Stem Cells that Control Skin and Hair Color


A research team at NYU Langone Medical Center has uncovered a pair of molecular signals that control the hair and skin color in mice and humans. Manipulation of these very signals may lead to therapies or even drugs to treat skin pigment disorders, such as vitiligo.

Vitiligo
Vitiligo

Vitiligo is somewhat disfiguring condition characterized by the loss of skin pigmentation, leaving a blotchy, white appearance. Finding ways to activate these two signaling pathways may provide clinicians with the means to mobilize the pigment-synthesizing stem cells that place pigment in skin structures and, potentially, repigment the pigment-bearing structures that were damaged in cases of vitiligo. Such treatment might also repigment grayed hair cells in older people, and even correct the discoloration that affects scars.

Workers in the laboratory of Mayumi Ito at the Ronald O. Perelman Department of Dermatology and the Department of Cell Biology showed that a skin-based stem cell population of pigment-producing cells, known as “melanocytes,” grow and regenerate in response to two molecular signals. The Endothelin receptor type B (EdnrB) protein is found on the surfaces of melanocytes. EdnrB signaling promotes the growth and differentiation of melanocyte stem cells (McSCs). Activation of EdnrB greatly enhances the regeneration of hair-based and epidermal-based melanocytes. However, EdnrB does act alone. Instead, the effect of EdnrB depends upon active Wnt signaling. This Wnt signal is initiated by the secretion of Wnt glycoproteins by the hair follicle cells.

This work was published in Cell Reports, April 2016 DOI: http://dx.doi.org/10.1016/j.celrep.2016.04.006.

Previous work on EdnrB has established that it plays a central role in blood vessel development. This work by Ito and his team is the first indication that pigment-producing melanocytes, which provide color to hair and skin, are controlled by this protein.

A lack of EdnrB signaling in mice caused premature graying of the hair. However, stimulating the EdnrB pathway resulted in a 15-fold increase in melanocyte stem cell pigment production, and by two months, the mice showed hyperpigmentation. In fact, wounded skin in these mice became pigmented upon healing.

Overexpression of Edn1 Promotes Upward Migration of McSCs and Generation of Epidermal Melanocytes following Wounding (A–D) Whole-mount image of X - gal - stained wound area of Dct-LacZ (control; A and B) and Tyr-CreER ; EdnrB fl/fl ; Dct-lacZ (C and D) at indicated days after re-epithelialization. (E–J) Double immunohistochemical staining of Dct and Ki67 in the bulge (E and H), upper hair follicle (F and I), and inter-follicular epidermis (G and J ) in control (E–G) and K14-rtTA ; TetO-Edn1-LacZ (Edn1; H–J) mice. (K–N) Whole - mount analyses of wound site (K and L) and de novo hair follicles (M and N) within wound site from control (K and M) and Edn1 mice (L and N) at 8 days after re-epithelialization. (O–R) Quantification of the number of Dct - LacZ+ cells in wound site (O), the percentage of Ki67+/Dct+ cells (P), the number of pigmented cells in wound site (Q), and the percentage of pigmented de novo hair (R), respectively. Dashed lines indicate periphery of wound site in (A) and (D) and boundary between epidermis and dermis in (E)–(J). Arrowheads show Dct - LacZ + cells in wound area in (A)–(D) and Ki67+/Dct+ cells (H)–(J). IFE, inter-follicular epidermis; UF, upper follicle. Data are presented as the mean ± SD. *p < 0.01; **p < 0.02; ***p < 0.05. The scale bar represents 1 mm in (A), 50 m m in (E), 200 m m in (K) and (L), and 100 m m in (M) and (N).
Overexpression of Edn1 Promotes Upward Migration of McSCs and Generation of Epidermal Melanocytes following Wounding (A–D) Whole-mount image of X-gal – stained wound area of
Dct-LacZ (control; A and B) and Tyr-CreER; EdnrB fl/fl; Dct-lacZ (C and D) at indicated days after
re-epithelialization. (E–J) Double immunohistochemical staining of Dct and Ki67 in the bulge (E and H), upper hair follicle (F and I), and inter-follicular epidermis (G and J) in control (E–G) and K14-rtTA;
TetO-Edn1-LacZ (Edn1; H–J) mice.  (K–N) Whole-mount analyses of wound site (K and L) and de novo hair follicles (M and N) within wound site from control (K and M) and Edn1 mice (L and N) at
8 days after re-epithelialization.  (O–R) Quantification of the number of Dct-LacZ+ cells in wound site (O), the percentage of Ki67+/Dct+ cells (P), the number of pigmented cells in wound site (Q),
and the percentage of pigmented de novo hair (R), respectively.  Dashed lines indicate periphery of wound site in (A) and (D) and boundary between epidermis and dermis in (E)–(J). Arrowheads show Dct-LacZ+ cells in wound area in (A)–(D) and Ki67+/Dct+ cells (H)–(J). IFE, inter-follicular epidermis; UF, upper follicle. Data are presented as the mean ± SD. *p < 0.01; **p < 0.02; ***p < 0.05.
The scale bar represents 1 mm in (A), 50 micrometer in (E), 200 micrometer in (K) and (L), and 100
micrometer in (M) and (N).

