Insulin resistance is the hallmark of T2D. It is widely accepted that the inability of pancreatic β cells to function in compensating for insulin resistance leads to the onset of clinical diabetes. Persistent metabolic stresses including glucotoxicity, lipotoxicity, chronic metabolic inflammation, oxidative stress and endoplasmic reticulum stress, cause progressive dysfunction of islet β cells and finally lead to the cellular death and absolute shortage of islet β cells in long-standing T2D subjects . The current phase 1/2 study demonstrates the safety and therapeutic efficacy of Stem Cell Educator therapy in the treatment of T2D. Insulin sensitivities were markedly increased after receiving Stem Cell Educator therapy, followed by the significant improvement of metabolic controls in these long-standing T2D patients. Notably, we found that T2D subjects in Group C (with the absolute shortage of islet β cells) significantly improved fasting C-peptide levels and β cell function. These data indicate that Stem Cell Educator therapy may open up a new avenue for the treatment of T2D.
Chronic inflammation of visceral adipose tissue (VAT) is a major contributor to insulin resistance mediated by adipose tissue-released adipokines (for example, IL-6, TNFα, MCP-1 and resistin) [40, 43]. Growing evidence strongly demonstrated that an accumulation of macrophages by metabolic stress in the sites of affected tissues (such as vasculature, adipose tissue, muscle and liver) has emerged as a key process in the chronic metabolic-stress-induced inflammation . Monocytes/macrophages, as one type of the professional antigen-presenting cells, play an essential role in controlling the Th1/Th2 immune responses and maintaining homeostasis through the co-stimulating molecules CD80/CD86 and released cytokines. Persistent destructive effects of lipid influx (for example, fatty acids and cholesterol) cause macrophage dysfunctions (including defective efferocytosis and unresolved inflammation), resulting in recruitment and activation of more monocytes/macrophages via MCP-1 and its receptor CCR2 . Consequently, inflammatory cytokines (for example, IL-6 and TNFα) produced by activated macrophages induce insulin resistance in major metabolic tissues [26, 44, 45]. To prove the action of macrophage in chronic inflammation and insulin resistance in T2D, conditional depletion of CD11c+ macrophages or inhibition of macrophage recruitment via MCP-1 knockout in obese mice resulted in a significant reduction in systemic inflammation and an increase in insulin sensitivity [46–48].
To clarify the modulation of Stem Cell Educator therapy on blood monocytes, we found that expression of CD86 and CD86+CD14+/CD80+CD14+ monocyte ratios have been markedly changed after receiving Stem Cell Educator therapy in T2D subjects. CD80 and CD86 are two principal co-stimulating molecules expressed on monocytes to skew the immune response toward Th1 or Th2 differentiation through their ligands CD28/CTLA4 [49, 50]. Due to the differences of expression levels and binding affinity between CD80 and CD86 with their ligands CD28/CTLA4, it is widely accepted that the interaction of CD86 with CD28 dominates in co-stimulating signals; conversely, the combination of CD80 and CTLA4 governs negative signaling [49–52]. The normalization of the CD86+CD14+/CD80+CD14+ monocyte ratio post-treatment may favor the immune balance of Th1/Th2 responses in diabetic subjects. Taken together with our in vitro study on the direct interaction between CB-SCs and purified CD14+ monocytes, these data indicate that restoration of monocyte functions (such as the expression of CD86, cytokine productions and chemokine productions) mainly contributes to anti-inflammation and reversal of insulin resistance following Stem Cell Educator therapy in T2D subjects.
