Stem Cells Heal Damaged Cells by Transferring Mitochondria


An Indian team from Delhi, India has identified a protein that increases the transfer of mitochondria from mesenchymal stem cells to lung cells, thus augmenting the healing of lung cells.

Stem cells like mesenchymal stem cells from bone marrow, fat, tendons, liver, skeletal muscle, and so on secrete a host of healing molecules, but they also form bridges to other cells and export their own mitochondria to heal damaged cells. Mitochondria are the structures inside cells that make energy. Damaged cells can have serious energy deficiencies and mitochondrial transfer ameliorates such problems (see Cárdenes N et al, Respiration. 2013;85(4):267-78).

This present work from the laboratory of Anurag Agrawal, who is housed in the Centre of Excellence in Asthma & Lung Disease, at the CSIR‐Institute of Genomics and Integrative Biology in Delhi, India has identified a protein called Miro1 that regulates the transfer of mitochondria to recipient cells.

Mitochondrial transfer has so many distinct benefits that stem cell scientists hope to engineer stem cells to transfer more of their mitochondria to damaged cells, and Miro1 might be a target for such stem cell engineering experiments.

Mitochondrial transfer between stem cells and other cells occurs by means of tunneling nanotubes, which are thread-like structures formed from the plasma membranes of cells that form bridges between different cell types. Under stressful conditions, the number of these nanotubes increases.

In the present study. stem cells engineered to express more Miro1 protein transferred mitochondria more efficiently than control stem cells. When used in mice with damaged lungs and airways, these Miro1-overexpressing cells were therapeutically more effective than control cells.

This study presents the first mechanistic insight into how Mesenchymal Stem Cells (MSC) act as mitochondrial donors during attenuation of lung inflammation and injury. Mitochondrial donation is an essential part of the MSC therapeutic effect in these models and is positively regulated by Miro1 / Rhot1 mitochondrial transport proteins.
This study presents the first mechanistic insight into how Mesenchymal Stem Cells (MSC) act as mitochondrial donors during attenuation of lung inflammation and injury. Mitochondrial donation is an essential part of the MSC therapeutic effect in these models and is positively regulated by Miro1 / Rhot1 mitochondrial transport proteins.

The hope is to use Miro1 manipulations to make better stem cell therapies for human diseases.

To summarize this work:

1. MSCs donate mitochondria to stressed epithelial cells (EC) that have malfunctioning mitochondrial.  Cytoplasmic nanotubular bridges form between the cells and Miro‐1 mediated mitochondrial transfer occurs unidirectionally from MSCs to ECs.

2. Other mesenchymal cells like smooth muscle cells and fibroblasts express Miro1 and can also donate mitochondria to ECs, but with low efficiency. ECs have very low levels of Miro1 and, as a rule, do not donate mitochondria.

3. Enhanced expression of Miro1 in mesenchymal cells increases their mitochondrial donor efficiency.  Conversely, cells lacking Miro1 do not show MSC mediated mitochondrial donation.

4. Miro1‐overexpressing MSCs have enhanced therapeutic effects in three different models of allergic lung inflammation and rat poison-induced lung injury.  Conversely, Miro1‐depleted MSCs lose much of their therapeutic effect.  Miro1 overexpression in MSCs may lead to more effective stem cell therapy.

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Physical Cues Push Mature Cells into Induced Pluripotent Stem Cells


Bioengineers from the laboratory of Song Li at UC Berkeley have used physical cues to help push mature cells to de-differentiate into embryonic-like cells known as induced pluripotent stem cells.

Essentially, Li and his coworkers grew skin fibroblasts from human skin and mouse ears on surfaces with parallel grooves 10 micrometers apart and 3 micrometers high, in a special culture medium. This procedure increased the efficiency of reprogramming of these mature cells four-fold when compared to cells grown on a flat surface. Growing cells in scaffolds of nanofilbers aligned in parallel had similar effects.

