Hair regrowth in alopecia areata patients following Stem Cell Educator therapy

Yanjia Li, Baoyong Yan, Hepeng Wang, Heng Li, Quanhai Li, Dong Zhao, Yana Chen, Ye Zhang, Wenxia Li, Jun Zhang, Shanfeng Wang, Jie Shen, Yunxiang Li, Edward Guindi, Yong Zhao, Yanjia Li, Baoyong Yan, Hepeng Wang, Heng Li, Quanhai Li, Dong Zhao, Yana Chen, Ye Zhang, Wenxia Li, Jun Zhang, Shanfeng Wang, Jie Shen, Yunxiang Li, Edward Guindi, Yong Zhao

Abstract

Background: Alopecia areata (AA) is one of the most common autoimmune diseases and targets the hair follicles, with high impact on the quality of life and self-esteem of patients due to hair loss. Clinical management and outcomes are challenged by current limited immunosuppressive and immunomodulating regimens.

Methods: We have developed a Stem Cell Educator therapy in which a patient's blood is circulated through a closed-loop system that separates mononuclear cells from the whole blood, allows the cells to briefly interact with adherent human cord blood-derived multipotent stem cells (CB-SC), and returns the "educated" autologous cells to the patient's circulation. In an open-label, phase 1/phase 2 study, patients (N = 9) with severe AA received one treatment with the Stem Cell Educator therapy. The median age was 20 years (median alopecic duration, 5 years).

Results: Clinical data demonstrated that patients with severe AA achieved improved hair regrowth and quality of life after receiving Stem Cell Educator therapy. Flow cytometry revealed the up-regulation of Th2 cytokines and restoration of balancing Th1/Th2/Th3 cytokine production in the peripheral blood of AA subjects. Immunohistochemistry indicated the formation of a "ring of transforming growth factor beta 1 (TGF-β1)" around the hair follicles, leading to the restoration of immune privilege of hair follicles and the protection of newly generated hair follicles against autoimmune destruction. Mechanistic studies revealed that co-culture with CB-SC may up-regulate the expression of coinhibitory molecules B and T lymphocyte attenuator (BTLA) and programmed death-1 receptor (PD-1) on CD8β(+)NKG2D(+) effector T cells and suppress their proliferation via herpesvirus entry mediator (HVEM) ligands and programmed death-1 ligand (PD-L1) on CB-SCs.

Conclusions: Current clinical data demonstrated the safety and efficacy of the Stem Cell Educator therapy for the treatment of AA. This innovative approach produced lasting improvement in hair regrowth in subjects with moderate or severe AA.

Trial registration: ClinicalTrials.gov, NCT01673789, 21 August 2012.

Figures

Figure 1
Figure 1
Diagram of Stem Cell Educator therapy for the treatment and follow-up studies.
Figure 2
Figure 2
Ex vivo studies of the immune modulation of CB-SCs on T cells. (A) Phase contrast microscopy shows the formation of cell clusters in human peripheral blood-derived lymphocytes that were activated with Dynabeads coupled with anti-CD3, anti-CD28, and anti-CD137 antibodies, 50 U/ml rIL-2, and 5 ng/ml rIL-7 for 5 days, in absence (left panel) and presence (right panel) of CB-SCs. Co-culture with lymphocytes (top right panel) served as control. Original magnification, × 100. (B) Cell proliferation was analyzed with CellTrace™ CFSE Cell Proliferation Kit. Untreated lymphocytes (left panel) served as control. (C) Multi-color flow cytometry on CD8+NKG2D+ T cells. The gated CD8+NKG2D+ T cells were further analyzed for the expression of coinhibitory molecules BTLA and PD-1. Isotype-matched IgG Abs served as control for flow cytometry. Mean fluorescence intensity (MFI) was presented for CD8+NKG2D+BTLA+PD-1+ T cells. Flow cytometry dot plots and the percentage of each population were representative of three independent experiments with similar results.
Figure 3
Figure 3
Flow cytometry analysis. (A) Expression of HVEM on CB-SCs. Isotype-matched IgG served as control. (B) Expression of HVEM on the gated CD45+Oct3/4+ CB-SCs. (C) Expression of BTLA and PD-1 on peripheral blood-derived immune cells. (D) The percentage of BTLA+ PD-1+ cells in each subpopulation. Each population was gated using the specific surface markers as described in Methods.
Figure 4
Figure 4
Regrowth of hair following Stem Cell Educator therapy. A subject with severe AA (patient 4 in Table 1) achieved complete hair regrowth at 12 weeks follow-up after receiving Stem Cell Educator therapy and maintained regrowth through the last follow-up (2 years).
Figure 5
Figure 5
Immune modulation of Stem Cell Educator therapy. Patient lymphocytes were isolated from peripheral blood by Ficoll-Hypaque technique (γ = 1.077) for flow cytometric analyses in AA patients at baseline and 4 weeks after Stem Cell Educator therapy. Isotype-matched IgG served as control. Histologic examination of alopecic skin (C and D). (A) Flow analysis of intracellular cytokines demonstrating differential effects on key interleukins at 4 weeks post-treatment. (B) Flow analysis of costimulating molecules demonstrating an increase of CD28 expression at 4 weeks post-treatment. Data are representative of preparations from all AA participants (n = 9) who received Stem Cell Educator therapy. (C) Fresh skin tissues were collected from the scalps via biopsy for immunohistochemistry testing in participants with alopecia totalis before treatment and 12 weeks after receiving Stem Cell Educator therapy. TGF-β1 staining surrounds a hair follicle of AA participants after receiving Stem Cell Educator therapy, with vertical section of hair follicle (top panels) and horizontal section of hair follicle. Isotype-matched mouse IgG1 served as a negative control for TGF-β1 immunostaining in a serial hair follicle section. Representative images were obtained from five experiments. Scale bar, 25 μm. (D) H&E staining of scalp tissues. Scale bar, 25 μm.

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