In Vivo Differentiation of Endogenous Bone Marrow-Derived Cells into Insulin-Producing Cells Using Four Soluble Factors

Seung-Ah Lee; Subin Kim; Seog-Young Kim; Jong Yoen Park; Hye Seung Jung; Sung Soo Chung; Kyong Soo Park

doi:10.4093/dmj.2024.0174

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Lee, Kim, Kim, Park, Jung, Chung, and Park: In Vivo Differentiation of Endogenous Bone Marrow-Derived Cells into Insulin-Producing Cells Using Four Soluble Factors

Brief Report

Type 1 Diabetes

Diabetes Metab J (Seoul) 2025;49(1):150-159.

Published online: 24 October 2024

DOI: https://doi.org/10.4093/dmj.2024.0174

In Vivo Differentiation of Endogenous Bone Marrow-Derived Cells into Insulin-Producing Cells Using Four Soluble Factors

Seung-Ah Lee^1,^*

, Subin Kim^2,^*

, Seog-Young Kim³, Jong Yoen Park⁴, Hye Seung Jung⁵, Sung Soo Chung³, Kyong Soo Park^1,^4,⁵

¹Genomic Medicine Institute, Medical Research Center, Seoul National University, Seoul, Korea

²Department of Translational Medicine, Seoul National University College of Medicine, Seoul, Korea

³Biomedical Research Institute, Seoul National University Hospital, Seoul, Korea

⁴Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, Seoul, Korea

⁵Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea

Corresponding author: Kyong Soo Park https://orcid.org/0000-0003-3597-342X Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, 101 Daehak-ro, Jongno-gu, Seoul 03080, Korea E-mail: kspark@snu.ac.kr

^* Seung-Ah Lee and Subin Kim contributed equally to this study as first authors.

Received 2 April 2024 Accepted 17 June 2024

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Four soluble factors—putrescine, glucosamine, nicotinamide, and signal transducer and activator of transcription 3 (STAT3) inhibitor BP-1-102—were shown to differentiate bone marrow mononucleated cells (BMNCs) into functional insulin-producing cells (IPCs) in vitro. Transplantation of these IPCs improved hyperglycemia in diabetic mice. However, the role of endogenous BMNC regeneration in this effect was unclear. This study aimed to evaluate the effect of these factors on in vivo BMNC differentiation into IPCs in diabetic mice. Mice were orally administered the factors for 5 days, twice at 2-week intervals, and monitored for 45–55 days. Glucose tolerance, glucose-stimulated insulin secretion, and pancreatic insulin content were measured. Chimeric mice harboring BMNCs from insulin promoter luciferase/green fluorescent protein (GFP) transgenic mice were used to track endogenous BMNC fate. These factors lowered blood glucose levels, improved glucose tolerance, and enhanced insulin secretion. Immunostaining confirmed IPCs in the pancreas, showing the potential of these factors to induce β-cell regeneration and improve diabetes treatment.

Keywords: Bone marrow cells, Cell differentiation, Insulin, Insulin-secreting cells, Regeneration

KEY FIGURE

Highlights

• Oral administration of putrescine, glucosamine, nicotinamide, and BP-1-102 induces BMNCs into functional IPCs.

• These factors improve insulin secretion and restore beta-cell mass in diabetic mice.

INTRODUCTION

Diabetes has become a challenging global healthcare burden; approximately 536.6 million patients (10.5%) were recorded in 2021, and this is expected to reach 783.2 million (12.2%) by 2045 [1]. Type 1 diabetes mellitus is an insulin-dependent autoimmune disorder characterized by insulin-producing pancreatic β-cell destruction, whereas type 2 diabetes mellitus results from insulin resistance in peripheral tissues (e.g., muscles, liver, and adipose tissues) [2-4], and progressive decline in β-cell secretion [5]. Although islet transplantation helps cure diabetes, this approach is limited by a shortage of suitable donors, poor function, and immune rejection [6,7]. Current regimens involving both anti-diabetic drugs and daily insulin injections also do not provide efficient blood glucose control in a manner similar to that of functional pancreatic β-cells, nor do these treatments prevent diabetes progression. Thus, a therapeutic approach that replenishes β-cell mass to enhance insulin secretion in patients with diabetes is an important unmet need for all patients with diabetes, regardless of the type of diabetes.

Bone marrow-derived cells are characterized by their ability to differentiate into multiple tissue-specific cell types, secrete various factors, modulate the local microenvironment, and activate endogenous progenitor cells [8-10]. Studies using cell lineage tracking methods have identified the differentiation of bone marrow-derived cells into specific cells residing in various tissues [11-13]. Tissue injury effectively enhances the recruitment of bone marrow-derived cells for a non-hematopoietic fate [13-16]. This may be due to the migration of bone marrow cells throughout the body that acts as a backup system to augment the intrinsic regenerative capacity of each organ. Thus, bone marrow-derived cells are considered potential tools for damaged tissue regeneration and as treatments for multiple diseases. The effect of bone marrow cells on alleviating diabetes has been reported in numerous animal models [17-21].