Increasing animal and clinical evidence demonstrate multiple immune cells contributing to the inflammation-induced insulin resistance in T2D, such as abnormalities of lymphocytes (including T cells, B cells and Tregs [53–57]), neutrophils , eosinophils , mast cells  and dendritic cells (DCs) [61, 62]. Specifically, B and T lymphocytes have emerged as unexpected promoters and controllers of insulin resistance . These adaptive immune cells infiltrate into the VAT, releasing cytokines (IL-6 and TNFα) and recruiting more monocytes/macrophages via MCP-1/CCR2 . Finally, this obesity-related inflammation leads to insulin resistance [57, 63]. Thus, a major challenge for treatment of T2D is to identify therapeutic approaches that fundamentally correct insulin resistance through targeting the dysfunctions of multiple immune cells. The valuable lessons from intensive research pressure over the past 25 years in T1D  highlight the difficulties in overcoming these multiple immune dysfunctions by utilizing conventional immune therapy. Stem Cell Educator therapy functions as “an artificial thymus” that circulates a patient’s blood through a blood cell separator , briefly co-cultures the patient’s blood mononuclear cells (such as T cells, B cells, Tregs, monocytes and neutrophils) with CB-SCs in vitro. During the ex vivo co-culture in the device, these mononuclear cells can be educated by the favorable microenvironment created by CB-SCs through: 1) the action of an autoimmune regulator (AIRE) expressed in CB-SCs ; 2) the cell-cell contacting mechanism via the surface molecule programmed death ligand 1 (PD-L1) on CB-SCs ; and 3) the soluble factors released by CB-SCs. Previous work  and current data indicate that CB-SC-derived NO mainly contributes to the immune modulation on T cells and monocytes. During the passage of monocytes and other immune cells through the device, NO, as a free radical released by CB-SCs, can quickly transmit into their cellular membrane, without the aid of dedicated transporters; 4) correcting the functional defects of regulatory T cells (Tregs) ; and 5) directly suppressing the pathogenic T cell clones . During this procedure, both peripheral and infiltrated immune cells in VAT can be isolated by a blood cell separator and treated by CB-SCs, leading to the correction of chronic inflammation, the restoration of the immune balance, and clinical improvements in metabolic control via increasing of insulin sensitivity. Additionally, TGF-β1 is a well-recognized cytokine with a pleiotropic role in immune modulation on multiple immune cells, such as the differentiation and function of Th1/Th2 cells and Tregs, as well as B cells, monocytes/macrophages, dendritic cells, granulocytes and mast cells [64–66]. These immune cells are involved in the inflammation-induced insulin resistance in T2D [53–62]. Therefore, the up-regulation of TGF-β1 level in peripheral blood of T2D subjects is another major mechanism underlying the immune modulation after receiving Stem Cell educator therapy.
During the procedure of Stem Cell Educator therapy, the mononuclear cells circulating in a patient’s blood are collected by a blood cell separator. Additionally, patients are required to move their hips, legs and turn to one side every 15 to 30 minutes during the treatment, in order to mobilize their immune cells from peripheral tissues (including adipose tissues) and organs entering into the blood circulation to be processed by a blood cell separator. Thus, the immune cells both in peripheral blood and in tissues can be isolated by a blood cell separator and treated by CB-SCs. The full blood volume is processed approximately twice during Stem Cell Educator therapy (approximately 10,000 ml whole blood) , which ensures a comprehensive approach to modulating essentially all circulating immune cells to address multiple immune dysfunctions and overcome global insulin resistance resulting from a variety of reasons. No other current medications and/or other approaches have yet been shown to achieve this unique therapy success. There are some pathogenic immune cells remaining in tissues and lymph nodes which fail to enter into the blood circulation during the procedure and may escape from the treatment by CB-SCs. These immune cells may migrate into the blood circulation and decrease the therapeutic effectiveness. Therefore, T2D subjects may need additional treatment six to nine months later after receiving the first treatment; however, this is yet to be explored in the phase 3 clinical trial.
We observed that the improvement of islet β cell function (C-peptide levels) progresses slowly over weeks after receiving Stem Cell Educator therapy, not disappearing with the progression of time. We reported similar data in previous T1D trials [18, 19]. If Stem Cell Educator therapy only temporarily corrects the immune dysfunctions, the clinical efficacy in metabolic control should disappear soon after receiving Stem Cell Educator therapy, because of the short lifespans of most immune cells, (for example, 5.4 days for neutrophils , 3 months for lymphocytes, 1 to 3 days for bone marrow-derived monocytes existing in blood and then migrating into tissues). Previous work demonstrated that CB-SCs showed the marked modulation of Th1-Th2-Th3 cell-related genes, including multiple cytokines and their receptors, chemokines and their receptors, cell surface molecules, along with signaling pathway molecules and transcription factors, as indicated by quantitative real time PCR array . Due to these fundamental immune modulations and induction of immune balance , this trial indicates that a single treatment with Stem Cell Educator therapy can give rise to long-lasting reversal of immune dysfunctions and improvement of insulin sensitivity in long-standing T2D subjects.