Induction of Skin Allograft Chimerism by Pharmacological Mobilization of Endogenous Bone Marrow-Derived Stem Cells

Abstract

Skin substitutes including allografts remain a standard therapeutic approach to promote healing of both acute and chronic large wounds. However, none have resulted in the regrowth of lost and damaged tissues and scarless wound healing. Here, we demonstrate skin allograft chimerism and repair through the mobilization of endogenous bone marrow-derived stem and immune cells in rats and swine. We show that pharmacological mobilization of bone marrow stem cells and immune cells into the circulation promotes host repopulation of skin allografts and restoration of the skin’s normal architecture without scarring and minimal contracture. When skin allografts from DA rats are transplanted into GFP transgenic Lewis recipients with a combination of AMD3100 and low-dose FK506 (AF) therapy, host-derived GFP-positive cells repopulate and/or regenerate cellular components of skin grafts including epidermis and hair follicles and the grafts become donor-host chimeric skin. Using AF combination therapy, burn wounds with skin allografts were healed by newly regenerated chimeric skin with epidermal appendages and pigmentation and without contracture in swine.

INTRODUCTION

Treatment of large full-thickness burns is a major challenge due to limitations of autogenous skin, problems with wound infection, severe metabolic stress, and other associated complications. The prolonged time to healing results in susceptibility to infection, prolonged pain, and long hospitalization. Human deceased donor skin allografts can provide skin to cover severe burns, but they are eventually rejected, requiring reoperations. While conventional treatment eventually provides survival from severe burns, it leaves scarring, disfiguration, loss of critical skin functions, and vulnerability to late cosmetic, psychological, and physical defects.^1–3 In response to this challenge, we tested a novel pharmacological therapy that mobilizes bone marrow stem cells into the circulation and assimilates them into the skin allograft.

The new pharmacological therapy that we have engineered is injected subcutaneously and acts systemically by mobilizing bone marrow-derived stem and immunoregulatory cells and incorporates them into the wound using a combination of two drugs (AMD3100 = A and FK506 = F). It enables long-term liver⁴ and kidney^5,6 allograft survival with freedom from immunosuppression through host repopulation from only short-term treatment,^4–7 but in addition, we discovered that it also produces marked improvement of skin wound healing.^8,9 AMD3100 is a CXCR4 antagonist, originally an anti-HIV medicine but is now used for the mobilization of stem cells in stem cell donors. Stem cells and a multitude of immune cells are anchored in the bone marrow niche through a strong interaction of stromal derived factor-1 (SDF-1) and CXCR4. AMD3100 disrupts this interaction thereby releasing these cells into the peripheral circulation. FK506 is an immunosuppressive drug widely used at higher doses in solid organ transplantation to prevent organ rejection. We have found a potent synergism between AMD3100 and low-dose FK506 (one-tenth of effective dosage to prevent rejection) in the mobilization and recruitment of stem cells and immunoregulatory cells.^4–6,8,9 AMD3100 treatment results in “pushing” stem cells from the bone marrow, while low-dose FK506 promotes “pulling” them into the injury sites via activation of M2 macrophages that produce SDF-1, which binds to CXCR4.^4–12 We hypothesized that in situ skin chimerism can be realized by host repopulation of skin allografts through pharmacological mobilization and local incorporation of endogenous host bone marrow stem cells with a mechanism similar to host repopulation of liver⁴ and kidney allografts.^5,6 This study consists of two separate experiments in two species. First, the goal was to demonstrate that AF combination therapy is able to prolong skin allograft survival by host repopulation of skin epithelium and skin appendages in the absence of burn-induced immunosuppression in a rat model of fully major histocompatibility complex-mismatched skin transplantation. Subsequently, a clinically relevant burn model in fully swine leukocyte antigen mismatch (SLA) minipigs was used to demonstrate AF’s ability to reproduce the findings in the rat model and to induce skin repair and regeneration after burns and skin transplantation.

