Stem Cell-Based Skin Graft for Severe Burns


Severe wounds are typically treated with full thickness skin grafts. Some new work by researchers from Michigan Tech and the First Affiliated Hospital of Sun Yat Sen University in Guangzhou, China might provide a way to use a patient’s own stem cells to make split thickness skin grafts (STSG). If this technique pans out, it would eliminate the needs for donors and could work well for large or complicated injury sites.

This work made new engineered tissues were able to capitalize on the body’s natural healing power. Dr. Feng Zhao at Michigan Tech and her Chinese colleagues used specially engineered skin that was “prevascularized, which is to say that Zhao and other designed it so that it could grow its own veins, capillaries and lymphatic channels.

This innovation is a very important one because on of the main reasons grafted tissues or implanted fabricated tissues fail to integrate into the recipient’s body is that the grafted tissue lacks proper vascular support. This leads to a condition called graft ischemia. Therefore, getting the skin to form its own vasculature is vital for the success of STSG.

STSG is a rather versatile procedure that can be used under unfavorable conditions, as in the case of patients who have a wound that has been infected, or in cases where the graft site possess less vasculature, where the chances of a full thickness skin graft successfully integrating would be rather low. Unfortunately, STSGs are more fragile than full thickness skin grafts and can contract significantly during the healing process.

In order to solve the problem of graft contraction and poor vascularization, Zhao and others grew sheets of human mesenchymal stem cells (MSCs) and mixed in with those MSCs, human umbilical cord vascular endothelial cells or HUVECs. HUVECs readily form blood vessels when induced, and growing mesenchymal stem cells tend to synthesize the right cocktail of factors to induce HUVECs to form blood vessels. Therefore this type of skin is truly poised to form its own vasculature and is rightly designated as “prevascularized” tissue.

Zhao and others tested their MSC/HUVEC sheets on the tails of mice that had lost some of their skin because of burns. The prevascularized MSC/HUVEC sheets significantly outperformed MSC-only sheets when it came to repairing the skin of these laboratory mice.

When implanted, the MSC/HUVEC sheets produced less contracted and puckered skin, lower amounts of inflammation, a thinner outer skin (epidermal) thickness along with more robust blood microcirculation in the skin tissue. And if that wasn’t enough, the MSC/HUVEC sheets also preserved skin-specific features like hair follicles and oil glands.

The success of the mixed MSC/HUVEC cell sheets was almost certainly due to the elevated levels of growth factors and small, signaling proteins called cytokines in the prevascularized stem cell sheets that stimulated significant healing in surrounding tissue. The greatest challenge regarding this method is that both STSG and the stem cell sheets are fragile and difficult to harvest.

An important next step in this research is to improve the mechanical properties of the cell sheets and devise new techniques to harvest these cells more easily.

According to Dr. Zhao: “The engineered stem cell sheet will overcome the limitation of current treatments for extensive and severe wounds, such as for acute burn injuries, and significantly improve the quality of life for patients suffering from burns.”

This paper can be found here: Lei Chen et al., “Pre-vascularization Enhances Therapeutic Effects of Human Mesenchymal Stem Cell Sheets in Full Thickness Skin Wound Re-pair,” Theranostics, October 2016 DOI: 10.7150/ thno.17031.

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Cultured Skin-Based Stem Cells Regenerate Hair Follicles and Sebaceous Glands


It has been over ten years since the development of cultured skin substitutes or CSSs. CSSs consist of cultured epidermis from the patients and dermal fibroblasts that can form a good epidermal layer. However, CSSs do not form hair follicles or sebaceous glands which makes for mighty dry skin. Therefore, it is highly preferable to make at skin substitute that can form such epidermal appendages.

Fortunately the skin possesses several types of stem cells for use in regenerative medicine. Epidermal stem cells (Epi-SCs) in the basal layer of the epidermis that constantly provide new cells to the epidermis (the uppermost layer of the skin). Unfortunately, adult Epi-SCs cannot form hair follicles, but they can if they are combined with embryonic or newborn dermal cells. Dermal papilla (DP) cells can induce hair follicles, but the availability of DP cells has greatly limited work with them. Adult dermal skin also contains multipotent SKPs or skin-derived precursors. Injection of SKPs underneath the skin of mice, leads to the induction of new hair follicles (Biernaskie et al., 2009, Cell Stem Cell 5:610-623). This suggests that SKPs might be applicable in a clinical setting to induce hair follicles in cultured skin substitutes.

