Getting Stem Cells to Engraft More Effectively – With A Little Help From My “Friends”


The old Beatles song, “With A Little Help from My Friends” begins:

What would you think if I sang out of tune
Would you stand up and walk out on me?
Lend me your ears and I’ll sing you a song
And I’ll try not to sing out of key
Oh I get by with a little help from my friends
Mm I get high with a little help from my friends
Mm going to try with a little help from my friends

For mesenchymal stem cells, a little help from circulating stem cells, that is, their “friends” can make all the difference.

Ruei-Zeng Lin, in the laboratory of Juan M. Melero-Martin at the Boston Children’s Hospital and Department of Surgery at Harvard Medical School, in Boston, Massachusetts, have made a profound discovery that was published in the Proceedings of the National Academy of Sciences USA. They have shown that cells called “endothelial colony-forming cells” or ECFCs that not only circulate throughout the bloodstream but also contribute to the formation of new blood vessels, can function as “nurse cells” that positively regulate the regenerative potential of human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) secrete a whole cocktail of healing molecules, but these cells also respond to several different molecules made by other cells, and ECFCs make some of these pro-MSC molecules.

In their experiment, Lin and others injected human MSCs isolated from white fat and bone marrow aspirates underneath the skin of immunodeficient mice in the presence or absence of ECFCs derived from human umbilical cord blood. The results were quite telling.

The engraftment of the MSCs (engraftment means the ability of the implanted stem cells to survive, differentiate and integrate into existing tissues) was regulated by a protein secreted by ECFCs called “platelet-derived growth factor BB” or PDGF-BB. When MSCs and ECFCs were transplanted together, the ECFCs significantly enhanced MSC engraftment. The MSCs not only survived better, showed much less cell death, but they also preserved the stem cell-character of the MSCs. THis is was established by the fact that when the implanted MSCs were removed and reimplanted into another mouse, these cells could repopulate secondary grafts. However, if MSCs were implanted without ECFCs, MSC engraftment was negligible. Also, if a drug called Tyrphostin AG1296 was used, MSCs engraftment was also negligible. Tyrphostin AG1296 inhibits the receptor for PDGF-BB and completely abrogates any EFCF-related enhancement of MSC function.  This shows that the enhancement of MSC engraftment by ECFCs is largely dependent on PDGF-BB-mediated signaling.

Strangely, transplanted MSCs that had been co-transplanted with ECFCs displayed fate-restricted differentiation in animals.  This simply means that the fat-based stem cells differentiated into fat and the bone marrow-derived MSCs differentiated into bone.  It seems that with the increased growth and stem cell function comes a more restricted differentiation program as well.  This could potentially prevent the phenomenon of “out-of-place” differentiation also known as heterotypic differentiation, which can cause the formation things like bone during fat transplantation or other such things.

These experiments show that blood-derived ECFCs can amplify the regenerative potential of MSCs via PDGF-BB – based signaling.  These data also suggest that the systematic use of ECFCs can improve MSC transplantation, and provides new insights into the therapeutic capabilities of ECFCs.  The authors add: “We foresee the use of ECFCs as a means to improve the outcome of MSC transplantation.”

This is a remarkable preclinical trial, but before it can work in humans, it must prove its efficacy and safety in human clinical trials and in other preclinical trials as well.

Adding One Gene to Cells can Regrow Hair, Cartilage, Bone and Soft Tissues


The reactivation of a gene called Lin28a, which is active in embryonic stem cells, can regrow hair and repair cartilage, bone, skin, and other soft tissues in mice.

This study comes from scientists at the Stem Cell Program at Boston Children’s Hospital who found that the Lin28a promotes tissue repair by enhancing metabolism in mitochondria, which are the energy-producing engines in cells. These data suggest that upregulation of common “housekeeping” functions might provide new ways to develop regenerative treatments.

George Q. Daley, the director of Boston Children’s Hospital Stem Cell Transplantation Program, said, “Efforts to improve wound healing and tissue repair have mostly failed, but altering metabolism provides a new strategy which we hope will prove successful.”

One of the first authors of this paper, Shyh-Chang Ng, added, “Most people would naturally think that growth factors are the major players in wound healing, but we found that the core metabolism of cells is rate-limiting in terms of tissue repair. The enhanced metabolic rate we saw when we reactivated Lin28a is typical of embryos during their rapid growth phase.”