MATERIALS AND METHODS

Animals

Male and female Lewis (RT1¹) and Dark Agouti (RT1A^a) weighing between 250 and 350 g rats were purchased from Charles River (Wilmington, MA) and Taconic Biosciences (Germantown, NY). Lewis full-body GFP+ rats were acquired from the Rat Recourse & Research Center (Columbia, MO). SLA-identified MGH-minipigs were acquired from the Massachusetts General Hospital. Animals were housed and cared for according to NIH guidelines in a pathogen-free facility. All experimental procedures were approved by the institutional animal care and use committee.

Skin transplantation

Full-thickness grafts (3.0 × 3.5 cm) were harvested from the dorsum of Dark Agouti (DA) rats and sutured in an interrupted fashion on the dorsum of Lewis (LEW) recipients using 3-0 silk sutures. Nonadherent bacteriostatic Xeroform sterile gauze was used as wound dressing and secured with cohesive flexible bandages (Thermo Fisher Scientific, Waltham, MA) using 3-0 silk sutures (Supplemental Figure 1). Skin grafts were assessed for rejection every week and bandages with Xeroform were renewed to keep the graft protected from the animal damaging it by scratching. Skin graft rejection was defined as over 80% necrosis of the graft. In case of rejection, bandages were permanently removed. Pictures were taken weekly to demonstrate the dynamic changes of skin graft rejection or chimerism.

Treatment protocol

Rats were randomly divided into two groups. AF-treated animals received 1.0 mg/kg AMD3100 (Plerixafor injection) and 0.1 mg/kg FK506 (Prograf, Astellas) fresh diluted in 0.9% sterile saline subcutaneously every other day for 8 weeks. Control animals received a similar volume of sterile saline.

Bone marrow transplantation

Adult wild-type LEW rats were lethally irradiated with a single dose of 12 Gy by a Gammacell 40 (Nordion, Ottawa, Canada). Adult GFP+ LEW rats were killed and bone marrow cells were collected by flushing the femurs and tibias with sterile Hanks’ Balanced Salt Solution (HBSS, Merck, St. Louis, MO) complemented with 2% fetal bovine serum (Merck, St. Louis, MO). A single cell suspension of 2–4 × 10⁸ GFP+ bone marrow cells was injected intravenously to wild-type LEW rats 24 h after lethal irradiation. After allowing sufficient time for full bone marrow reconstitution by GFP+ cells (8–10 weeks), rats underwent a skin transplant as described previously. Before the skin transplant, tail blood was drawn and red blood cells were lysed. PBMCs were analyzed for their GFP+ expression by FACS analysis.¹³

Swine burn model

A female pig served as a fully SLA mismatched skin donor for male recipients. Burns were created in temporarily anesthetized pigs by pressing a hot steaming iron at 204 °C for 40 s onto the skin of the lateral side of the thorax. Afterward pigs were returned to their cages with optimal pain management. Recipient pigs underwent necrectomy of burnt skin creating an optimal wound bed for the skin graft 48 h after burns to mimic the clinical situation. Split skin grafts were harvested with a dermatome set and placed in cold saline 4C until grafting. Skin grafts were placed on necrectomized healthy subcutaneous tissue and stapled to the recipient. The skin graft was covered with xeroform sterile gauze and pressurized with sterile gauze and 1-0 prolene sutures. To further protect the grafts, pigs were dressed with a protective cotton jacket and returned to modified protective cages. Pigs were weekly sedated for graft visualization by photography and dressing changes until full rejection (>80% graft loss) had occurred. Animals that lost their protective pressurized grafting or wound manipulation during the first 4 weeks were excluded from the study.