A new paper by Yaojiong Wu and coworker from the Shenzhen Key Laboratory of Health Sciences and Technology, in collaboration with Edward E. Tredget from University College Dublin has successfully induced the growth of hair follicles and sebaceous glands in a cultured skin substitute. They combined cultured Epi-SCs and SKPs, from mice and humans, and embedded them into a hydrogel. These stem cell-embedded hydrogels were then implanted into immunodeficient mice with substantial skin wounds.

The results were remarkable. The implants able to induce the formation of new epidermis with hair follicles. Furthermore, when Epi-SCs and SKPs taken from human scalps were used in these experiments, they worked just as well as those taken from mice.

Hair neogenesis with cultured epidermal stem cells (Epi-SCs) and skin-derived precursors (SKPs). (A): Putative epidermal stem cells residing in the basal layer of neonatal mouse epidermis expressed CD49f (red) in immunofluorescence stain, and mature keratinocytes in the top layers of the epidermis expressed cytokeratin (CK)6 (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole. (B–E): Cultured Epi-SCs derived from neonatal mice (B) were positive for CD49f (C) and CK15 (D) in immunofluorescence stain; fluorescence-activated cell sorting analysis of the Epi-SCs indicated high levels of surface CD29 and CD49f (E). (F): The expression level of CD49f decreased progressively upon successive passages (P) in culture as determined by immunofluorescence analysis (in relation to the fluorescence intensity of P0 cells). Triple wells were used for each of the above experiments, and each experiment was repeated three times with similar results (∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001). (G): Hair genesis of cultured Epi-SCs in different passages. Cultured Epi-SCs derived from neonatal mice in different passages (P0 to P5) were implanted into excisional wounds in nude mice in combination with freshly isolated neonatal dermal cells (fresh D) in Matrigel; dermal cells alone or freshly isolated neonatal epidermal cells plus dermal cells (fresh E+D) were used as controls. Hair shafts generated 20 days posttransplant were counted (n = 6; ∗, p < .05; ∗∗∗, p < .001). (H–J): SKPs derived from neonatal mice in spheroid culture (H) expressed nestin, fibronectin (I), and BMP6 (J) in immunofluorescence analysis. (K): Hair genesis of SKPs in different passages. SKPs in P0 to P5 were implanted into excisional wounds in nude mice in combination with freshly isolated neonatal mouse epidermal cells (fresh E), and freshly isolated neonatal mouse epidermal cells alone or in combination with freshly isolated neonatal mouse dermal cells (fresh E+D) were used as controls. Twenty days posttransplant, hairs generated were counted (n = 6; ∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001). (L-N): Cultured Epi-SCs and SKPs in hair genesis. Combinations of cultured neonatal mouse Epi-SCs (P0 to P3) and SKPs (P0 to P3) were engrafted into excisional wounds in nude mice, and the number of hairs generated were counted 20 days posttransplant (n = 3, ∗, p < .05). (L). A representative image of hairs generated 20 days after a transplantation of P1 Epi-SCs and SKPs (M). Immunofluorescence analysis of the skin tissue with hair genesis showed densely populated hair follicles and sebaceous glands (N). Scale bars = 50 μm. Abbreviations: BM, basement membrane; BMP6, bone morphogenetic protein 6; CK, cytokeratin; DAPI, 4′,6-diamidino-2-phenylindole; Derm, dermis; Epi, epidermis; Epi-SC, epidermal stem cells; FITC, fluorescein isothiocyanate; fresh D, freshly isolated neonatal dermal cells; fresh D+E, freshly isolated neonatal epidermal cells plus dermal cells; HF, hair follicle; HS, hair shafts; P, passage; PE, phycoerythrin.