Li’s study could significantly advance the protocols for making induced pluripotent stem cells (iPSCs). Normally iPSCs are made by genetically engineering adult cells so that they overexpress four different genes: Oct-4, Sox-2, Klf-4, and c-Myc. To put these genes into the cells, genetically modified viruses are used, or plasmids (small circles of DNA). Initially, Shinya Yamanaka, the scientist who invented iPSCs, and his co-workers used retroviruses that contained these four genes. When fibroblasts were infected with these souped-up retroviruses, the viruses inserted their viral DNA into the genomes of the host cells and expressed these genes.

retrovirus_life_cycle

Shinya Yamanaka won the Nobel Prize for this work in Physiology or Medicine in 2012 for this work. Unfortunately, retroviruses and can cause insertional mutations when they integrate into the genome (Zheng W., et al., Gene. 2013 Apr 25;519(1):142-9), and for this reason they are not the preferred way of making iPSCs. There are other viral vectors that do not integrate into the genome of the host cell (e.g., Sendai virus; see Chen IP, et al., Cell Reprogram. 2013 Dec;15(6):503-13). There are also techniques that use plasmids, which encode the four genes but do not integrate into the genome of the host cell. Finally, synthetic messenger RNAs that encode these four genes have also been used to make iPSCs (Tavernier G,, et al., Biomaterials. 2012 Jan;33(2):412-7).

The use of physical cues to make iPSCs may replace the need for gene overexpression, just as the use of particular chemicals can replace the need for particular genes (Zhu, S. et al. Cell Stem Cell 7, 651–655 (2010); Li, Y. et al. Cell Res. 21, 196–204 (2011)). If physical cues can replace the need for the overexpression of particular genes, then this discovery could revolutionize iPSC derivation; especially since the overexpression of particular genes in mature cells tends to cause genome instability in cells (Doris Steinemann, Gudrun Göhring, and Brigitte Schlegelberger. Am J Stem Cells. 2013; 2(1): 39–51).

“Our study demonstrates for the first time that the physical features of biomaterials can replace some of these biochemical factors and regulate the memory of a cell’s identity,” said study principal investigator Song Li, UC Berkeley, Professor of bioengineering. “We show that biophysical signals can be converted into intracellular chemical signals that coax cells to change.”

a, Scanning electron micrograph of PDMS membranes with a 10 μm groove width. All grooves were fabricated with a groove height of 3 μm. b, The top row shows phase contrast images of flat and grooved PDMS membranes with various widths and spacings. The bottom row shows fibroblast morphology on various PDMS membranes. Images are fluorescence micrographs of the nucleus (DAPI, blue) and actin network (phalloidin, green; scale bars, 100 μm). c, Reprogramming protocol. Colonies were subcultured and expanded or immunostained and quantified by day 12–14. d, Fluorescence micrograph showing the morphology of iPSC colonies generated on flat and grooved membranes (scale bar, 1 mm). Groove dimensions were 10 μm in width and spacing, denoted as 10 μm in this and the rest of the figures. Double-headed arrow indicates microgroove orientation of alignment. e, Reprogramming efficiency of fibroblasts transduced with OSKM and cultured on PDMS membranes with flat or grooved microtopography. The number of biological replicates, n, used for this experiment was equal to 6. Groove width and spacing were varied between 40, 20 and 10 μm. Differences of statistical significance were determined by a one-way ANOVA, followed by Tukey’s post-hoc test. * indicates significant difference (p<0.05) compared with the control flat surface. f, Reprogramming efficiency in fibroblasts transduced with OSK (n = 4). *p<0.05 (two-tailed, unpaired t-test) compared with the control flat surface. Error bars represent one standard deviation. g, Immunostaining of a stable iPSC line expanded from colonies generated on 10 μm grooves. These cells express mESC-specific markers Oct4, Sox2, Nanog and SSEA-1 (scale bar, 100 μm). h, The expanded iPSCs in g were transplanted into SCID mice to demonstrate the formation of teratomas in vivo (scale bar, 50 μm).
a, Scanning electron micrograph of PDMS membranes with a 10 μm groove width. All grooves were fabricated with a groove height of 3 μm. b, The top row shows phase contrast images of flat and grooved PDMS membranes with various widths and spacings. The bottom row shows fibroblast morphology on various PDMS membranes. Images are fluorescence micrographs of the nucleus (DAPI, blue) and actin network (phalloidin, green; scale bars, 100 μm). c, Reprogramming protocol. Colonies were subcultured and expanded or immunostained and quantified by day 12–14. d, Fluorescence micrograph showing the morphology of iPSC colonies generated on flat and grooved membranes (scale bar, 1 mm). Groove dimensions were 10 μm in width and spacing, denoted as 10 μm in this and the rest of the figures. Double-headed arrow indicates microgroove orientation of alignment. e, Reprogramming efficiency of fibroblasts transduced with OSKM and cultured on PDMS membranes with flat or grooved microtopography. The number of biological replicates, n, used for this experiment was equal to 6. Groove width and spacing were varied between 40, 20 and 10 μm. Differences of statistical significance were determined by a one-way ANOVA, followed by Tukey’s post-hoc test. * indicates significant difference (p<0.05) compared with the control flat surface. f, Reprogramming efficiency in fibroblasts transduced with OSK (n = 4). *p