Flow cytometry

Freshly heparinized blood was obtained 3 h after injecting rats with AF or saline. After diluting the whole blood in a 1:1 ratio with PBS, peripheral blood mononuclear cells were obtained by gradient centrifugation with Ficoll-Hypaque (1.077 g/L) (Sigma-Aldrich, St. Louis, MO). Before staining, a single cell suspension (1.0 × 10⁶ cells) was incubated with fixable viability dye eFluor 780 (Thermofisher, Waltham, MA) for 30 min on ice. Cells were fixed and permeated with Intracellular Fixation & Permeabilization Buffer Set (eBioscience) according to the manufacturer’s instructions. Subsequently, cells were first blocked with anti-rat CD32 (D34-485) for 30 min and stained with fluorochrome-labeled antibodies against CD45 (OX-1), CD3 (1F4), αßTCR (R73), CD4 (OX-45), CD8 (OX-8), Foxp3 (FJK-16S), CD45RA (OX-33), Thy1 (OX-7) from BD (San Jose, CA) and CD133 (PROM1, Novus Biologicals, Centennial, CO) diluted in brilliant stain buffer for 30 min. Data were acquired using a BD FACSCelesta (BD, San Jose, CA) and analyzed with DIVA software (BD, San Jose, CA).

FK506 measurement

Five rats were injected with 0.1 mg/kg FK506 (Prograf, Astellas, Japan) subcutaneously (sc), and five rats with 0.5 mg/kg FK506 sc. After 12 h, rats were killed and whole blood was collected in EDTA collection tubes (BD) and sent to measure FK506 trough levels to the Johns Hopkins department of pathology. The samples were measured for tacrolimus levels by liquid chromatography and tandem mass spectrometry (LC/MS/MS) technique with a detection range between 2.0 and 40.0 ng/mL.

Histopathological analysis

All skin tissues were fixed in 10% formalin, embedded in paraffin blocks, and cut in 5 µm sections. The sections were stained with hematoxylin and eosin (H&E) to assess histology and morphology. Paraffin sections were deparaffinized and rehydrated for immunohistochemistry staining. Nonspecific antibody binding was blocked with 10% normal goat serum (MilliporeSigma) for 30 min and subsequently incubated with the following primary antibodies at 4 °C overnight: anti-CD133 (Novus Biologicals), anti-GFP (Abcam, Cambridge, MA), anti-FOXP3+ (Cell Signaling, Danvers, MA), anti-OCT4 (Santa Cruz Biotechnology, Dallas, TX). After overnight incubation, endogenous peroxidase and alkaline phosphatase were blocked with dual endogenous enzyme-blocking reagent (Dako) for 10 min at room temperature. Sections were then incubated with secondary anti-mouse or anti-rabbit IgG horseradish peroxidase (Dako) for 30 min at room temperature. Diaminobenzidine tetrahydrochloride and blue alkaline phosphatase (Vector Laboratories) were used as the chromogen and hematoxylin was used for counterstaining.

RESULTS

Full-thickness donor skin grafts (black) from DA rats were used with Lewis rats (white) as recipients. To keep the graft moisturized and protected from the animal damaging it by scratching, nonadherent bacteriostatic Xeroform sterile gauze was used as a wound dressing and secured with cohesive flexible bandages using 3-0 silk sutures (Supplementary Figure 1). Recipient rats were randomly divided into two groups (n = 6/group) following wounding and skin transplantation and they received AF combination therapy (AMD3100 1 mg/kg and FK506 0.1 mg/kg) or saline (1 mL/kg) by subcutaneous injection every other day for 10 weeks. In a separate study, we have confirmed that AF combination therapy mobilized stem cells and immunoregulatory cells into peripheral blood (Supplementary Figures 2 and 3) and trough levels of FK506 were no more than 2.8 ng/mL (Supplementary Figure 4). Skin allografts survived in all recipient rats on day 7 following transplantation except in rats with surgical failures (skin grafts that had not been taken on day 7 due to losing the bandage that protects the skin graft). Skin allografts on saline controls were rejected (desiccated and shrunken) at 2 weeks, and were completely lost with resultant wounds at 3 weeks (Figure 1A, upper panels). Skin wounds were completely healed with scars at 6–8 weeks. But in contrast, skin allografts in the AF test group remained soft without shrinking at 2 weeks, and the outer epidermal layer became loose and could be easily removed by forceps at 3 weeks (Figure 1A, lower panels). Interestingly, in these test group wounds, the epidermal layer beneath (black) was covered by newly generated epithelium (white). Except for the easily removable superficial epidermal layers, the skin allografts survived and healed without a scar at 6 weeks, and white hairs (recipient phenotype) appeared in the skin allograft although some black hairs (indicating chimerism for donor phenotype) remained (Figure 1A, lower and right panels) at 8 weeks following transplantation. At 10 weeks posttransplantation (Figure 1B), 6 recipients were killed and their skin was studied histologically. Scars were identified macroscopically in the wound areas of saline-treated recipients and H&E staining demonstrated the presence of scar tissue in the center of healed skins (Figure 1B, upper panels). In contrast, in the test group, H&E staining demonstrated normal skin with hair follicles in the center of skin allografts (Figure 1B, lower panels). Interestingly, hair follicles containing either black or white hair appeared in the histological sections (Figure 1B, lower and right panels) indicating the formation of chimeric donor (black) and the recipient (white) skin.