If the Wnt signaling pathway was blocked, stem cell growth and maturation sputtered and stalled and never got going, even when the EdnrB pathway was working properly. These mice had unpigmented fur (see E in figure below).

Loss of beta-catenin Function Suppresses Edn1-Mediated Effects on McSC Proliferation, Differentiation, and Upward Migration (A) Experimental scheme for treatment of Tyr-CreER ; b -catenin fl/fl ; K14-rtTA ; TetO-Edn1-LacZ ( b -cat cKO; Edn1 ) mice and control K14-rtTA ; TetO-Edn1- LacZ ( Edn1 ) mice. (B–E) Gross appearance of Edn1 (B and D) and b -cat cKO; Edn1 mice (C and E) at second (B and C) and third telogen (D and E). (F–K) Immunohistochemistry for indicated markers (F, G, I, and J) and bright- field image (H and K) of bulge/sHG region in skin sections from Edn1 mice (F–H) and b -cat cKO; Edn1 mice (I–K) at anagen II. (L–Q) Bright-field image (L–N) and Dct immunostaining of whole-mount wound site (O–Q) from Tyr-CreER ; b -catenin fl/fl ( b -cat cKO; L and O), Edn1 (M and P), and b -cat cKO; Edn1 mice (N and Q). (R and S) Quantification of the percentage of Dct+ cells positive for Ki67, Tyr, and pigmentation (R) and the number of Dct+ cells in wounded site (S). Dashed lines indicate border between hair follicle and dermis. Arrow- heads indicate double positive cells for indicated markers (F and G) and pigmented cells (H). Data are presented as the mean ± SD.*p<0.05;**p< 0.02; ***p < 0.001. The scale bar represents 1 cm in (B)–(E), 10 m min(F), and 200 m min(L). 1
Loss of beta-catenin Function Suppresses Edn1-Mediated Effects on McSC Proliferation, Differentiation, and Upward Migration
(A) Experimental scheme for treatment of
Tyr-CreER; beta-catenin fl/fl; K14-rtTA; TetO-Edn1-LacZ (beta-cat cKO; Edn1) mice and control K14-rtTA; TetO-Edn1-LacZ (Edn1) mice.
(B–E) Gross appearance of Edn1 (B and D) and
beta-cat cKO; Edn1 mice (C and E) at second (B and C) and third telogen (D and E). (F–K) Immunohistochemistry for indicated markers (F, G, I, and J) and bright-field image (H and K) of bulge/sHG region in skin sections from Edn1 mice (F–H) and beta-cat cKO; Edn1
mice (I–K) at anagen II. (L–Q) Bright-field image (L–N) and Dct immunostaining of whole-mount wound site (O–Q) from
Tyr-CreER; beta-catenin fl/fl (beta-cat cKO; L and O), Edn1 (M and P), and beta-cat cKO; Edn1 mice (N and Q). (R and S) Quantification of the percentage of Dct+ cells positive for Ki67, Tyr, and pigmentation (R) and the number of Dct+ cells in wounded site (S).
Dashed lines indicate border between hair follicle and dermis. Arrow-heads indicate double positive cells for indicated markers (F and G) and pigmented cells (H). Data are presented as the mean ± SD.*p<0.05;**p<
0.02; ***p < 0.001. The scale bar represents 1 cm in (B)–(E), 10 micrometers in (F), and 200 micrometer  in (L).

