Yanling Liao

Human Cord Blood-Derived Unrestricted Somatic Stem Cells Promote Wound Healing and Have Therapeutic Potential for Patients With Recessive Dystrophic Epidermolysis Bullosa

Human umbilical cord blood (CB)-derived unrestricted somatic stem cells (USSCs) have previously been demonstrated to have a broad differentiation potential and regenerative beneficial effects when administered in animal models of multiple degenerative diseases. Here we demonstrated that USSCs could be induced to express genes that hallmark keratinocyte differentiation. We also demonstrated that USSCs express type VII collagen (C7), a protein that is absent or defective in patients with an inherited skin disease, recessive dystrophic epidermolysis bullosa (RDEB). In mice with full-thickness excisional wounds, a single intradermal injection of USSCs at a 1-cm distance to the wound edge resulted in significantly accelerated wound healing. USSC-treated wounds displayed a higher density of CD31+ cells, and the wounds healed with a significant increase in skin appendages. These beneficial effects were demonstrated without apparent differentiation of the injected USSCs into keratinocytes or endothelial cells. In vivo bioluminescent imaging (BLI) revealed specific migration of USSCs modified with a luciferase reporter gene, from a distant intradermal injection site to the wound, as well as following systemic injection of USSCs. These data suggest that CB-derived USSCs could significantly contribute to wound repair and be potentially used in cell therapy for patients with RDEB.

Efficacy of Human Placental‐Derived Stem Cells in Collagen VII Knockout (Recessive Dystrophic Epidermolysis Bullosa) Animal Model

Recessive dystrophic epidermolysis bullosa (RDEB) is a devastating inherited skin blistering disease caused by mutations in the COL7A1 gene that encodes type VII collagen (C7), a major structural component of anchoring fibrils at the dermal‐epidermal junction (DEJ). We recently demonstrated that human cord blood‐derived unrestricted somatic stem cells promote wound healing and ameliorate the blistering phenotype in a RDEB (col7a1−/−) mouse model. Here, we demonstrate significant therapeutic effect of a further novel stem cell product in RDEB, that is, human placental‐derived stem cells (HPDSCs), currently being used as human leukocyte antigen‐independent donor cells with allogeneic umbilical cord blood stem cell transplantation in patients with malignant and nonmalignant diseases. HPDSCs are isolated from full‐term placentas following saline perfusion, red blood cell depletion, and volume reduction. HPDSCs contain significantly higher level of both hematopoietic and nonhematopoietic stem and progenitor cells than cord blood and are low in T cell content. A single intrahepatic administration of HPDSCs significantly elongated the median life span of the col7a1−/− mice from 2 to 7 days and an additional intrahepatic administration significantly extended the median life span to 18 days. We further demonstrated that after intrahepatic administration, HPDSCs engrafted short‐term in the organs affected by RDEB, that is, skin and gastrointestinal tract of col7a1−/− mice, increased adhesion at the DEJ and deposited C7 even at 4 months after administration of HPDSCs, without inducing anti‐C7 antibodies. This study warrants future clinical investigation to determine the safety and efficacy of HPDSCs in patients with severe RDEB. Stem Cells Translational Medicine 2018;7:530–542

Cord Blood-Derived Stem Cells Suppress Fibrosis and May Prevent Malignant Progression in Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin fragility disorder caused by mutations in the Col7a1 gene. Patients with RDEB suffer from recurrent erosions in skin and mucous membranes and have a high risk for developing cutaneous squamous cell carcinoma (cSCCs). TGFβ signaling has been associated with fibrosis and malignancy in RDEB. In this study, the activation of TGFβ signaling was demonstrated in col7a1−/− mice as early as a week after birth starting in the interdigital folds of the paws, accompanied by increased deposition of collagen fibrils and elevated dermal expression of matrix metalloproteinase (MMP)‐9 and MMP‐13. Furthermore, human cord blood‐derived unrestricted somatic stem cells (USSCs) that we previously demonstrated to significantly improve wound healing and prolong the survival of col7a1−/− mice showed the ability to suppress TGFβ signaling and MMP‐9 and MMP‐13 expression meanwhile upregulating anti‐fibrotic TGFβ3 and decorin. In parallel, we cocultured USSCs in a transwell with RDEB patient‐derived fibroblasts, keratinocytes, and cSCC, respectively. The patient‐derived cells were constitutively active for STAT, but not TGFβ signaling. Moreover, the levels of MMP‐9 and MMP‐13 were significantly elevated in the patient derived‐keratinocytes and cSCCs. Although USSC coculture did not inhibit STAT signaling, it significantly suppressed the secretion of MMP‐9 and MMP‐13, and interferon (IFN)‐γ from RDEB patient‐derived cells. Since epithelial expression of these MMPs is a biomarker of malignant transformation and correlates with the degree of tumor invasion, these results suggest a potential role for USSCs in mitigating epithelial malignancy, in addition to their anti‐inflammatory and anti‐fibrotic functions. Stem Cells 2018;36:1839–12