Figure 1.

AF Combination Therapy Prolonged Skin Allograft Survival and Induces Chimeric Skin in Rats. Full-Thickness Donor Skin Grafts (Black) From DA Rats Were Used With Lewis rats (White) as Recipients. (A) Representative Photographs of Skin Allografts in Rats Following Skin Transplantation. Recipient Rats Treated With Saline (Upper Panels) or AF Combination. Skin Allografts Were Completely Lost and Results in Wounds at 3 Weeks and Healed With Scars at 8 Weeks (Upper Panels), While Skin Allografts Remained Intact (Except the Epidermal Layer) in AF-Treated Rats and Both White and Black Hairs Appeared in the Skin Allograft at 8 Weeks (Right Lower Panel). (B) H&E Staining of Skin Allografts at 10 Weeks Following Transplantation. Scars Were Identified Microscopically in the Wound Areas of Saline-treated Recipients (Upper Panels), While Normal Skin Structure With Hair Follicles Appeared in the Center of Skin Allografts (Lower Panels)

To determine if skin allografts were repopulated by host-derived cells, skin grafts from DA rats were transplanted into Green Fluorescent Protein (GFP) transgenic Lewis recipients. Immunohistochemistry staining demonstrated that the epidermis, hair follicles, sweat glands, and sebaceous glands were GFP positive in the skin from GFP transgenic Lewis rats, while none of the components in the skin from DA rats were positive for GFP expression (Figure 3A). Transplanted rats were treated with AF combination or placebo (saline) s.c. every other day. Three recipients in each group were killed earlier, at 25 days posttransplantation, and skin allograft survival was confirmed by H&E staining. Macroscopic pictures showed open wounds in saline-treated control rats after allograft rejection (Figure 2A), and H&E staining demonstrated loss of skin allograft and skin wounds with inflammation. Immunohistochemistry staining showed GFP-positive epidermis and GFP/CD133 double-positive hair follicles in the intact skin (GFP Lewis recipient), while there was no GFP-positive epidermis or GFP/CD133 double-positive hair follicles in the control wound areas, although some GFP-positive cells appeared. In contrast, skin allografts remained soft without shrinking in AF combination-treated rats at 25 days (Figure 2B). Biopsies from different areas of these test skin allografts (H&E staining) showed newly generated thin epithelium beneath the epidermal layer of skin allograft and the structure of the skin remained intact despite mild/moderate inflammation. Immunohistochemistry staining showed GFP-positive epidermis and hair follicles in these skin allografts (Figure 2B, right panels). Interestingly, some sebaceous gland cells and CD133 positive hair follicle cells co-stained with GFP antibody (Figure 2C) and CD133/GFP double-positive hair follicle containing a black hair (Figure 2C, middle panel) suggesting the repopulation of skin components by host-derived cells. Some GFP-negative hair follicles containing black hairs (from the donor) remained in the skin allograft (Figure 2C, left panel) indicating the formation of donor-recipient chimeric skin. At 10 weeks posttransplantation of GFP-negative DA skin into GFP transgenic Lewis recipients (Figure 3A), the epidermis and the majority of hair follicles and sweat/sebaceous glands were GFP positive confirming in the test group the host repopulation of long-lived skin allografts (Figure 3B and C).

Figure 3.

