Human Stem Cell-Derived Skin Progenitors Produce Alpha 2-HS Glycoprotein (Fetuin): A Revolutionary Cosmetic Ingredient.

These studies were designed to determine the effect of stem cell-derived skin lineage precursor secretions on the intrinsic and extrinsic symptoms of human skin aging.
Human stem cells cultivated in balanced conditions were differentiated into skin lineage precursors, and shown to secrete large amounts of fetuin as well as multiple growth factors beneficial for human skin development and maintenance. The cell secretions were incorporated in two simple cosmetic formulations (serum and lotion) and investigated in an IRB-approved 12-week human trial that included 25 subjects in each group. Subjects were examined at 2, 4, 8, and 12 weeks by a dermatologist to evaluate safety, trans-epidermal water loss, wrinkles, firmness, radiance, texture, softness, and overall appearance. A sub-group of subjects from each group consented for biopsies for histological analyses.
Protein analyses in the cell secretions revealed a high concentration of the multifunctional alpha 2-HS glycoprotein (fetuin) along with a multitude of protein factors involved in the development and maintenance of healthy human skin. Clinical investigation demonstrated significant amelioration of the clinical signs of intrinsic and extrinsic skin aging, findings that were confirmed by significant changes in skin morphology, filaggrin, aquaporin 3, and collagen I content.
Our data strongly support our hypothesis that cosmetic application of stem cell-derived skin lineage precursor secretions containing fetuin and growth factors beneficial for human skin development and maintenance, positively influence intrinsic and extrinsic aging.

J Drugs Dermatol. 2016;15(5):583-598.

A personal COVID-19 dendritic cell vaccine made at point-of-care: Feasibility, safety, and antigen-specific cellular immune responses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a world-wide pandemic. Internationally, because of availability, accessibility, and distribution issues, there is a need for additional vaccines. This study aimed to: establish the feasibility of personal dendritic cell vaccines to the SARS-CoV-2 spike protein, establish the safety of a single subcutaneous vaccine injection, and determine the antigen-specific immune response following vaccination. In Phase 1, 31 subjects were assigned to one of nine formulations of autologous dendritic cells and lymphocytes (DCL) incubated with 0.10, 0.33, or 1.0 µg of recombinant SARS-CoV-2 spike protein, and admixed with saline or 250 or 500 µg of granulocyte-macrophage colony-stimulating factor (GM-CSF) prior to injection, then assessed for safety and humoral response. In Phase 2, 145 subjects were randomized to one of three formulations defined by incubation with the same three quantities of spike protein without GM-CSF, then assessed for safety and cellular response. Vaccines were successfully manufactured for every subject at point-of-care. Approximately 46.4% of subjects had a grade 1 adverse event (AE); 6.5% had a grade 2 AE. Among 169 evaluable subjects, there were no acute allergic, grade 3 or 4, or serious AE. In Phase 1, anti-receptor binding domain antibodies were increased in 70% of subjects on day-28. In Phase 2, in the 127 subjects who did not have high levels of gamma interferon-producing cells at baseline, 94.4% had increased by day 14 and 96.8% by day 28. Point-of-care personal vaccine manufacturing was feasible. Further development of such subject-specific vaccines is warranted.

Genomic, proteomic, and immunologic associations with a durable complete remission of measurable metastatic melanoma induced by a patient-specific dendritic cell vaccine.