Lin28a was first discovered in worms, but the Lin28a gene is found in all animals. It is abundantly expressed in embryonic stem cells and during early embryonic development. Stem cell scientists have even used Lin28a to help reprogram adult cells into induced pluripotent stem cells. Lin28a encodes an RNA-binding protein that regulates the translation of messenger RNAs into protein.

To express more of this protein in mice, Daley and his colleagues attached the Lin28a gene to a piece of DNA that would drive expression when the mice were fed the drug doxycycline. Ng and others noticed that one of the targets of Lin28a was a small RNA molecule called Let-7, which is known to promote aging and cell maturation. Let-7 is a member of a class of non-coding RNA molecules called micro-RNAs that bind to messenger RNAs and prevent their translation.  Let-7 is made as a larger precursor molecule that is processed to a smaller molecule that is functional.  LIN28 binds specifically to the primary and precursor forms of Let-7, and inhibits Let-7 processing.

Lin28a function

Ng said, “We were confident that Let-7 would be the mechanism, but there was something else involved.”

Let-28a is known to activate the translation of several different genes that play a role in basic energy metabolism (e.g., Pfkp, Pdha1, Idh3b, Sdha, Ndufb3, and Ndufb8), Activation of these genes enhances oxidative metabolism and promotes an embryonic bioenergetic state.

In their Lin28a transgenic mice, Daley, Ng and others noticed that Lin28a definitively enhanced the production of metabolic enzymes in mitochondria, and that these “revved up” the mitochondria so that they generated the energy needed to stimulate and grow new tissues.

PIIS0092867413012786.fx1.lrg

“We already know that accumulated defects in mitochondria can lead to aging in many cells and tissues,” said Ng. We are showing the converse: enhancement of mitochondrial metabolism can boost tissue repair and regeneration, recapturing the remarkable repair capacity of juvenile animals. ”

Further experiments showed that bypassing Lin28a and directly activating mitochondrial metabolism with small molecules had the same effect on wound healing. This suggests that pharmaceuticals might induce regeneration and enhance tissue repair.

“Since Lin28 itself is difficult to introduce into cells, the fact that we were able to activate mitochondrial metabolism pharmacologically gives us hope,” said Ng.

Lin28a did not cause universal regeneration of all tissues. Heart tissue, for example, was poorly aided by Lin28a. Also, Lin28a induced the regeneration of severed finger tips in newborn mice, but not in adult mice.

Nevertheless, Lin28a could be a key factor in constituting a kind of healing cocktail, in combination with other embryonic factors yet to be found.

Growing Skeletal Muscle in the Laboratory


Skeletal muscle – that type of voluntary muscle that allows movement – has proven difficult to grow in the laboratory. While particular cells can be differentiated into skeletal muscle cells, forming a coherent, structurally sound skeletal muscle is a tough nut to crack from a research perspective.

Another problem dogging muscle research is the difficulty growing new muscle in patients with muscle diseases such as muscular dystrophy or other types of disorders that weaken and degrade skeletal muscle.

Now research groups at the Boston Children’s Hospital Stem Cell Program have reported that they can boost the muscle mass and even reverse the disease of mice that suffer from a type of murine muscular dystrophy. To do this, this group use a combination of three different compounds that were identified in a rapid culture system.

This ingenious rapid culture system uses the cells of zebrafish (Danio rerio) embryos to screen for these muscle-inducing compounds. These single cells are placed into the well of a 96-well plate, and then treated with various compounds to determine if those chemical induce the muscle formation. To facilitate this process, the zebrafish embryo cells express a very special marker that consists of the myosin light polypeptide 2 gene fused to a red-colored protein called “cherry.” When cells become muscle, they express the myosin light polypeptide 2 gene at high levels. Therefore, any embryo cell that differentiates into muscle should glow a red color.

(A) myf5-GFP;mylz2-mCherry double-transgenic expression recapitulates expression of the endogenous genes. myf5-GFP is first detected at the 11-somite stage. mylz2-mCherry expression is not observed until 32 hpf. Scale bars represent 200 mm. (B) myf5-GFP;mylz2-mCherry embryos were dissociated at the oblong stage and cultured in zESC medium. Images were taken 48 hr after plating. Scale bars represent 250 mm.
(A) myf5-GFP;mylz2-mCherry double-transgenic expression recapitulates expression of the endogenous genes. myf5-GFP is first detected at the 11-somite
stage. mylz2-mCherry expression is not observed until 32 hpf. Scale bars represent 200 mm.
(B) myf5-GFP;mylz2-mCherry embryos were dissociated at the oblong stage and cultured in zESC medium. Images were taken 48 hr after plating. Scale bars
represent 250 mm.