However, perhaps the most exciting finding for Ito and his colleagues was that Wnt-dependent, EdnrB signaling rescued the defects in melanocyte regeneration caused by loss of the Mc1R receptor. This is precisely the receptor that does not function properly in red-heads, which causes them to have red hair and very light skin that burns easily in the sun. These data suggest that Edn/EdnrB/Wnt signaling in McSCs can be used therapeutically to promote photoprotective-melanocyte regeneration in those patients with increased risk of skin cancers due to their very lightly colored skin.

Melanocyte Stem Cell Modeld

Umbilical Cord Blood Mesenchymal Stem Cells Relieve the Symptoms of Interstitial Cystitis by Activating the Wnt Pathway and EGF Receptor


Interstitial tissue refers to the tissue that lies between major structures in an organ. For example, the tissue between muscles is an example of interstitial tissue.

Interstitial cystitis, otherwise known as painful bladder syndrome is a chronic condition that causes bladder pressure, bladder pain and sometimes pelvic pain, ranging from mild discomfort to severe pain.

The bladder is a hollow, muscular organ that stores urine and expands until it is full, at which time it signals the brain that it is time to urinate, communicating through the pelvic nerves. This creates the urge to urinate for most people. In the case of interstitial cystitis, these signals get mixed up and you feel the need to urinate more often and with smaller volumes of urine than most people. Interstitial cystitis most often affects women and can have a long-lasting impact on quality of life. Unfortunately no treatment reliably eliminates interstitial cystitis, but medications and other therapies may offer relief. There is no sign of bacterial infection in the case of bacterial cystitis.

A new study evaluated the potential of umbilical cord blood-derived mesenchymal stem cells or (UCB-MSCs) to treat interstitial cystitis (IC). In this study, Dr. Miho Song and colleagues from the Asan Medical Center, Seoul, South Korea, established a rat model of IC in 10-weeks-old female Sprague-Dawley rats by instilling 0.1M HCl or PBS (sham). After 1-week, human UCB-MSCs (IC+MSCs) or PBS (IC) were directly injected into the submucosal layer of the bladder.

To clarify this part of the experiment, the urinary bladder is made of several distinct tissue layers: a) The innermost layer of the bladder is the mucosa layer that lines the hollow lumen. Unlike the mucosa of other hollow organs, the urinary bladder is lined with transitional epithelial tissue that is able to stretch significantly to accommodate large volumes of urine. The transitional epithelium also provides protection to the underlying tissues from acidic or alkaline urine; b) Surrounding the mucosal layer is the submucosa, a layer of connective tissue with blood vessels and nervous tissue that supports and controls the surrounding tissue layers; c) The visceral muscles of the muscularis layer surround the submucosa and provide the urinary bladder with its ability to expand and contract. The muscularis is commonly referred to as the detrusor muscle and contracts during urination to expel urine from the body. The muscularis also forms the internal urethral sphincter, a ring of muscle that surrounds the urethral opening and holds urine in the urinary bladder. During urination, the sphincter relaxes to allow urine to flow into the urethra.

Bladder histology

Now a single subcutaneous injection of human UCB-MSCs significantly attenuated the irregular and decreased voiding interval in the IC group. In addition, the denudation of the epithelium that is characteristic of IC and increased inflammatory responses, mast cell infiltration, neurofilament production, and angiogenesis observed in the IC bladders were prevented in the IC+MSC group. Therefore, the injected UBC-MSCs prevented the structural changes in the bladder associated with the pathology of IC.

How did these cells do this? Further examination showed that the injected UCB-MSCs successfully engrafted to the stromal and epithelial tissues of the bladder and activated the Wnt signaling cascade. In fact, if the Wnt activity of these infused cells was blocked, the positive effects of the UCB-MSCs were also partially blocked. Additionally, activation of the epidermal growth factor receptor (EGFR) also helped UCB-MSCs heal the bladder. If the activity of the EGF receptor was inhibited by small molecules, then the benefits of MSC therapy were also abrogated. Also if both the Wnt pathway and EGFR were inhibited, the therapeutic capacities of UCB-MSCs were completely wiped out.

These data show the therapeutic effects of MSC therapy against IC in an orthodox rat animal model. However, this study also elucidates the molecular mechanisms responsible for these therapeutic effects. Our findings not only provide the basis for clinical trials of MSC therapy to IC, but also advance our understanding of IC pathophysiology.