To boost the efficiency of mature cell reprogramming, scientists also use a chemical called valproic acid, which dramatically affects global DNA structure and expression.

“The concern with current methods is the low efficiency at which cells actually reprogram and the unpredictable long-term effects of certain imposed genetic or chemical manipulations,” said the lead author of this study Timothy Downing. “For instance, valproic acid is a potent chemical that drastically alters the cell’s epigenetic state and can cause unintended changes inside the cell. Given this, many people have been looking at different ways to improve various aspects of the reprogramming process.”

This new study confirms and extends previous studies that showed that mechanical and physical cues can influence cell fate. Li’s group showed that physical and mechanical cues can not only affect cell fate, but also the epigenetic state and cell reprogramming.

a, Scanning electron micrograph of nanofibres showing fibre morphology in aligned and random orientations (scale bar, 20 μm). Confocal fluorescence micrograph of fibroblasts cultured on nanofibres (DAPI (blue) and phalloidin (green) staining; scale bar, 100 μm). b, Western blotting analysis for fibroblasts cultured on random and aligned nanofibres for three days. c, Fibroblasts were transduced with OSKM and seeded onto nanofibre surfaces, followed by immunostaining for Nanog expression (red) at day 12. Nuclei were stained with DAPI in blue; scale bar, 500 μm. d, Quantification of colony numbers in c (n = 5). *p<0.05 (two-tailed, unpaired t-test) compared with the control surface with random nanofibres. e, Fibroblasts were micropatterned into single-cell islands of 2,000 μm2 area with a CSI value of 1 (round) or 0.1 (elongated). After 24 h, cells were immunostained for AcH3, H3K4me2 or H3K4me3 (in green). Phalloidin staining (red) identifies the cell cytoskeleton for cell shape accuracy. The white arrowhead indicates the location of the nucleus (scale bars, 20 μm). f, Quantification of fluorescence intensity in e (n = 34, 20 and 34 for AcH3, H3K4me2 and H3K4me3, respectively). *p<0.05 (two-tailed, unpaired t-test) compared with the circular micropatterned cells (CSI = 1). Error bars represent one standard deviation.
a, Scanning electron micrograph of nanofibres showing fibre morphology in aligned and random orientations (scale bar, 20 μm). Confocal fluorescence micrograph of fibroblasts cultured on nanofibres (DAPI (blue) and phalloidin (green) staining; scale bar, 100 μm). b, Western blotting analysis for fibroblasts cultured on random and aligned nanofibres for three days. c, Fibroblasts were transduced with OSKM and seeded onto nanofibre surfaces, followed by immunostaining for Nanog expression (red) at day 12. Nuclei were stained with DAPI in blue; scale bar, 500 μm. d, Quantification of colony numbers in c (n = 5). *p

“Cells elongate, or example, as they migrate throughout the body,” said Downing, who is a research associate in Li’s lab. “In the case of topography, where we control the elongation of a cell by controlling the physical microenvironment, we are able to more closely mimic what a cell would experience in its native physiological environment. In this regard, these physical cues are less invasive and artificial to the cell and therefore less likely to cause unintended side effects.”

Li and his colleagues are studying whether growing cells on grooved surfaces eventually replace valproic acid and even replace other chemical compounds in the reprogramming process.

“We are also studying whether biophysical factors could help reprogram cells into specific cell types, such as neurons,” said Jennifer Soto, a UC Berkeley graduate student in bioengineering who was also a co-author on this paper.