This report describes efforts to understand the immune mechanism of action that led to a complete response in a patient with progressive, refractory, metastatic melanoma after treatment with a therapeutic vaccine consisting of autologous dendritic cells (DC) loaded with autologous tumor antigens (ATA) derived from cells that were self-renewing in cell culture. Her histocompatibility type proved to be HLA B27 with extensive mutations in the HLA-A locus. Exomic analysis of proliferating tumor cells revealed more than 2800 non-synonymous mutations compared to her leukocytes. Histology of resected tumor lesions showed no evidence of an existing or suppressed immune response. In in vitro mixed cell cultures, DC loaded with ATA induced increased IL-22 expression, and a four-fold increase in CD8 + T lymphocytes. Cryopreserved blood samples obtained at week-0, 1 week before the first of three-weekly vaccine injections, and at week-4, 1 week after the third dose, were analyzed by protein array and compared for 110 different serum markers. At baseline, she had marked elevations of amyloid A, IL-12p40, IL21, IL-22, IL-10, IL-16, GROa, TNF-alpha, IL-3, and IL-2, and a lesser elevation of IL-15. One week after 3 weekly vaccinations she had a further 80% increase in amyloid A, a further 66% increase in IL-22, a 92% decrease in IL12p40, a 45% decrease in TGF-ß and 26% decrease in IL-10. The data suggested that by 3 weeks after the first DCV injection, vaccine-induced changes in this particular patient were most consistent with enhanced innate and Th1 immune responses rather than Th2 or Th17.

Preliminary observations on soluble programmed cell death protein-1 as a prognostic and predictive biomarker in patients with metastatic melanoma treated with patient-specific autologous vaccines.

Because of its role as an immune checkpoint, levels of soluble programmed cell death protein-1 (sPD-1) could be useful as a prognostic biomarker or predictive biomarker in cancer patients treated with vaccines. Very low levels of sPD-1 may indicate lack of an existing anti-cancer immune response; very high levels may indicate an active immune response that is suppressed. In between these extremes, a decrease in PD-1 following injections of an anti-cancer vaccine may indicate an enhanced immune response that has not been suppressed. Blood samples obtained during a randomized trial in patients with metastatic melanoma were tested from 22 patients treated with a tumor cell vaccine (TCV) and 17 treated with a dendritic cell vaccine (DCV). Survival was better in DCV-treated patients. sPD-1 was measured at week-0, one week before the first of three weekly subcutaneous injections, and at week-4, one week after the third injection. The combination of a very low baseline sPD-1, or absence of a very high PD-1 at baseline followed by a decline in sPD-1 at week-4, was predictive of surviving three or more years in DCV-treated patients, but not TCV-treated. Among DCV-treated patients, these sPD-1 criteria appropriately classified 8/10 (80%) of 3-year survivors, and 6/7 (86%) of patients who did not survive three years. These preliminary observations suggest that sPD-1 might be a useful biomarker for melanoma patients being considered for treatment with this DCV vaccine, and/or to predict efficacy after only three injections, but this would have to be confirmed in larger studies.

Derivation of high purity neuronal progenitors from human embryonic stem cells.

The availability of human neuronal progenitors (hNPs) in high purity would greatly facilitate neuronal drug discovery and developmental studies, as well as cell replacement strategies for neurodegenerative diseases and conditions, such as spinal cord injury, stroke, Parkinson's disease, Alzheimer's disease, and Huntington's disease. Here we describe for the first time a method for producing hNPs in large quantity and high purity from human embryonic stem cells (hESCs) in feeder-free conditions, without the use of exogenous noggin, sonic hedgehog or analogs, rendering the process clinically compliant. The resulting population displays characteristic neuronal-specific markers. When allowed to spontaneously differentiate into neuronal subtypes in vitro, cholinergic, serotonergic, dopaminergic and/or noradrenergic, and medium spiny striatal neurons were observed. When transplanted into the injured spinal cord the hNPs survived, integrated into host tissue, and matured into a variety of neuronal subtypes. Our method of deriving neuronal progenitors from hESCs renders the process amenable to therapeutic and commercial use.

Histological and functional benefit following transplantation of motor neuron progenitors to the injured rat spinal cord.

