umbilical cord blood cells into neuron-like cells. These new cells could prove valuable for the treatment of a wide variety of neurological conditions, including stroke, traumatic brain injury and spinal cord injury.

Physicians have used umbilical cord blood for more than 20 years to treat many different types of illnesses, including cancer, immune disorders, and blood and metabolic diseases. However, these Salk Institute researchers demonstrated that cord blood (CB) cells can be differentiated into cell types from which brain, spinal and nerve cells arise.

Juan Carlos Izpisua Belmonte, a professor in Salk’s Gene Expression Laboratory, who led the research team, said: “This study shows for the first time the direct conversion of a pure population of human cord blood cells into cells of neuronal lineage by the forced expression of a single transcription factor.”

Izpisua Belmonte’s group used an engineered retrovirus to introduce a gene called Sox2, a transcription factor that acts as a switch inside cells that converts them into neurons. Therefore, by introducing Sox2 into CB cells, and culturing them in the lab, the cells formed colonies that expressed genes normally found in neurons.

Were these cells actual neurons or faux neurons? Cells might make neuron-specific genes, but they do not assemble those gene products into neuron-specific machinery, then they are not neurons. To if such cells are neurons, they should be able to manipulate the electrical charges across their cell membranes. But subjecting cells to electrophysiological tests, they determined that these new cells, which they called induced neuronal-like cells or iNCs, could transmit electrical impulses. This shows that the iNCs were mature and functional neurons. Next, they implanted these Sox2-transformed CB cells to a mouse brain and found that they integrated into the existing mouse neuronal network and were capable of transmitting electrical signals like mature functional neurons.

Mo Li, a scientist in Belmonte’s lab and a co-first author on the paper, said: “We also show that the CB-derived neuronal cells can be expanded under certain conditions and still retain the ability to differentiate into more mature neurons both in the lab and in a mouse brain. Although the cells we developed were not for a specific lineage-for example, motor neurons or mid-brain neurons-we hope to generate clinically relevant neuronal subtypes in the future.”

Scientists can use these cells in the future to model neurological diseases such as autism, schizophrenia, Parkinson’s or Alzheimer’s disease.

CB cells offer several advantages over other types of stem cells. First, they are not embryonic stem cells and are not controversial. They are more plastic, or flexible, than adult stem cells from sources like bone marrow, which may make them easier to convert into specific cell lineages. The collection of CB cells is safe and painless and poses no risk to the donor, and they can be stored in blood banks for later use.

“If our protocol is developed into a clinical application, it could aid in future cell-replacement therapies,” said Li. “You could search all the cord blood banks in the country to look for a suitable match.”

X (Chromosome) Marks the Plot

In female mammalian embryos, the X chromosome represents a problem. Since mammalian females have two X chromosomes, the embryo contains twice as much of the gene products of the X chromosome as opposed to male mammalian embryos, which only have one copy of the X chromosome. How is this problem solved? X chromosome inactivation (XCI). XCI occurs very early during female mammalian development, and it occurs on a cell-by-cell basis, and occurs randomly. The embryo has some cells that have one copy of the X chromosome inactivated and all the other cells have the other copy of the X chromosome inactivated. This is the reason the bodies of mammalian females are mosaics in which some cells have one copy of the X chromosome inactivated and yet other cells in which the other copy of the X chromosome is inactivated. Thus genetic diseases that map to the X chromosome will affect the entire body of the mammalian male but only a portion of the mammalian female’s body.

What does this mean for stem cells? Quite a bit. Embryonic stem cells are derived from the inner cell mass of the blastocyst-stage embryo. This is precisely the time when the cells of the embryo begin to randomly select a copy of the X chromosome to inactivate. The timing of XCI differs slightly from one species to another. In mice, for example, both copies of the X chromosome are active in mouse embryonic stem cells (ESCs) (Fan and Tran, Hum Genet 130 (2011):217-22; Chaumeil, et al., Cytogenet Genome Res 99 (2002):75-84), and XCI occurs when the cells differentiate (Murakami, et al., Development 138 (2011):197-202). Human ESCs, however, vary tremendously (Dvash and Fan, Epigenetics 4 (2009):19-22), with a few hESC lines showing activation of both copies of the X chromosome and many others showing inactivation of one or the other copy of the X chromosome. Human induced pluripotent stem cells (iPSCs) are derived from adult cells that already have one copy of the X chromosome inactivated. Therefore, de-differentiation of adult cells into iPSCs undoes XCI and activates both copies of the X chromosome (Maherali, et al., Cell Stem Cell 1 (2007):55-70 & Hanna, et al., PNAS 107 (2010):9222-7).

XCI is a process that is linked to pluripotency. The genes necessary for the maintenance of pluripotency (OCT4, Sox2, Nanog) all repress genes necessary for XCI (Xist) and activate genes that repress XCI (Tsix). Therefore, XCI seems to be a factor in the down-regulation of pluripotency in early embryonic cells.

There is a new study that underscores this link between XCI and pluripotency. Researchers at the Gladstone Institutes at the University of California, San Francisco have expanded upon the so-called Kyoto method for making iPSCs. The Kyoto method uses an animal cell line that grows in the culture dish and makes a protein called LIF (leukemia inhibitory factor). LIF activates the growth of cultured iPSCs and allows them to grow and establish an iPSC line.

According to Kiichiro Tomoda from the Gladstone Institute, iPSC derivation on LIF-making feeder cells always produces IPSCs that have two active copies of the X chromosome. However, if iPSCs are derived on feeder cells that do not make LIF, the result is very poor iPSCs derivation and the resultant iPSCs only have one active copy of the X chromosome. Furthermore, by passaging iPSCs that were derived from non-LIF-making feeder cells on LIF-making feeder cells, the inactivated X chromosome became active. This shows that iPSC derivation is highly sensitive to the environment in which the cells are derived. If also shows how to make iPSCs that more closely resemble early embryonic cells.