Timothy Downing, et al., Nature Materials 12, 1154–1162 (2013).  

Reducing the Heart Scar After a Heart Attack


After a heart attack, inflammation in the heart kills off heart muscle cells and fibroblasts in the heart make a protein called collagen, which forms a heart scar. The heart scar does not contract and does not conduct electrochemical signals. The scar will contract over time, but its presence can lead to abnormal heart rhythms, also known as arrhythmias. Arrythmias can be fatal, since they can cause a heart attack. To prevent a heart attack, physicians will treat heart attack patients with a group of drugs called beta-blockers that slow down the heart rate and protect the heart from the deleterious effects of norepinephrine (secreted by the sympathetic nerve inputs to the heart). An alternative treatment is digoxin or digitalis, which is a chemical found in foxglove. Digitalis inhibits ion pumps in heart muscle cells and slows the heart and the force of its contractions. Digitalis, however, interacts with a whole shoe box fill of drugs, has a very long half-life, and is hard to dose. Therefore it is not the first choice.

Given all this, helping the heart to make a smaller heart scar is a better strategy for treating a heart after a heart attack. To accomplish this, you need to inhibit the heart fibroblasts that make the heart scar in the first place. Secondly, you must move something into the place of the dead cells. Otherwise, the heart could burst or scar tissue will move into the area anyway.

To that end, Yigang Wang and his colleagues at the University of Cincinnati Medical Center in Ohio have published an ingenious paper in which they tried two different strategies to reduce the size of the heart scar, which concomitantly increased the colonization of the heart by induced pluripotent stem cells engineered to express a sodium-calcium exchange pump.

Previously, Wang and his colleagues used a patch to heal the heart after a heart attack. The patch consisted of endothelial cells, which make blood vessels, induced pluripotent stem cells engineered to make a sodium-calcium exchange pump called NCX1, and embryonic fibroblasts. This so-called tri-cell patch makes new blood vessels, establishes new heart muscle, and the foundational matrix molecules to form a platform for beating heart muscle.

In order to get these cells to spread throughout the injured heart, Wang and others used a reagent that specifically inhibits heart fibroblasts. They used a small non-coding RNA molecule. A group of microRNAs called miR-29 family are downregulated after a heart attack. As it turns out, these microRNAs inhibit a group of genes that involved in collagen deposition. Therefore, by overexpressing miR-29 microRNAs, they could prevent collagen deposition and reduce scar formation.

The experimental design in this paper is rather complex. Therefore, I will go through it slowly. First, they tried to overexpress miR-29 microRNAs in cultured heart fibroblasts and sure enough, they inhibited collagen synthesis. Cells overexpressing miR-29 made less than a third of the collagen of their normal counterparts. When they placed these fibroblasts into the heart and induced heart attacks, again, they made significantly less collagen when they were expressing miR-29.

Then they used their miR-29 RNAs by injecting them directly into the heart before inducing a heart attack, and then after the heart attack, they applied the tri-patch. Their results were significant. The scar size was smaller (almost one-third the size of the controls), and the density of blood vessels was much higher in the tri-patched hearts treated with miR-29. The induced pluripotent stem cells differentiated into heart muscle cells and spread throughout the heart. Heart function measures also consistently went up too.  The echiocardiograph before more normal, the ejection fraction went up, the % shortening of the heart muscle fibers was increased, and the relaxation phase of the heart (diastole) also was not so puffy (see graphs and figures below).