BACKGROUND: Motor neuron loss is characteristic of cervical spinal cord injury (SCI) and contributes to functional deficit. METHODOLOGY/PRINCIPAL FINDINGS: In order to investigate the amenability of the injured adult spinal cord to motor neuron differentiation, we transplanted spinal cord injured animals with a high purity population of human motor neuron progenitors (hMNP) derived from human embryonic stem cells (hESCs). In vitro, hMNPs displayed characteristic motor neuron-specific markers, a typical electrophysiological profile, functionally innervated human or rodent muscle, and secreted physiologically active growth factors that caused neurite branching and neuronal survival. hMNP transplantation into cervical SCI sites in adult rats resulted in suppression of intracellular signaling pathways associated with SCI pathogenesis, which correlated with greater endogenous neuronal survival and neurite branching. These neurotrophic effects were accompanied by significantly enhanced performance on all parameters of the balance beam task, as compared to controls. Interestingly, hMNP transplantation resulted in survival, differentiation, and site-specific integration of hMNPs distal to the SCI site within ventral horns, but hMNPs near the SCI site reverted to a neuronal progenitor state, suggesting an environmental deficiency for neuronal maturation associated with SCI. CONCLUSIONS/SIGNIFICANCE: These findings underscore the barriers imposed on neuronal differentiation of transplanted cells by the gliogenic nature of the injured spinal cord, and the physiological relevance of transplant-derived neurotrophic support to functional recovery.

Reduced susceptibility to azoxymethane-induced aberrant crypt foci formation and colon cancer in growth hormone deficient rats.

OBJECTIVES: To evaluate the role of GH in colon carcinogenesis, we examined the formation of aberrant crypt foci (ACFs) and tumor development in wild type (WT) and GH-deficient, spontaneous dwarf rats (SDRs) exposed to the carcinogen azoxymethane (AOM). DESIGN: ACF were quantified by stereomicroscopy and tumor number and weights were recorded for each animal. Cell proliferation was measured by vincristine metaphase arrest, flow cytometry, and bromodeoxyuridine (BrdU) incorporation. Apoptosis was measured by TUNEL staining and cleaved caspase-3 immunohistochemistry. IGF-I was measured by radioimmunoassay (RIA). Hexokinase activity was measured by spectrophotometric assay. PARP cleavage, and IGF-IR, and p27(kip/cip) expression were measured by Western blotting. RESULTS: ACFs detected by stereomicroscopy were markedly reduced ( approximately 85%) in SDRs vs. WT rats at 10, 25, and 28 weeks after AOM. Tumor incidence, number, and weight also were reduced in SDR vs. WT animals. AOM treatment increased cell proliferation in the distal colon (where tumors occur) of WT rats but not SDRs, and these changes corresponded to increased ACF and tumor formation. Apoptosis rates were similar in AOM-treated WT and SDRs. Alterations in serum IGF-I levels may contribute to differences in the proliferative response to AOM and decreased ACF formation in SDR vs. WT rats. CONCLUSIONS: We conclude that early neoplastic lesions (ACFs) were reduced in GH-deficient animals. This effect corresponds with differences in AOM-induced proliferation, but not apoptosis. These data indicate that GH is required for the full effect of AOM on colon ACF and tumor development, and that the SDR rat is a promising model for studies regarding the role of GH/IGF system in the initiation and promotion of colon cancer.

Functions of p21 and p27 in the regenerating epithelial linings of the mouse small and large intestine.

The epithelial linings of the small and large intestine are rapidly turned over and provide an ideal system for exploring links between differentiation and regulation of cell cycle exit. We utilized wild type, p21-/-, p27-/- and p21/p27-/- mice to address contributions of the Cdk inhibitors p21 and p27 to proliferation and differentiation in the mouse gastrointestinal tract. We did not detect any significant differences in proliferation, and all differentiated epithelial cell lineages were represented in all four genotypes. These data indicate that p21 and p27 do not play essential roles in the regulation of normal epithelial renewal in the intestine. These Cdk inhibitors are not needed in vivo for either assembly of Cdk/Cyclin complexes that drive active proliferation, or inhibition of Cdk/Cyclin complexes during cell cycle exit. However, expression of Cyclin D2 and to a lesser degree Cyclin D3 was reduced in p27-/- and p21/p27-/- mice, indicating a unique role for p27 in the regulation of these specific D-type Cyclins in vivo. In the absence of p27, reduced levels of Cyclin D2 and D3 may help to counteract increased proproliferative signals in the intestine.