Contribution of Cytokeratin 19-Expressing Cells towards Islet Regeneration Induced by Multipotent Stromal Cell Secreted Proteins

Endocrinology Immunology Bioengineering Basic Medical Sciences Cell Biology Biochemistry Life Sciences Medicine
Cytokeratin 19 Multipotent stromal cells Islet regeneration Diabetes Lineage tracing Ductal endocrine progenitor cells

Background Introduction

Diabetes, especially type 1 diabetes (T1D), is a chronic autoimmune disease characterized by the persistent destruction of pancreatic β cells by the immune system, resulting in the loss of the ability to regulate blood glucose. Patients with type 1 diabetes typically require lifelong insulin injections; however, long-term insulin replacement cannot perfectly mimic the body’s own pancreatic function. Consequently, these patients often experience severe glucose fluctuations, as well as cardiovascular and renal complications, leading to significantly decreased quality of life. Although current medical approaches can help patients manage the disease relatively well, achieving regeneration of pancreatic β cells to fundamentally restore endogenous islet function remains a long-standing goal in the field of diabetes.

In recent years, researchers in the “Joslin Medalist” study have found that in some patients diagnosed with T1D for over 50 years, residual insulin-positive cells and continuous secretion of C-peptide can still be detected in the pancreas, suggesting the existence of certain endogenous β cell regeneration mechanisms in the human body. However, due to the persistence of autoimmune responses and the unclear cellular sources and mechanisms underpinning islet regeneration in humans, effectively activating and leveraging endogenous regenerative capacity remains a challenge and an important research frontier.

During pancreatic development and repair, a wealth of literature suggests that a variety of progenitor cells—including pancreatic ductal epithelial cells, acinar cells, and pre-existing β cells—may participate in β cell regeneration. However, owing to the diversity of animal models, lineage tracing techniques, and injury methods, there remains significant debate over the origin of newly formed β cells. To further address these questions, scientists have begun to focus on how exogenous interventions can stimulate or reprogram these potential primitive cells, promoting their differentiation into β cells, ultimately facilitating islet regeneration.

Currently, mesenchymal stem cells (MSCs) and the growth factors they secrete are considered to have significant roles in tissue repair and immune regulation. Based on this, the present study further investigates whether secreted proteins derived from MSCs (conditioned media, CDM)—particularly under conditions of Wnt pathway activation—can induce and accelerate islet regeneration, and clarifies the true cellular sources of regenerated β cells.

Paper Source and Author Information

This research paper, titled “Contribution of cytokeratin 19-expressing cells towards islet regeneration induced by multipotent stromal cell secreted proteins,” was completed by Nazihah Rasiwala, Gillian I Bell, Anargyros Xenocostas, David A Hess, and others. The primary institutions involved were the Department of Physiology and Pharmacology at Western University, the Robarts Research Institute, and the Department of Hematology at London Health Sciences Center, all in Canada. The paper was published in 2025 by Oxford University Press in an academic journal and has been made open access, allowing for widespread academic dissemination and reuse.

Detailed Research Process

Overall Design

The study aimed to clarify whether cytokeratin 19 (CK19)-expressing cells can contribute to β cell regeneration under the stimulation of MSC secretions—particularly Wnt pathway-activated conditioned media (Wnt+ CDM)—as well as to determine the actual cellular origin of regenerated β cells. The research utilized a combination of advanced techniques, including β cell ablation, direct pancreatic injection of CDM, and genetic lineage tracing, comprising the following key experimental procedures:

1. MSC Culture and CDM Preparation

  • Source and Culture of MSCs: Bone marrow-derived MSCs from 8 human donors were cultured to passage 4 using AmnioMax™ medium supplemented with 15% fetal calf serum.
  • Wnt Pathway Activation: The Wnt pathway was activated by adding 10 μM GSK3 inhibitor CHIR99021; the control group received DMSO.
  • CDM Collection and Concentration: After 24 hours, both Wnt+ CDM and control CDM were collected and concentrated 20-fold using a 3kDa ultrafiltration spin column; total protein concentration was quantified at 0.1–0.25 μg/μl and frozen for future use.
  • MSC Phenotype Verification: Flow cytometry was used to confirm expression of MSC markers CD73, CD90, and CD105, with less than 1% expressing CD34 or CD45 to ensure MSC purity; ELISA and cellular fractionation confirmed upregulation of nuclear β-catenin by Wnt signaling.