(A): M-mode echocardiograph data in three groups. (B): Quantification analysis for heart function. Quantitative data for LVDd (B-1), LVDs (B-2), EF (B-3), and FS (B-4) 4 weeks after Tri-P implantation. *p,0.05 vs. Ctrl+MI+Tri-P group; {p,0.05 vs. Anti-29b+MI+Tri-P group. LVDd, left ventricular enddiastolic diameters; LVDs, left ventricular end-systolic diameters; EF, ejection fraction index; FS, fractional shortening. All values expressed as mean 6 SEM. n = 6 for each group. (C): Two-D mode echocardiograph data in three groups, analyzed by long-axis and short-axis views. *p,0.05 vs. Ctrl+MI+Tri-P group; {p,0.05 vs. miR-29b+MI+Tri-P group. Ctrl, control mimic pretreatd rat with Tri-cell patch graft; miR-29b, miR- 29b mimic pretreated rat with Tri-cell patch graft; Anti-29b, miR-29b inhibitor pretreated rat with Tri-cell patch graft. White dotted lines indicate endocardium and epicardium.
(A): M-mode echocardiograph data in three groups. (B): Quantification analysis for heart function. Quantitative data for LVDd (B-1), LVDs (B-2), EF (B-3), and FS (B-4) 4 weeks after Tri-P implantation. *p,0.05 vs. Ctrl+MI+Tri-P group; {p,0.05 vs. Anti-29b+MI+Tri-P group. LVDd, left ventricular enddiastolic diameters; LVDs, left ventricular end-systolic diameters; EF, ejection fraction index; FS, fractional shortening. All values expressed as mean 6 SEM. n = 6 for each group. (C): Two-D mode echocardiograph data in three groups, analyzed by long-axis and short-axis views. *p,0.05 vs. Ctrl+MI+Tri-P group; {p,0.05 vs. miR-29b+MI+Tri-P group. Ctrl, control mimic pretreatd rat with Tri-cell patch graft; miR-29b, miR-29b mimic pretreated rat with Tri-cell patch graft; Anti-29b, miR-29b inhibitor pretreated rat with Tri-cell patch graft. White dotted lines indicate endocardium and epicardium.

There is a cautionary note to this study. Inhibiting collagen formation after a heart attack could create soft fragile regions of the heart that are subject to rupture should the vascular systolic pressure increase. While that threat was not observed in this study, human hearts, which are much larger, would be much more susceptible to such a mishap. Therefore, while this study is interesting and suggest a strategy in humans, it requires more testing and refinement before anyone can even think about applying it to humans.

Induced Pluripotent Stem Cells Improve Hearts


Mayo Clinic Investigators have shown that induced pluripotent stem cells, which were made from particular cells in the skin called “fibroblasts,” were differentiated into heart muscle cells and used to treat mice with heart disease. This proof-of-principle study shows that is might be possible to use iPSCs to fix hearts after a heart attack with iPSCs.

Timothy Nelson, the principal author of this study, said that this study “establishes the real potential for using iPS cells in cardiac treatment. iPSCs have already been used to treat sickly cell anemia, Parkinson’s disease and hemophilia A in laboratory mice. This experiment, which was also done in mice, further extends the clinical conditions that iPSCs might treat.

This is an exciting result, but there is a caveat I must mention. Christine Mummery and her colleagues have shown that even though human embryonic stem cell transplantation improves the condition of the heart after a heart attack in rodents after four weeks, examination of these same rodents twelve weeks after the transplants reveals that the improvements have largely disappeared.  In this study, the mice were examined after four weeks and not after twelve. Therefore, this study might be in the same category as those done with human embryonic stem cells. If the improvements could be shown to last even up to twelve weeks after the transplantations, then I think we would have something really to crow about. However, as it is, while this result is interesting, it is simply not conclusive.

Induced Pluripotent Stem Cells


Embryonic stem cells might provide the means to heal a variety of physical ailments. However the problem with embryonic stem cells is not necessarily in their use, but in their derivation. In order to make embryonic stem cell lines, human embryos are destroyed.

The following video shows Alice Chen from Doug Melton’s laboratory at Harvard University destroying embryos to make embryonic stem cells:  http://www.jove.com/index/details.stp?ID=574.

Now that federal funding is available to not only work with existing embryonic stem cell lines but to MAKE new lines, there is nothing to stop researchers from thawing and (I’m sorry to be so blunt) killing human embryos. Can we have our “cake and eat it too?” Can we have the benefits of embryonic stem cells and not destroy embryos? Perhaps we can.