Abstract 3155: Neuroblastoma (NB), Medulloblastoma (MB), and Ewing's sarcoma (ES) express ROR1 and can be effectively targeted with NK cells modified to express an anti ROR1 chimeric antigen receptor (CAR)

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Metastatic pediatric neuroectodermal solid tumors especially NB, MB, and ES have a dismal prognosis (Perkins et al, PLoS One, 2014; Smoll, Cancer, 2012). Targeted cellular therapy with T or NK cells modified with CARs is a novel approach to chemo-resistant childhood solid tumors (Grupp SA, Clin Cancer Res, 2012; Mackall C,Front Oncol, 2012). NK cells can be significantly expanded by co-culture with genetically engineered K562 cells overexpressing mb-IL21 (Lee D, PLOS, 2012). ROR1 has been identified as a novel target on B cell tumors in which CARs can be developed and utilized for targeted cellular therapy (Hudecek et al., Blood, 2010). Objective: To evaluate the in-vitro cytotoxic activity and function of PBNK expanded ex-vivo with K562 mb-IL21 and nucleofected with mRNA encoding an anti ROR1-CAR against NB, MB, and ES. Material and Methods: PBNK were expanded with irradiated K562 Clone 9.mb-IL21 (generously provided by Lee D, MD, PhD, MD Anderson, TX). Ex-vivo expanded PBNK (ExPBNK) cells were electroporated with anti ROR1-CD28-41BBl-CD3ζ-tEGFR-mRNA. The cell surface expression of the ROR1-CAR on NK cells was detected using anti-mouse IgG, F(ab’)2. Cytotoxicity of ROR1 CAR-NK cells was investigated against NB (SKNBE2, SKNFI & SHSY5Y), MB (DAOY) and ES (TC71, EWS 502 & A673) cell lines by DELFIA cytotoxicity assay at an E:T ratio of 10:1. Intracellular staining of CD107a, interferon gamma, perforin and granzyme B was performed using a 10:1 E:T ratio of ROR1-CAR-NK cells against tumor targets and analyzed on the MACSQuant flow cytometer. Results: NB, MB, and ES cell lines expressed ROR1 (50.2±15.6%), (55.5±5.1%), and (31.5±12%), respectively. Expansion of NK cells was significantly increased 3988 ± 435 fold (p = 0.00001) at day 14 vs day 0. CAR expression after nucleofection was measured by F(ab’)2 staining and showed a significant increase in anti-ROR1-CAR- (88.3±1.7%) vs mock-electroporated NK cell populations (8.1± 6.9%) p = 0.0001 at 36-48 hours. Anti-ROR1-CAR-NK cells exhibited significantly increased lysis of ROR1 expressing tumor cell lines compared to mock NK cells (93±4.6% vs 63.6±7.4%) p = 0.00001 at an E:T of 10:1. Similarly, expression of CD107a (46.1±9.1 vs 27.6±2.4%) p = 0.001, interferon gamma (34.1±11.6 vs 16.7±6.7%) p = 0.003, granzyme B (68.5±8.9 vs 46±7.2%) p = 0.002, and perforin (51.3±7.7 vs 30.3±11.9%) p = 0.002 were significantly increased in anti-ROR1-CAR-NK cells vs Mock-NK cells at 10:1 E:T ratio against the ROR1 expressing targets. Conclusion: Anti-ROR1-CAR-ex-PBNK cells had significant enhanced cytotoxicity and significantly increased CD107a, interferon gamma, perforin, and granzyme B activity against ROR1 expressing tumors. Future directions include investigating the ex-PBNK anti ROR1-CAR cells in-vivo against ROR1 expressing pediatric solid tumors. Citation Format: Mona Elmacken, Aradhana Awasthi, Janet Ayello, Carmella VanDeVen,, Wen Luo, Yanling Liao, Stanley Riddell, Mitchell S. Cairo. Neuroblastoma (NB), Medulloblastoma (MB), and Ewings sarcoma (ES) express ROR1 and can be effectively targeted with NK cells modified to express an anti ROR1 chimeric antigen receptor (CAR). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3155. doi:10.1158/1538-7445.AM2015-3155