A previous study showed that grafting bone marrow mononucleated cell (BMNC)-induced insulin-producing cells (IPCs) using four differentiation-inducing factors, namely, putrescine, glucosamine, nicotinamide, and signal transducer and activator of transcription 3 (STAT3) inhibitor BP-1-102, ameliorated hyperglycemia in streptozotocin (STZ)-induced diabetic mice, which was characterized by numerous newly formed IPCs [22]. However, the newly formed IPCs were not fully functional as blood glucose-regulating cells, and glucose-stimulated secreted insulin levels were not completely normalized. Approaches aimed at promoting the regeneration and maturation of β-cells require further investigation.

Therefore, this study aimed to investigate whether a differentiation cocktail (diff. cocktail) constituting the four extrinsic soluble factors could directly differentiate endogenous bone marrow-derived cells into IPCs and stimulate β-cell regeneration in a diabetic mouse model to determine whether islet function could be restored.

METHODS

Animals

All mice experiments were approved by the Institutional Animal Care and Use Committee of Seoul National University (SNU-191022-3-5). Efforts were made to minimize animal suffering and the number of animals used in the experiments.

Wild-type C57BL/6 mice (Orient-Bio Co. Ltd., Seoul, Korea) and C57BL/6-Tg (insulin 2 [Ins2]-Luc/enhanced green fluorescent protein [EGFP]/TK) 300 Kauf/J (Jackson Laboratories, Bar Harbor, ME, USA; referred to as MIP-Luc/GFP in this study) were maintained on a 12-hour light/dark cycle and had ad libitum access to both diet and water in a specific pathogen-free facility at the Biomedical Center for Animal Resource Development of Seoul National University. MIP-Luc/GFP mice characteristics have been described previously [20].

Induction of diabetes

Diabetes was induced in male C57BL/6 mice (aged 8 to 12 weeks) fasted for 6 hours through a single intraperitoneal injection of 150 mg/kg STZ (Sigma-Aldrich, St. Louise, MI, USA) dissolved in 0.1 mL of 0.1 M citrate buffer (pH 4.5). Mice injected with STZ were considered diabetic when blood glucose levels exceeded 350 mg/dL for 2 consecutive days. Blood glucose levels were monitored using a standard glucometer (AccuChek Active, Roche Diagnostics, Mannheim, Germany) during daytime hours (9:00 to 11:00 AM) in mice fed ad libitum.

Administration of mice with differentiation cocktail

Non-diabetic (normal) or diabetic animals were subjected to oral gavage of physiologically relevant doses of differentiation-inducing factors (200 mg/kg of putrescine, 200 mg/kg of glucosamine, 500 mg/kg of nicotinamide, 3 mg/kg of BP-1-102; referred to as 1×diff. cocktail) or its vehicle for 5 consecutive days twice at 2-week intervals, and the mice were monitored for 55 days. Random blood glucose levels in each mouse were determined using a glucometer, as described previously. Food and water intake and body weight were measured every 3 to 4 days.

Glucose tolerance test and in vivo glucose-stimulated insulin secretion

Mice were fasted overnight, and blood glucose was measured following intraperitoneal administration of glucose (2 g/kg body weight) at the indicated time points. The area under the curve (AUC) of glucose was calculated. For in vivo glucose-stimulated insulin secretion (GSIS), blood samples were collected from the tail vein at baseline insulin levels (0 minute) and 60 minutes after glucose loading. Plasma insulin levels were measured using the Mouse Ultrasensitive Insulin enzyme-linked immunosorbent assay (ELISA) (ALPCO, Salem, NH, USA), according to the manufacturer’s instructions.

Immunohistochemistry

Tissue biopsies were fixed overnight in 10% (v/v) neutral buffered formalin, embedded in paraffin, sectioned, and stained with anti-insulin (Cell Signaling, Danvers, MA, USA), pancreatic and duodenal homeobox 1 (PDX1; Abcam, Cambridge, UK), and NK6 homeobox 1 (NKX6.1; Cell Signaling) antibodies. All histochemical examinations were performed at the Pathology Core Facility at the Seoul National University Hospital Biomedical Research Institute, and images were captured using the ECLIPSE Ci-L microscope (Nikon Instruments Inc., Melville, NY, USA).

Pancreatic insulin contents

One-half of the pancreas was placed into acidic ethanol (1.5% HCl in 70% ethanol) and incubated overnight at –20°C. The tissues were homogenized and incubated overnight at –20°C. After centrifugation at 2,000 rpm for 15 minutes, the aqueous solution was neutralized with an equal volume of 1 M Tris (pH 7.5). Insulin levels were measured using the mouse insulin ELISA kit (ALPCO), and the insulin content was normalized to the amount of protein in the tissue homogenates.