2. Animal Model Establishment & Lineage Tracing

  • Lineage Tracing Mouse Model: CK19-CreERT and Rosa26-mTomato transgenic mice were crossed to generate animals with specific, inducible tracing of CK19+ cells.
  • Induction of Labeling: Each mouse received oral tamoxifen at 6 mg per day for 2 consecutive days, 7 and 6 days before the experiment, inducing Cre recombinase nuclear entry to permanently activate mTomato fluorescent protein expression.
  • β Cell Ablation Model: Five consecutive days of intraperitoneal injection with 50 mg/kg streptozotocin (STZ) were used to ablate β cells; only mice with non-fasting blood glucose >12 mmol/L were included as experimental subjects.

3. Intrapancreatic CDM Injection and Animal Monitoring

  • CDM Injection Groups: In STZ-induced hyperglycemic mice, on day 10 (short-term, 21-day monitoring) or day 14 (long-term, 42-day monitoring), 20 μl of Wnt+ CDM, control CDM, or standard culture medium (containing 2–5 μg total protein) was directly injected into the pancreas.
  • Physiological Monitoring: Non-fasting blood glucose and body weight were measured twice weekly; on day 42, a glucose tolerance test was performed (intravenous glucose bolus followed by periodic blood glucose assessment).
  • Cell Proliferation Assay: After CDM injection, some mice received daily intraperitoneal injections of 5-ethynyl-2’-deoxyuridine (EdU) for 3 days to label proliferating cells.

4. Histopathology and Immunohistochemistry

  • Section Preparation and Staining: Pancreatic tissue was cryosectioned and stained with multiplex immunofluorescence for insulin, glucagon, CK19, and the acinar marker MPX1.
  • Quantification of β Cell Quality: Calculated as the area of insulin-positive cells / total pancreas area × pancreas weight; islet number and β/α cell ratio were also assessed.
  • Lineage Transdifferentiation Quantification: The proportion of tdTomato+/insulin+ double-positive cells within islets was determined to assess the contribution of CK19-derived cells to β cells.
  • Flow Cytometry Analysis: Pancreatic tissue was digested into a single-cell suspension and analyzed by flow cytometry to identify CK19, INSULIN, and tdTomato triple-labeled cell populations for high-resolution quantification.

5. Statistical Analysis

  • Data Processing: GraphPad Prism 9 was used, employing two-way ANOVA, one-way ANOVA with Tukey’s multiple comparison; significance set at *p<0.05, **p<0.01, ***p<0.001.

Main Experimental Results

1. Analysis of MSC Secretions and Wnt+ CDM Composition

  • Stimulation of MSCs with CHIR99021 significantly upregulated nuclear β-catenin, verifying strong Wnt pathway activation.
  • Both Wnt+ and control CDM extractions maintained excellent MSC phenotype and viability post-collection.

2. CK19+ Cell Lineage Tracing Efficiency and Specificity

  • Following tamoxifen dose optimization, up to 8% of total pancreatic cells and 21.7% of CK19+ cells were successfully tdTomato labeled.
  • A portion of acinar cells (approximately 17% of MPX1+ cells) also displayed low-level labeling, and 2.2% of insulin+ cells were baseline tdTomato+, suggesting some “leakage” in labeling.
  • Nonetheless, CK19 protein expression remained mainly confined to pancreatic ductal epithelium, providing a solid foundation for lineage tracing.

3. CDM Promotes Islet Regeneration and β Cell Recovery

  • 21-Day Short-Term Analysis: Mice injected with Wnt+ CDM and control CDM showed clear downward blood glucose trends and significant β cell mass restoration compared to basic medium group (Wnt+ 0.45 mg vs control 0.39 mg vs basal medium 0.19 mg).
  • 42-Day Long-Term Analysis: The Wnt+ CDM group exhibited better glycemic control than basal medium controls, with significant improvements in glucose tolerance. Female mice showed more pronounced β cell mass improvement under Wnt+ CDM stimulation.
  • Flow Cytometric Analysis: At 21 days, Wnt+ group showed an increase in insulin+/tdTomato+ double-positive cells from 1% to 5%; the control CDM group reached 4.3%, while the basic medium group remained at 1%.