In 2001, Masako Tada reported the fusion of embryonic stem cells with a connective tissue cell called a fibroblast. This fusion reprograms the fibroblasts so that they behave like embryonic stem cells (Current Biology 11, no. 9 (2001): 1553–8). This suggests that something within embryonic stem cells can redirect the machinery of somatic cells to become more like that of embryonic stem cells. In 2006 Kazutoshi Takahashi and Shinya Yamanaka were able to generate embryonic stem cell lines by introducing four specific genes into mouse skin fibroblasts. These “induced pluripotent stem cells” (iPSCs) shared many of the properties of embryonic stem cells derived from embryos, but when transplanted into mouse embryos, they were not able to participate in the formation of an adult mouse (Cell 126, no. 4 (2006): 663–76). This experiment showed that it is possible to convert adult cells into something that resembles an embryonic stem cell. Could we push adult cells further? In 2007, three different research groups used retroviruses to transfer four different genes (Oct3/4, Sox2, c-Myc and Klf4) into mouse skin fibroblasts and completely transformed them into cells that had all the features and behaviors of embryonic stem cells (Cell Stem Cell 1, no. 1 (2007): 55–70; Nature 448 (2007): 313–7; Nature 448 (2007): 318–24.).

These experiments drew a great deal of excitement, but there were several safety concerns that had to be addressed before iPSCs could be used in human clinical trials.  Scientists used engineered retroviruses to introduce genes into adult cells in order to reprogram them into iPSCs (Current Topics in Microbiology and Immunology 261 (2002): 31-52).  Retroviruses insert a DNA copy of their genome into the chromosomes of the host cell they have infected.  If that viral DNA inserts into a gene, it can disrupt it and cause a mutation.  This can have dire consequences (see Folia Biologia 46 (2000): 226-32; Science 302 (2003): 415-9).  Fortunately this is not an intractable problem.  The conversion of adult cells into iPSCs only requires the transient expression of the inserted genes.  Secondly, scientists have created retroviruses that self-inactivate after their initial insertion (Journal of Virology 72 (1998): 8150-7; Virology 261, (1999).  One laboratory has also discovered a way to make iPSCs with a virus that does not insert into host cell chromosomes (Science 322 (2008): 945-9).  Other researchers have designed ingenious ways to move the necessary genes into adult cells without using viruses (Science 322 (2008): 949-53).  Both procedures avoid the dangers associated with the use of retroviruses.

A second concern involves the genes used to convert re-program adult cells into iPSCs.  One of these genes, c-Myc, is found in multiple copies in human and animal tumors.  Thus increasing the number of copies of the c-Myc gene might predispose such cells to form tumors (Recent Patents on Anticancer Drug Discovery 1 (2006): 305-26; Seminars in Cancer Biology 16 (2006): 318-30). Indeed, the increased ability of iPSCs made by Yamanaka to cause tumors in laboratory animals underscore this concern (Hepatology 46, no 3 (2009): 1049-9).  Several groups, however, have succeeded in making iPSCs from adult cells without the use of the c-Myc gene (Science 321 (2008): 699­-702; Nature Biotechnology 26 (2008): 101-6; Science 318 (2007): 1917–20), although the conversion is much less efficient.  Additionally, several groups have established that particular chemicals, in combination with the addition of a subset of the four genes originally used, can effectively transform particular cells into iPSCs (Cell Stem Cell 2 (2008): 525-8).   Thus the larger safety concerns facing iPSCs have been largely solved.

Finally, patient-specific iPSCs have been made in several labs, even though they have not been used in clinical trials to date.  Here is a short list of some of the diseases for which patient-specific iPSCs have been made:

Amylotrophic Lateral SclerosisScience 321 (2008): 1218­21.

Spinal Muscular AtrophyNature 457 (2009): 277­81.

Parkinson’ DiseaseCell 136, no. 5 (2009): 964­77.

Adenosine deaminase deficiency-related severe combined immunodeficiency – Cell 134, no. 5 (2008): 877­86.

Shwachman-Bodian-Diamond syndrome – Cell 134, no. 5 (2008): 877­86.

Gaucher disease – Cell 134, no. 5 (2008): 877­86.

Duchenne and Becker muscular dystrophy – Cell 134, no. 5 (2008): 877­86.

Huntington disease – Cell 134, no. 5 (2008): 877­86.

Juvenile-onset type 1 diabetes mellitus – Cell 134, no. 5 (2008): 877­86.

Down syndrome – Cell 134, no. 5 (2008): 877­86.

Lesch-Nyhan syndromeCell 134, no. 5 (2008): 877­86.

Thus iPSCs represent an exciting, embryo-free alternative to embryonic stem cells that provide essentially all of the opportunities for regenerative medicine without destroying embryos.