Skin Regeneration Through Host Bone Marrow Stem Cell Repopulation of Skin Allografts in Recipient Rats Treated with AF Combination. Full-Thickness Skins From DA Rats Were Transplanted into GFP Transgenic Lewis Recipients or GFP Bone Marrow Transplanted Wild-type Lewis Recipients. (A) Immunohistochemistry Staining of GFP in Skin Tissue Sections from DA or GFP Lewis rats. (B and C) Immunohistochemistry Staining of GFP in Skin Allografts from GFP Lewis Recipients Treated with AF Combination at 10 Weeks Following Transplantation. (D and E) Full-Thickness Skins from DA Rats Were Transplanted onto GFP Bone Marrow Transplanted Wild-Type Lewis Recipients. (D) Macroscopic Photographs of Skin Allografts (Upper Panels) and Immunohistochemistry Staining of GFP in Tissue Sections of Skin Allografts at 3 Weeks Posttransplantation (Lower Panels). (E) H&E Staining (Upper Panels) and Immunohistochemistry Double Staining for GFP and CD133 in Tissue Sections of Skin Allografts at 10 Weeks Posttransplantation. Representative Pictures From 3 or 4 Rats Per Group

Figure 2.

Host-Derived Cells Repopulate Epidermal and Hair Follicles of Skin Allografts in GFP Lewis Rats with AF Combination Therapy at Day 25 Following Transplantation. Full-Thickness Skins From DA Rats Were Transplanted into GFP Transgenic Lewis Recipients. Skin Allografts Were Collected for Histological Analysis at 25 Days Following Transplantation. (A) Saline Controls. H&E Staining of Wounds Following Skin Allograft Rejection and Immunohistochemistry Double Staining for GFP (Brown) and CD133 (Blue). (B) AF-Treated Group. Tissue Samples were Collected from Different Areas of Skin Allograft and Tissue Sections were Stained with H&E, GFP, and CD133 Antibodies. (C) Immunohistochemistry Double Staining for GFP and CD133 Double Staining in Skin Allografts Recovered from GFP Lewis Recipient With AF Combination Therapy. Representative Pictures From 3 to 6 Rats Per Group

To determine the critical role of mobilized bone marrow-derived stem cells in repopulating the skin allografts by AF combination therapy, bone marrow cells (3–4 × 10⁷) recovered from GFP+ Lewis rats were transplanted into lethally irradiated eight-week-old wild-type Lewis rats. Recipient rats were used as recipients of DA skin allograft for at least 10 weeks following bone marrow transplantation (BMT). In this model, flow cytometry analysis demonstrated that all bone marrow-derived cells were GFP positive at 2 months following BMT.¹³

Skin allografts were collected at 3 weeks or 10 weeks following transplantation and evaluated for bone marrow-derived cells (GFP positive) during the repopulation process. Skin allografts were desiccated or lost in control rats treated with saline at 3 weeks after transplantation and host bone marrow-derived GFP-positive cells (likely inflammatory cells) were accumulated in the wound beds (Figure 3D, left panels). In contrast, skin allografts in the AF combination test group remained soft without shrinking at 3 weeks, and clusters of GFP-positive cells appeared in the allograft tissues and most GFP+ cells localized in the hair follicles (Figure 3D, right panels). At 10 weeks post-skin transplantation, wounds healed with scars in rats treated with saline and some GFP-positive cells appeared in the scar tissues and blood vessels beneath the dermis (Figure 3E, left panels), while wounds healed with skin allografts in rats treated with AF combination and the skin allografts showed normal architecture of skin with hair follicles and glands (Figure 3E, right upper panel). Interestingly, most hair follicular papilla and bulb, inner and outer root sheath were GFP positive suggesting these cells derived from bone marrow (Figure 3E, right lower panels). These results show that pharmacologically mobilized bone marrow stem cells and their progeny are principal contributors to host repopulation of skin allografts.