Hair neogenesis with cultured epidermal stem cells (Epi-SCs) and skin-derived precursors (SKPs). (A): Putative epidermal stem cells residing in the basal layer of neonatal mouse epidermis expressed CD49f (red) in immunofluorescence stain, and mature keratinocytes in the top layers of the epidermis expressed cytokeratin (CK)6 (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole. (B–E): Cultured Epi-SCs derived from neonatal mice (B) were positive for CD49f (C) and CK15 (D) in immunofluorescence stain; fluorescence-activated cell sorting analysis of the Epi-SCs indicated high levels of surface CD29 and CD49f (E). (F): The expression level of CD49f decreased progressively upon successive passages (P) in culture as determined by immunofluorescence analysis (in relation to the fluorescence intensity of P0 cells). Triple wells were used for each of the above experiments, and each experiment was repeated three times with similar results (∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001). (G): Hair genesis of cultured Epi-SCs in different passages. Cultured Epi-SCs derived from neonatal mice in different passages (P0 to P5) were implanted into excisional wounds in nude mice in combination with freshly isolated neonatal dermal cells (fresh D) in Matrigel; dermal cells alone or freshly isolated neonatal epidermal cells plus dermal cells (fresh E+D) were used as controls. Hair shafts generated 20 days posttransplant were counted (n = 6; ∗, p < .05; ∗∗∗, p < .001). (H–J): SKPs derived from neonatal mice in spheroid culture (H) expressed nestin, fibronectin (I), and BMP6 (J) in immunofluorescence analysis. (K): Hair genesis of SKPs in different passages. SKPs in P0 to P5 were implanted into excisional wounds in nude mice in combination with freshly isolated neonatal mouse epidermal cells (fresh E), and freshly isolated neonatal mouse epidermal cells alone or in combination with freshly isolated neonatal mouse dermal cells (fresh E+D) were used as controls. Twenty days posttransplant, hairs generated were counted (n = 6; ∗, p < .05; ∗∗, p < .01; ∗∗∗, p < .001). (L-N): Cultured Epi-SCs and SKPs in hair genesis. Combinations of cultured neonatal mouse Epi-SCs (P0 to P3) and SKPs (P0 to P3) were engrafted into excisional wounds in nude mice, and the number of hairs generated were counted 20 days posttransplant (n = 3, ∗, p < .05). (L). A representative image of hairs generated 20 days after a transplantation of P1 Epi-SCs and SKPs (M). Immunofluorescence analysis of the skin tissue with hair genesis showed densely populated hair follicles and sebaceous glands (N). Scale bars = 50 μm. Abbreviations: BM, basement membrane; BMP6, bone morphogenetic protein 6; CK, cytokeratin; DAPI, 4′,6-diamidino-2-phenylindole; Derm, dermis; Epi, epidermis; Epi-SC, epidermal stem cells; FITC, fluorescein isothiocyanate; fresh D, freshly isolated neonatal dermal cells; fresh D+E, freshly isolated neonatal epidermal cells plus dermal cells; HF, hair follicle; HS, hair shafts; P, passage; PE, phycoerythrin.