Once a cocktail of muscle-inducing chemicals were identified in this assay, those same chemicals were used to treat induced pluripotent stem cells made from cells taken from patients with muscular dystrophy.  Those iPSCs were treated with the combination of chemicals identified in the zebrafish embryo screen as muscle inducing agents.

Zebrafish embryo culture system

The results were outstanding.  Leonard Zon from the Division of Hematology/Oncology, Children’s Hospital Boston and Dana-Farber Cancer Institute and his colleagues showed that a combination of basic Fibroblast Growth Factor, an  adenylyl cyclase activator called forskolin, and the GSK3β inhibitor BIO induced skeletal muscle differentiation in human induced pluripotent stem cells (iPSCs).  Furthermore, these muscle cells produced engraftable myogenic progenitors that contributed to muscle repair when implanted into mice with a rodent form of muscular dystrophy.

Representative hematoxylin and eosin staining (H&E) images and immunostaining on TA sections of preinjured NSG mice injected with 1 3 105 iPSCs at day 14 of differentiation. Muscles injected with BJ, 00409, or 05400 iPSC-derived cells stain positively for human d-Sarcoglycan protein (red). Fibers were counterstained with Laminin (green). No staining is observed in PBS-injected mice or when 00409 fibroblast cells were transplanted. Because the area of human cell engraftment could not be specifically distinguished on H&E stained sections, which must be processed differently from sections for immunostaining, the H&E images shown do not represent the same muscle region as that shown in immunofluorescence images. Scale bars represent 100 mm, n = 3 per sample.
Representative hematoxylin and eosin staining
(H&E) images and immunostaining on TA sections
of preinjured NSG mice injected with 1 3 105
iPSCs at day 14 of differentiation. Muscles injected
with BJ, 00409, or 05400 iPSC-derived cells
stain positively for human d-Sarcoglycan protein
(red). Fibers were counterstained with Laminin
(green). No staining is observed in PBS-injected
mice or when 00409 fibroblast cells were transplanted.
Because the area of human cell engraftment
could not be specifically distinguished on
H&E stained sections, which must be processed
differently from sections for immunostaining, the
H&E images shown do not represent the same
muscle region as that shown in immunofluorescence
images. Scale bars represent 100 mm, n = 3
per sample.

Zon hopes that clinical trials can being soon in order to translate these remarkable results into patients with muscle loss within the next several years.  Zon and his co-workers are also screening compounds to address other types of disorders beyond muscular dystrophy.

This paper represents the application of shear and utter genius.  However, there is one caveat.  The mice into which the muscles were injected were immunodeficient mice whose immune systems are unable to reject transplanted tissues.  In human patients with muscular dystrophy, an immune response against dystrophin, the defective protein, has been an enduring problem (for a review of this, see T. Okada and S. Takeda, Pharmaceuticals (Basel). 2013 Jun 27;6(7):813-836).  While there have been some technological developments that might circumvent this problem, transplanting large quantities of muscle cells might be beyond the pale.  Muscular dystrophy results from disruption of an important junction between the muscle and substratum to which the muscle is secured.  This connection is mediated by the “dystrophin-glycoprotein complex.”  Structural disruptions of this complex (shown below) lead to unanchored muscle that cannot contract properly, and eventually atrophies and degrades.

Dystrophin-glycoprotein complex. Molecular structure of the dystrophin-glycoprotein complex and related proteins superimposed on the sarcolemma and subsarcolemmal actin network (redrawn from Yoshida et al. [5], with modifications). cc, coiled-coil motif on dystrophin (Dys) and dystrobrevin (DB); SGC, sarcoglycan complex;SSPN, sarcospan; Syn, syntrophin; Cav3, caveolin-3; N and C, the N and C termini, respectively; G, G-domain of laminin; asterisk indicates the actin-binding site on the dystrophin rod domain; WW, WW domain.
Dystrophin-glycoprotein complex. Molecular structure of the dystrophin-glycoprotein complex and related proteins superimposed on the sarcolemma and subsarcolemmal actin network (redrawn from Yoshida et al. [5], with modifications). cc, coiled-coil motif on dystrophin (Dys) and dystrobrevin (DB); SGC, sarcoglycan complex;SSPN, sarcospan; Syn, syntrophin; Cav3, caveolin-3; N and C, the N and C termini, respectively; G, G-domain of laminin; asterisk indicates the actin-binding site on the dystrophin rod domain; WW, WW domain.
This is a remarkable advance, but until the host immune response issue is satisfactorily addressed, it will remain a problem.