Finally, we tested our approach in a swine model of burns. Full-thickness burns (22 cm × 15 cm) were created on the back of temporarily anesthetized SLA (swine leucocyte antigens)-characterized miniature swine using an electric iron and SLA mismatched skin allografts were transplanted at 48 h after excision of burned tissues (full-thickness skin). The animal was treated with AF combination (AMD3100: 1 mg/kg and FK506: 0.02 mg/kg, subcutaneous injection) or saline immediately after transplantation and every other day for 8 weeks. Figure 4A (left panels) shows that saline-treated control allografts were desiccated/rejected at 3 weeks, wounds formed a scab at 6 weeks, and healed with contracture scar at 12 weeks. By contrast, in a pig treated with the AF combination, epidermal allografts were gradually lost over 3–4 weeks after transplantation while wound beds were filled with newly generated tissue, and the newly generated tissue was covered by a thin membrane-like epithelium with reddish-purple color (Figure 4A, middle panels). At 6 weeks, the wounds were fully covered by the newly generated epidermis without contracture. By 10 weeks, new hair and pigmentation were observed in newly generated skin and beginning at 9 months the burn wounds were covered by newly generated skin with hair and host (black) pigmentation indicating regeneration with allograft (Figure 4A, right lower panels). Histological study of biopsies from wound areas at 9 months demonstrated normal architecture of skin in swine treated with AF combination, and thickened epidermis and scar tissue in saline-treated swine (Figure 4B).

Figure 4.

Skin Regeneration Through Skin Allografts and AF Combination Therapy in a Pig Model of Skin Burn Wounds. Full-Thickness Burns (22 cm × 15 cm) Were Created on the Back of SLA (Swine Leucocyte Antigens) Characterized Miniature Swine Using an Electric Iron and SLA Mismatched Skin Allografts Were Transplanted 48 h After the Burn. (A) Macroscopic Analysis of Skin Allografts. Saline-Treated Control Skin Allografts were Rejected With Contracture Scars at 12 Weeks (Left Panels). In a Pig Treated With AF Combination, Skin Allografts Were Replaced by a Thin Membrane-like Epithelium With Reddish-Purple Color at 3 Weeks. By 10 Weeks, New Hair and Pigmentation Were Observed in Newly Generated Skin and Beginning at 9 Months the Burn Wounds Were Covered by Newly Generated Skin With Hair and Host (Black) Pigmentation (Middle Panels). (A) Long-Term Outcome of the Burn Wounds in Saline (Right Upper Panel) and AF Combination (Right Lower Panels) Treated Swine. (B) H&E Staining of Biopsy Samples from the Center of Burn Wounds at 9 Months. (C) Schematic Representation of the Potential Therapeutic Mechanism of AF Combination Therapy in Host Repopulation of Skin Allografts

DISCUSSION

Our previous studies demonstrated that AF combination therapy mobilized bone marrow stem cells/endothelial progenitor cells, and regulatory T cells into circulation and increased stromal cell-derived factor-1 (SDF-1) or/and HGF expression in macrophages that pulled circulating stem cells/monocytes into the wound sites^8,9,11,12 or the allograft.^4–6 Recruiting regulatory T cells and M2 macrophages into the allograft or the wound sites not only mutes the rejection response but may also facilitate stem cell differentiation and tissue repair. Similarly, AF combination therapy for skin allografting may release M2 macrophages and bone marrow stem cells into circulation and induce SDF-1 expression in the allograft (Figure 4C). Tregs are released and may also mute the rejection response and may facilitate epithelial stem cell differentiation and wound healing. These host monocyte cells are pulled into the skin graft and replace allograft cells with host-derived dermal/epidermal cells. Other host cell types are also induced by AF treatment, including vascular endothelial cells that provide microvasculature to carry circulating cells into the graft and nurture the dermal elements, such as replacing allograft hair follicles with follicles of host-derived cells and hair follicle neogenesis (Figure 4C). During this gradual process, the beneficial covering feature of the graft is retained.