Additionally, when the ability of Epi-SCs to differentiate into sebaceous glands was examined, Wu and others showed that Epi-SCs can form the precursors to sebaceous glands, sebocytes. Additionally, the oils secreted by these Epi-SC-derived sebocytes were chemically similar to sebaceous glands from native skin.

Thus, a combination of Epi-SCs and SKPs from human or mouse skin were sufficient to generate newly formed hair follicles and functional sebaceous glands. These results provide knowledge that is potentially transferable to clinical applications for regenerating damaged skin.

Two Proteins Safeguard Skin Stem Cell Function


Ever scraped your knees or elbows? It’s a good thing that they didn’t stay that way, since human skin readily renews, heals wounds, and regenerates the hair that covers it thanks to a resident population of stem cells. These cells continually produce new ones. Depending on someone’s age, the complete skin is renewed every 10-30 days.

A new study led by Salvador Aznar Benitah (Institute for Research in Biomedicine, Barcelona, Spain) has identified two proteins that are integral to the conservation of skin stem cells. Without these proteins these skin-based skin cells are lost.

The proteins identified, Dnmt3a and Dnmt3b, trigger the first step of the genetic program that leads to stem cell renewal and regeneration of the skin. “Without them (i.e. Dnmt3a & Dnmt3b), this program is not activated and the stem cells collapse and disappear from the tissue,” said Benitah.

Dnmt3a & 3b are enzymes that attach methyl groups (-CH3) to the cytosines in DNA molecules.  The full name of these enzymes, DNA (cytosine-5)-methyltransferase 3A, catalyze the transfer of methyl groups to specific CpG structures in DNA.  This process is known as “DNA methylation.”  These particular DNA methyltransferases participate in de novo DNA methylation.  They must be distinguished from so-called “maintenance DNA methylation,” which ensures the fidelity of replication of inherited epigenetic patterns.

Epigenetics refers to cellular and physiological trait variations that result from external or environmental factors that switch genes on and off and affect how cells express genes, but do not involve changes in nucleotide sequences, but in chemical modifications to DNA or higher-order structures of DNA.

DNMT3A forms part of the family of DNA methyltransferase enzymes that includes DNMT1, DNMT3A and DNMT3B.  While de novo DNA methylation modifies the information passed on by parents to their progeny, it enables key epigenetic modifications essential for processes such as cellular differentiation and embryonic development, transcriptional regulation, heterochromatin formation, X-inactivation, imprinting and genome stability.

Lorenzo Rinaldi, a graduate student in Benitah’s laboratory who was also the first author of this study, has mapped the regions of the genome that houses the genes that encodes these two proteins. Rinaldi and others have shown that Dnmt3a & 3b affect gene expression by methylating “gene enhancers” and “superenhancers.” Gene enhancers and superenhancers are sequences that tend to be far away from genes but still have the ability to increase the speed of gene expression up to 200-fold.

“It was surprising to see that two proteins that have always been associated with gene repression through DNA methylation are activated in the most transcriptionally active regions of stem cells. We had never observed this activity because we were unable to study the global distribution of Dnmt3a and Dnmt3b at the genomic level. Thanks to advances in sequencing techniques, more researchers are observing the very mechanism that we have described,” said Rinaldi.

Of the 12,000 gene enhancers in the genome, about 300 are superenhancers related to stem cell activity. Dnmt3a & 3b activate expression of the approximately 1,000 genes that are required for the self-renewing capacity of stem cells. By methylating the superenhancer, these proteins trigger the first step of the machinery that leads to the amplified expression of these essential genes for the stem cell.  Dnmt3a and Dnmt3b clearly associate with the most active enhancers in human epidermal SCs.

Graphical_Done

The expression of Dnmt3a & 3b is also altered in cancer cells. Cancer cells tend to show altered DNA methylation and altered gene enhancers that affect gene expression. The mass sequencing of tumor cell genomes has provided these observations. Dnmt3a and Dnmt3b activities are altered in many types of tumor, including leukemias, and cancers of the lung and the colon.

“Each of these three components is associated with the development of various kinds of cancer. Given that these proteins activate gene expression enhancers through DNA methylation, we believe that it would be of interest to study them in cancer cells in order to determine whether they participate in tumor development,” said Benitah.

This work appeared in: Lorenzo Rinaldi et al., “Dnmt3a and Dnmt3b Associate with Enhancers to Regulate Human Epidermal Stem Cell Homeostasis,” Cell Stem Cell, July 2016 DOI: 0.1016/j.stem.2016.06.020.

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

Plant Polyphenol May Help Improve Wound Healing By Activating Mesenchymal Stem Cells


Akito Maeda and his coworkers from Osaka University in Osaka, Japan have discovered that a plant-based polyphenol promotes the migration of mesenchymal stem cells (MSCs) in blood circulation. This same plant polyphenol also causes MSCs to accumulate in damaged tissues and improve wound healing.

This compound, cinnamtannin B-1, might be a candidate drug for stem cell treatments for cutaneous disorders associated with particular diseases and lesions.

Cinnamtannin B-1
Cinnamtannin B-1

Cinnamtannin B-1, a flavonoid, seems to activate membrane-bound enzymes; specifically the Phosphatidylinositol-3-kinase enzyme, which is an integral enzyme in the phosphoinositol signal transduction pathway, which culminates in the mobilization of intracellular calcium stores and profoundly alters cell behavior and function.