4. Contribution of CK19+ Lineage to β Cell Regeneration

  • After Wnt+ CDM injection, a small number of CK19+ lineage-traced cells acquired insulin expression and integrated into islets by day 11, mainly within larger, mature islet structures, with low expression in newly formed small islets.
  • Simultaneously, transdifferentiation of CK19+ cells was also observed in the control CDM group, indicating that factors secreted by MSCs—rather than Wnt activation alone—play a fundamental role in islet regeneration.
  • By day 42, the proportion of CK19+/INSULIN+ double-positive cells within islets decreased, suggesting that their contribution diminishes in later stages, possibly with other cell types dominating regeneration.

5. Pancreatic Duct-Islet Association and Mechanism of β Cell Proliferation

  • The frequency of direct contact between islets and ducts was not significantly different among groups, but at 42 days, the proportion of CK19+ cells within islets was higher in the Wnt+ CDM and control CDM groups.
  • EdU labeling of new β cell proliferation: approximately 1% in all three groups, with no significant difference among control and CDM groups. This indicates that β cell regeneration is not simply dominated by self-proliferation of pre-existing β cells.

Conclusions and Value

This study, for the first time, combines an efficient lineage tracing system with comprehensive animal experiments and molecular/cellular assays to confirm that CK19+ ductal/acinar-derived cells can partially transdifferentiate into insulin-secreting β cells under MSC secretory factor stimulation, directly participating in islet regeneration in damaged mouse pancreas. MSC secretions, especially after Wnt pathway activation, act as a novel biotherapeutic with high regenerative efficacy, low immunogenicity, and obviate the need for direct MSC transplantation, showing notable effects on tissue regeneration and glucose metabolism improvement.

The research also demonstrates that, while CK19+ lineage contribution is definite, only about 5% of new β cells arise from such transdifferentiation, implying that other important cellular sources/pathways exist for islet regeneration. Future studies tracing routes such as alpha-to-beta or acinar-to-beta transdifferentiation are warranted. Moreover, optimization and precise characterization of MSC secretome components lay the theoretical and experimental foundation for future development of cell-free regenerative drugs for diabetes.

Highlights and Innovations

  1. First Quantitative Demonstration of CK19+ Lineage Contribution to β Cell Regeneration: Provides direct animal experimental evidence for the differentiation of ductal/acinar cells into functional β cells.
  2. MSC Secretome-induced Regeneration Paradigm: Proposes a feasible path toward secretome-based biological drugs as alternatives to live cell transplantation, reducing safety and regulatory concerns for clinical translation.
  3. Innovative Application of Lineage Tracing: Utilizes CK19-CreERT × Rosa26-mTomato dual transgenic model combined with flow cytometric sorting for high-precision tracking of de novo β cell lineage sources.
  4. Wnt Signaling Enhances MSC Regenerative Capacity: Shows that modulation of the Wnt pathway can significantly boost MSC secretome production of regenerative proteins, providing a new approach for standardizing personalized cell products.
  5. Model Transformation in Experimental Animals: Demonstrates the regenerative effect of MSC secretions not only in immunodeficient but also in immunocompetent mice, enhancing clinical relevance of the findings.

Other Valuable Information

  • The study is well-designed, with ample sample size, results presented as mean±SD, and rigorous statistical methods, providing strong support for the reliability of the findings.
  • Results suggest sex-dependent differences in response to CDM regenerative effects, offering insights for future personalized medicine research.
  • The research team is further exploring the immunomodulatory effects of MSC secretions in autoimmune diabetes models (such as NOD mice) and tracing additional potential transdifferentiation lineages, providing new evidence for the multi-origin theory of β cell regeneration.

Scientific Significance and Impact

This study deepens the understanding of pancreatic β cell regeneration mechanisms at the cellular and molecular level, advancing regenerative medicine and molecular diabetes therapy, and provides a solid theoretical foundation for clinical development of safe, effective, and well-defined islet regenerative drugs such as MSC secretome preparations. Addressing the current incurable status of type 1 diabetes, this research challenges the traditional notion that β cell regeneration derives solely from self-duplication, and points toward future strategies of functional islet reconstruction by manipulating endogenous progenitor cells and coordinating multi-lineage regeneration. Overall, this research brings new hope for a cure for type 1 diabetes, enriches theoretical frameworks in tissue regeneration and cellular differentiation, and holds significant scientific and practical application value.