It should be noted that not all skin allografts survived and became repopulated in the AF-treated animals. This could be the attributed to two potential causes. Firstly, from a mechanistic view, not all rats can mobilize their stem cells and immunoregulatory cells to the same degree. There are ample clinical reports in humans where stem cell mobilization fails with GM-CSF and Plerixafor.¹⁴ This is often attributed to age, genetics, and prior treatment. Reports indicate that roughly one third of all plerixafor treated patients fails to mobilize sufficient numbers of stem cells to the peripheral blood.^14,15 In order to reduce this possibility of failure in clinic, a CD34+ stem cell count can be established after the initial treatment to measure the presence or lack of increase in this key stem cell population. This measurement can be used as biomarker to detect successful mobilization. In case stem cells are not mobilized to a satisfactory degree, the dosage can be increased to establish sufficient mobilization.

Secondarily, as with all surgical models, some failures can be attributed to technical issues. A moist environment is of paramount importance in this model for retention of biological fluids over the graft preventing desiccation of the denuded dermis and allowing faster reepithelization and prevention of skin infections.¹⁶ Rats develop itch at the skin graft site and start to scratch off the bandage within 2 weeks posttransplantation and without a moist bandage, the skin graft will dry out and die. However, none of the skin allografts survived over 2 weeks in saline-treated animals with perfect bandage protection.

In addition, more studies in small and large animals are required to gauge the long-term outcomes of chimeric skins. Is it yet to be confirmed, whether the skin chimerism and host repopulation will maintain over the course of years or gradually decline. The current available knowledge in solid organ transplantation indicates that the effects may be sustained for long-term. When the same treatment was applied in a rat kidney and liver transplant model, no further immunosuppressive medication was needed and no rejection occurred during a 6-month follow-up period.^4,5 This phenomenon was further confirmed in a minipig model of maximally swine leukocyte antigen (SLA) mismatched model of kidney transplantation where the animals received a short course of AF treatment. These animals demonstrated kidney host repopulation ranging from 40% to 50% which increased over time.⁶ It remains to be investigated whether the same can applied to skin transplants that turn chimeric. Important of note is, whether the chimeric skin graft can survive indefinitely when treatment has been ceased.

Burns affect >11 million people worldwide annually.¹⁷ Skin grafting remains a standard therapeutic approach for large wounds. Autologous skin grafts including split-thickness skin grafts and full-thickness skin grafts have the capacity for full integration into the donor site and are preferred for skin wounds. However, they suffer from contracture during healing.¹⁸ Also, autografts are inherently limited to the size of available donor sites and are insufficient for global burn injuries. Human deceased donor skin allografts represent a suitable and commonly used temporizing option for skin cover following severe burn injury. These allografts, while offering large amounts of coverage, undergo immunologic rejection with time and thus serve only as temporary biological dressings.¹⁹ The limitations of living skin grafts have promoted the development of a variety of transplantable bioengineered skin substitutes that are now approved for human clinical use. These bioengineered skin equivalents can be classified into epidermal, dermal, or composite categories according to their structure and the degree of their resemblance to normal skin. The engineered skin equivalents are life-saving and capable of covering the entire surface area of the body. However, like living skin allografts, they ultimately undergo rejection in 3 to 4 weeks.¹⁹ Our approach is also a departure from common stem cell therapies, where one needs to collect a source of stem cells from the bone marrow or adipose tissue, grown and expand them in the laboratory, and inject them back to patients. AF combination therapy can be readily used as a subcutaneous injection that mobilizes the patients own stem and immune cell reserves without the need for extraction and expansion ex vivo.

Our approach induced skin chimerism through host repopulation of skin allografts by mobilizing and recruiting bone marrow stem cells. Skin allografts occasionally survive in patients with deep burns for long-term without rejection²⁰ and limited host repopulation has been reported in long-term survival of human skin allografts in kidney transplanted patients with immunosuppression.²¹ The actual numbers may be higher than investigated in burn victims who have developed chimeric skin after transplantation with cadaveric skin. This could be the case especially in burn victims that were treated with the Alexander skin graft technique.²² With this surgical procedure, widely expanded autograft skin is covered with cadaver skin, acting as a physiological and mechanical barrier able to stimulate engraftment and offers a good environment for host repopulation. As the underlying wound bed re-epithelializes, the allograft slowly separates and, in some cases, may be partially retained. However, this small degree of host repopulation and chimerism is very limited. If the limited host repopulation of skin allografts that is currently observed^20,21 can be facilitated clinically by our new stem cell mobilizing therapy, conversion to a predominantly host phenotype will generate autochthonous skin, albeit perhaps partially of allogeneic origin.