Phosphoinositol pathway signaling

Flow cytometry analysis of mouse blood established that administration of cinnamtannin B-1 increased the release of MSCs from bone marrow. Laboratory experiments with cultured MSCs showed that cinnamtannin B-1 treatment activated MSC migration and recruitment to wounds. This seems to suggest that the enhanced healing caused by cinnamtannin B-1 treatment is due to enhanced MSC migration and homing to damaged tissues.

Imaging analysis of whole animals that had MSCs that expressed the firefly luciferase enzyme showed that cinnamtannin B-1 treatment increased the homing of MSCs to wounds and accelerated healing in a diabetic mouse model.

When Maeda and his colleagues treated MSCs with small molecules that inhibited phosphatidylinositol-3-kinase, those cells no longer responded to cinnamtannin B-1, which confirms the role of the phosphoinositol signal transduction pathway in cinnamtannin B-1 activation of MSCs.

Thus, cinnamtannin B-1 promotes MSC migration in culture and accelerates wound healing in mice. In addition, cinnamtannin B-1-induced migration of MSCs seems to be mediated by specific signaling pathways.

RepliCel Skin Rejuvenation and Tendon Repair Trials With Hair Follicle Stem Cells Underway


RepliCel Life Sciences has enrolled subjects for their skin rejuvenation and tendon repair trial.  The primary goal of these trials is to determine the safety of their cell therapeutic products.

NBDS-Cells-Follicle

In the first trial will test a product called RCS-01, which consists of cells derived from non-bulbar dermal sheath (NBDS) cells, which are taken from the outer regions of hair follicles.  NBDS cells express type 1 collagen, a protein that is steadily degraded in aged skin (hence the formation of wrinkles).  Therefore, RepliCel is confident that RCS-01 injections underneath the skin has the potential to rejuvenate aged or damaged skin.  The trial will examine male and female subjects, between 50-65 years old, and will address the inherent deficit of active fibroblasts required for the production of type 1 collagen, elastin and other critical extracellular dermal matrix components found in youthful skin. The trial will be conducted at the IUF Leibniz-Institut für umweltmedizinische Forschung GmbH in Dusseldorf, Germany.  Originally, RepliCel wished to enrolled 15 men and 15 women, but the large number of female subjects and paucity of men persuaded the company to move forward with the trial despite only enrolling a few men and all the projected women.

The second trial will test the safety and efficacy of RCT-01 in the repair of damaged Achilles tendons.  RCT-01 also consists of NBDS cells and this trial is a phase 1/2 clinical trial that examines the ability of NBDS cells to treat chronic tendinosis caused by acute and chronic tensile overuse.  This trial will take place at the University of British Columbia in Vancouver, BC, and will only treat 10 subjects.  Even though RepliCell wishes to originally test 28 participants, the company shorted the trial in order to have safety data by the end of 2016.

Darrell Panich, RepliCel Vice President of Clinical Affairs, said that the company had a late start on its trials, and therefore truncated the recruitment process in order to have safety data for analysis by the end of 2016.  Despite the small size of these trials (and they are small), the company is hopeful that their safety data will provide the impetus for moving forward with larger phase 2 trials.

Panich said, “We have adjusted our plans for the RCT-01 clinical trial in part because it started later in 2015 and enrolled slower than originally anticipated. While the trial did not meet projected enrollment targets, we are confident the safety and preliminary efficacy data obtained by year-end will provide a signal of the product’s potential to regenerate chronically injured tendon that has failed to respond to other treatments. This will allow our teams to effectively plan larger phase 2 trials in 2017 which are powered to be statistically significant for clinical efficacy (evidence the product works as intended).”

“Future trials involving products from our non-bulbar dermal sheath (NBDS) platform will be designed to investigate the efficacy of these products at different dose levels and treatment frequencies while continuing to collect other data that will be used to support eventual RCS-01 and RCT-01 marketing applications by our commercial partners.”