Growing Intestinal Stem Cells


Researchers from MIT and Brigham and Women’s Hospital in Boston, MA have discovered a protocol that allows them to grow unlimited quantities of intestinal stem cells. These intestinal stem cells can then be induced to differentiate into pure populations of various types of mature intestinal cells. Scientists can used these cultured intestinal cells to develop new drugs and treat gastrointestinal diseases, such as Crohn’s disease or ulcerative colitis.,

The small intestine has a small repository of adult stem cells that differentiate into mature adult cells that have specialized functions. Until recently, there was no good way to grow large numbers of these intestinal stem cells in culture. Intestinal stem cells, you see, only retain their immature characteristics when they are in contact with supportive cells known as Paneth cells.

paneth cells

In order to grow intestinal stem cells in culture, researchers from the laboratories of Robert Langer at the MIT Koch Institute for Integrative Cancer Research and Jeffrey Karp from the Harvard Medical School and Brigham and Women’s Hospital, determined the specific molecules that Paneth cells make that keep the intestinal stem cells in their immature state. Then they designed small molecules that mimic the Paneth cell-specific molecules. When Langer and Karp’s groups grew the intestinal stem cells in culture with those small molecules, the cells remained immature and grew robustly in culture.

Langer said, “This opens the door to doing all kinds of thing, ranging from someday engineering a new gut for patients with intestinal diseases to doing drug screening for safety and efficacy. It’s really the first time this has been done.”

The inner mucosal layer of the intestine has several vital functions: the absorption of nutrients, the secretion of mucus of create a barrier between our own cells and the bacteria and viruses and habitually inhabit our bowels, and alerting the immune system to the presence of potential disease-causing agents in the bowel.

The intestinal mucosa is organized into a collection of folds with small indentations called “intestinal crypts.”  At the bottom of each crypt is a small pool of intestinal stem cells that divide to routinely replace the specialized cells of the intestinal epithelium.  Because the cells of the intestinal epithelium show a high rate of turnover (they only last for about five days), these stem cells must constantly divide to replenish the intestine.

INTESTINES COMPARED

Once these intestinal stem cells divide, they can differentiate into any type of mature intestinal cell type.  Therefore, these intestinal stem cells provide a marvelous example of a “multipotent stem cell.”

Obtaining large quantities of intestinal stem cells could certainly help gastroenterologists  treat gastrointestinal diseases that damage the epithelial layer of the gut.  Fortunately, recent studies in laboratory animals have demonstrated that the delivery of intestinal stem cells can promote the healing of ulcers and regeneration of new tissue, which offers a new way to treat inflammatory bowel diseases like ulcerative colitis.

This, however, is only one of the many uses for cultured intestinal stem cells.  Researchers are literally salivating over the potential of studying things like goblet cells, which control the immune response to proteins in foods to which many people are allergic.  Alternatively, scientists would like to investigate the properties of enteroendocrine cells, which secrete hunger hormones and play a role in obesity.  I think you can see, that large numbers of intestinal stem cells could be a boon to gastrointestinal research.

Karp said, “If we had ways of performing high-throughput screens of large numbers of these very specific cell types, we could potentially identify new targets and develop completely new drugs for diseases ranging from inflammatory bowel disease to diabetes.”

The laboratory of Hans Clevers in 2007 identified a molecule that is specifically made by intestinal stem cells called Lgr5.  Clevers is a professor at the Hubrecht Institute in the Netherlands and he and his co-workers have just identified particular molecules that enable intestinal stem cells to grow in synthetic culture.  In culture, these small clusters of intestinal stem cells differentiate and form small sphere-like structures called “organoids,” because they consist of a ball of intestinal cells that have many of the same organizational properties of our own intestines, but are made in culture.

Clevers and his colleagues tried to properly define the molecules that bind Paneth cells and intestinal stem cell together.  The purpose of this was to mimic the Paneth cells in culture so that the intestinal stem cells would grow robustly in culture.  Clevers’ team discovered that Paneth cells use two signal transduction pathways (biochemical pathways that cells use to talk to each other) to coordinate their “conversations” with the adjacent stem cells.  These two signal transduction pathways are the Notch and Wnt pathways.