To date, skin wounds heal with these therapies, but they do not restore normal human skin. Rather, they heal as a facsimile of normal skin with a fibrotic (scarred) dermis, effaced epidermis, and an organ lacking sweat glands, oil glands, and hair. This facsimile of normal skin has considerable morbidity, lacks critical skin functions, and is highly vulnerable to further injury.¹⁹ The fibrosis of large burn wounds across limbs and body parts induces contractures that can limit the mobility and functionality of the victim. These all have been hazards of surviving serious burns.

The AF combination treatment dubbed as "MRG-001" has been further developed into a clinical-grade drug by MedRegen LLC and has completed its phase I trial in healthy subjects (NCT04646603) under FDA auspices.²³ The phase I trial demonstrated excellent safety and tolerability across all dose ranges. It further confirmed our findings in animal models. The drug combination was able to mobilize significant numbers of CD34+ hematopoietic and CD34+CD133+CD31+ endothelial progenitor cells, CD4+FOXP3+ and CD8+FOXP3+ cells into the peripheral circulation. RNA-sequencing of the mobilized peripheral blood cells showed significant pathway-related down-regulation of key inflammatory pathways such as the inflammatory response, IL2 STAT5 signaling, TGFβ, TNF-α, and allograft rejection pathway confirming that this drug has immunomodulatory and anti-inflammatory properties. Notably, the drug did not reach any immunosuppressive tacrolimus concentrations, confirming that immunosuppression is not the cause of the down-regulation of inflammatory pathway changes.

Most recently, a phase IIa study investigating the effect of this novel drug combination has been approved by the FDA to study its potential to accelerate full-thickness wound healing and increase the tensile strength in incisional scars in patients undergoing abdominoplasty (NCT05844527). The drug is also expected to be tested in a phase II trial in patients with limited burns requiring autografting in 2023-2024.

CONCLUSION

By applying AF combination treatment in skin allografts, replicating large burn scenarios where current treatment options are profoundly limited, we found gradual rejection of skin allograft, host repopulation, and restoration of the skin’s normal architecture without scarring and minimal contracture. This finding demonstrates a new type of skin repair and provides hope for victims with deep burns or large wounds.

Funding:

This research was partially funded by the National Institute of Health grant number R01AR082388 and partially funded by MedRegen LLC.

Author Contributions:

Ali Reza Ahmadi (Conceptualization: Lead; Formal analysis: Lead; Investigation: Lead; Methodology: Lead; Visualization: Lead; Writing – original draft: Lead; Writing – review & editing: Lead), Russell Wesson (Methodology: Equal; Supervision: Supporting; Writing – review & editing: Supporting), Jinny Huang (Data curation: Supporting; Formal analysis: Supporting; Investigation: Supporting; Writing – review & editing: Supporting), John Harmon (Investigation: Supporting; Methodology: Supporting; Resources: Supporting; Writing – review & editing: Supporting), James Burdick (Supervision: Equal; Writing – review & editing: Supporting), Andew Cameron (Supervision: Supporting; Writing – review & editing: Supporting), Zhaoli Sun (Conceptualization: Equal; Data curation: Supporting; Formal analysis: Supporting; Funding acquisition: Lead; Investigation: Supporting; Methodology: Supporting; Project administration: Lead; Resources: Lead; Supervision: Lead; Writing – original draft: Equal; Writing – review & editing: Equal)

Conflict of Interest Statement:

Z.S. is a cofounder of and holds equity in MedRegen LLC. Additionally, Z.S. is an inventor of technology and participates in research funded by MedRegen LLC that intends to further develop the technology. J.B. is also a stock holder at MedRegen LLC and works as the Chief Medical Officer. Other authors have nothing to declare.