“The delivery of clinical data when promised is important to management”, said R. Lee Buckler, President & CEO, RepliCel Life Sciences Inc. “We have made critical decisions to keep our commitment to the financial community and we believe the data from these trials will facilitate us closing a licensing and co-development deal on one or both of these products similar to the kind we have in place with Shiseido Company for our RCH-01 product,” he added.

RepliCel is confident that their NBDS fibroblast platform will address numerous indications where impaired tissue healing has been stalled due to a paucity of active fibroblasts, which are required for tissue remodeling and repair.  NBDS fibroblasts, isolated from the hair follicles of healthy individuals, are a rich source of fibroblasts and are unique in their ability to express high levels of type 1 collagen and elastin to push-start the healing process.

RepliCel is also developing products from this same platform to address larger commercial markets in the areas of musculoskeletal and skin-related conditions.

A Stem Cell Treatment for Hair Loss


Male and female pattern baldness involves the receding of the hair-line, hair loss and the thinning the hair covering on the scalp. Hair loss is also called alopecia and is a common problem among the elderly, those afflicted with certain diseases of the scalp, or those who take certain medicines. Hair life is not the end of life, but it can change someone’s appearance, affect their self-image, and affect someone’s emotional state. People with hair loss can use topical monoxidil (Rogaine), take an oral drug called finasteride (Propecia), or undergo a hair transplant. The drugs, however, must be used constantly or they stop working and hair transplants are horribly expensive.

Can stem cell-based treatment restore hair after it has been lost? Fortunately, a stem cell population called dermal papilla cells (DPCs), which are a type of mesenchymal stem cell population in hair follicles, have been identified and even characterized to some extent. DPCs are responsible for the formation of hair-follicles and play a very role in the process of hair cycling in which the hair shaft grows, is shed, is reestablished, and grows again. DPC might be useful for treating alopecia, but they do not survive when cultured outside the body, and this limitation has limited developing stem cell-based treatments for hair loss.

Now collaboration between two scientific research teams from Canada and China have resulted in a new way to use stem cells to treat hair loss.

A research team from the Nanfang Hospital of Southern Medical University, China, led by Zhi-Qi Hu and a Canadian team from the University of Manitoba, Canada, led by Malcolm Xing have designed a three-layered tunic that feeds and protects the DPCs and allows them to grow outside the body.

Xing describes this nutritive tunic as a “nutritious nano-clothing,” made of gelatin and alginate. These molecules can self-assemble and Hu and Xing and their coworkers encapsulated the cells within an inner layer of gelatin, a middle layer of alginate loaded with fibroblast growth factor-2, and an outer layer of gelatin. They call this method of encapsulation “layer-by-layer (LBL) nano-coating.”  This gelatin/alginate coating creates a protective microenvironment for cultured DPC and provides them with a significant source of a growth factor called fibroblast growth factor-2 (FGF2), which enhances proliferation of the DPCs and induces hair cell fates. The use of these three-layered tunics keeps the inductive signals close to the DPCs and circumvents the difficulties encountered in regenerating new hair follicles on bald skin.

DPC - hair follicle regeneration

When the Xing and Hu teams implanted these encased DPCs into the skin of nude mice, the implanted encapsulated cells generated the growth of abundant hair. The hair produced by these cells also was rooted in hair follicles that were normal in their appearance and function. The coating improvised by these teams greatly augmented the therapeutic capabilities of the DPCs by recapitulating the niche in which these cells are normally found. This stem cell niche induces the cells to secrete the native extracellular matrix that typically surrounds the cells and release the growth factors that keep the cells growing and in the proper stage of the cell cycle.

According to Xing, the most difficult part of this research project was “optimizing the concentrations of the coated polymers and manufacturing conditions to make the cells happy and healthy.”

Regenerative medicine researcher Oommen Varghese, from Uppsala University in Sweden, who was not involved in this work, said, “This is fascinating science that has enormous potential for clinical translational of stem cell based regenerative medicine. Such a coating could also protect cells from innate immunity, thereby improving the in vivo survivability. This is a major challenge in stem cell based translational research.”

Xing and his collaborators and colleagues would like to transform this technique from a laboratory bench to a clinical application that can be tested in human clinical trials.