Fortunately, two molecules could be used to induce intestinal stem cell proliferation and prevent their differentiation: valproic acid and CHIR-99021.  When Clevers and others grew mouse intestinal stem cells in the presence of these two compounds, they found that large clusters of cells grew that consisted of 70-90 percent pure stem cells.  When they used inhibitors of the Notch and Wnt pathway, they could drive the cells to form particular types of mature intestinal cells.

“We used different combinations of inhibitors and activators to drive stem cells to differentiate into specific populations of mature cells,” said Xiaolei Yin, first author of this paper.  Yin and others were able to get this strategy to work with mouse stomach and colon cells, and that these small molecules also drove the proliferation of human intestinal stem cells.

Presently, Clevers’ laboratory is trying to engineering intestinal tissues for potential transplantation in human patients and for rapidly testing the effects of drugs on intestinal cells.

Ramesh Shivdasani from Harvard Medical School and Dana-Farber Cancer Institute would like to use these cells to investigate what gives stem cells their ability to self-renew and differentiate into other cell types.  “There are a lot of things we don’t know about stem cells,” said Shivdasani.  “Without access to large quantities of these cells, it’s very difficult to do any experiments.  This opens the door to a systematic, incisive, reliable way of interrogating intestinal stem cell biology.”

X. Yi, et al. “Niche-independent high-purity cultures of Lgr5 intestinal stem cells and their progeny.” Nature Methods 2013; DOI:10.1038/nmeth.2737.

A Molecular Switch that Causes Stem Cell Aging


A study from the Cincinnati Children’s Hospital Medical Center, in collaboration with the University of Ulm in Germany has discovered a molecular switch that causes the aging of blood stem cells. This same work suggests a therapeutic strategy to delay stem cell aging.

Hematopoietic stem cells (HSCs) reside in the bone marrow and make all the red and white blood cells that populate the bloodstream. Proper HSC function is absolutely vital to the ongoing production of different types of blood cells that allow the immune system to fight infections and organs to receive adequate quantities of oxygen.

Hartmut Geiger from the Cincinnati Children’s Hospital Medical Center and the University of Ulm was the senior researcher on this project. Dr. Geiger said, “Although there is a large amount of data showing that blood stem cell function declines during aging, the molecular processes that cause this remain largely unknown. This prevents rational approaches to attenuate stem cell aging. This study puts us significantly closer to that goal through novel findings that show a distinct switch in a molecular pathway is very critical to the aging process.”

The pathway to which Dr. Geiger referred is the Wnt signaling pathway, which plays a foundational role in animal development, cell-cell communication, tissue generation, and is also involved in the pathology of various diseases.

Crystal structure of XWnt8
Crystal structure of XWnt8

Analysis of mouse models and cultured HSCs showed that under normal conditions, Wnt signaling in HSCs occurred through the so-called “canonical” Wnt signaling pathway. The canonical Wnt signaling pathway utilizes the typical components of Wnt signaling that were first identified in the fruit fly and then isolated and characterized in vertebrates (shown below).

Canonical Wnt signaling

However, Wnt proteins can also signaling through other, distinct signal transduction pathways, and these types of pathways are collectively known as “noncanonical” Wnt signaling pathway. In aging HSCs, a switch from canonical Wnt signaling to noncanonical Wnt signaling marked the onset of HSC aging.  See below for one example of non-canonical Wnt signaling.

Non-canonical Wnt signaling

To test this observation, Geiger’s group overexpressed Wnt5 in HSCs (a Wnt protein known to induced signaling through noncanonical Wnt signaling pathways), and immediately, the HSCs began to show the signs of aging.

One of the targets of Wnt5 signaling is a protein called Cdc42, which influences the cytoskeleton of cells.  Therefore, Geiger and his crew asked if Cdc42 was activated in those HSCs that overexpressed Wnt5.  The answer to this question was a clear “yes.”  Then they treated cultured HSCs with a molecule that inhibited Cdc42 activity.  This treatment reversed the aging process in HSCs.

To test their hypothesis in a living animal, Geiger and others removed a copy of the Wnt5 gene from HSCs in laboratory mice.  Mice that lacked functional Wnt5 protein in HSCs, showed rejuvenation of the aged HSCs.  Mice that lacked both copies of the Wnt5 gene showed a delayed aging process in their HSCs.

Even though this study has definitely made an important contribution to understanding HSC aging, more work is needed before a therapeutic strategy is in place.