Generation of chimeric diabetic mice

Chimeric mice were generated by transferring BMNCs from MIP-Luc/GFP mice via the retro-orbital vein into lethally irradiated C57BL/6 mice. Briefly, recipient C57BL/6 mice (8 to 12 weeks old) were placed in a pie chamber and exposed to a lethal dose of irradiation (1,000 cGy) at two equal doses of 500 cGy at 4-hour intervals. One day after the irradiation of the recipient mice, BMNCs were prepared by flushing the femurs and tibias of MIP-Luc/GFP donor mice. Whole BMNCs were suspended in BD Pharm Lyse buffer (BD Biosciences, San Jose, CA, USA) to remove red blood cells, washed, and resuspended in connaught medical research laboratories medium without glucosamine (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (ThermoFisher Scientific) and 1% penicillin-streptomycin (ThermoFisher Scientific). The irradiated recipient C57BL/6 mice were intravenously administered with 1×10⁶ cells/recipient from donors via the retro-orbital vein using a 27-G needle and maintained in neomycin sulfate water (2 mg/mL) for 2 weeks to prevent infection. After BMNC reconstitution (4 to 5 weeks), the recipient mice were intraperitoneally injected with 50 mg/kg STZ for 5 consecutive days to induce diabetes. Recipient mice administered STZ were considered diabetic when their blood glucose levels exceeded 300 mg/dL for 3 consecutive days. Chimeric mice with STZ-induced hyperglycemia were orally administered the diff. cocktail for 5 consecutive days, twice at 2-week intervals (Fig. 1A), and monitored for 45 days (Fig. 2A).

Immunofluorescence staining

Sectioned tissues were washed three times with phosphate-buffered saline (PBS), permeabilized with PBS containing 0.3% Triton X-100 (Sigma-Aldrich), and blocked with PBS containing 10% normal donkey serum (NDS; Jackson ImmunoResearch, West Grove, PA, USA) for 1 hour at room temperature (20°C to 22°C). The sections were then incubated with primary antibodies (Supplementary Table 1) in 1% NDS overnight at 4°C, washed three times with PBS containing 1% NDS, and incubated with secondary antibodies in 1% NGS for 2 hours at 20°C to 22°C. The cells were counterstained with 4´,6-diamidino-2-phenylindole (DAPI, 1:2,500; Molecular Probes, Eugene, OR, USA) and observed using a confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with appropriate filters. The islet areas in pancreatic tissue images were manually traced and analyzed using the Metamorph Image Analysis tool (Molecular Devices, San Jose, CA, USA).

Quantitative real-time polymerase chain reaction

Total RNA was isolated from the cells using TRIzol reagent (Life Technologies, Waltham, MA, USA). cDNA was synthesized from 1 µg total RNA using SuperScript II Reverse Transcriptase (Promega, Madison, WI, USA), per the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (qPCR) was performed using SYBR Premix ExTaq (Takara, Shiga, Japan) and ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Each cycle threshold (Ct) value was subtracted from the glyceraldehyde 3-phosphate dehydrogenase (Gapdh) Ct value of the same samples (dCt) and then from the dCt value of each control set (ddCt). Relative mRNA expression levels are expressed as 2^–ddCt. The primer sequences are listed in Supplementary Table 2.

Statistical analysis

Data are presented as the mean±standard error of the mean, unless otherwise specified. Statistical significance was determined using the unpaired Student t-test. All statistical analyses were performed using GraphPad Prism version 10 (GraphPad Software Inc., San Diego, CA, USA). A P value of <0.05 was considered significant.

RESULTS

Oral administration of the four differentiation-inducing factors improved hyperglycemia in diabetic mice

The STZ-treated mice administered with diff. cocktail (Fig. 1A) showed a modest increase in body weight, but the difference was not significant (Fig. 1B). Random blood glucose levels were elevated in the STZ-treated mice. Compared with the STZ-only treated mice, STZ-induced diabetic mice with oral administration of the diff. cocktail had significantly lower blood glucose levels from day 17 following oral administration to the end of the study (Fig. 1C). The aberrant water intake behavior was significantly better in STZ-induced diabetic mice administered diff. cocktail than in vehicle-administered STZ-induced diabetic mice. However, food intake did not change, regardless of the presence of diff. cocktail (Supplementary Fig. 1). Moreover, in the STZ-treated group, diff. cocktail-administered mice showed markedly lower fasting blood glucose levels and better glucose tolerance 42 days after oral administration than the matched controls. The glucose clearance AUC was reduced following an intraperitoneal glucose challenge for diff. cocktail-treated mice in the STZ-induced diabetic group (Fig. 1D). In vivo GSIS showed significantly higher levels of plasma insulin at 1 hour post-glucose administration in diff. cocktail-administered mice than in corresponding controls in the STZ-treated group (Fig. 1E).

Increased expression of β-cell markers in the pancreas of STZ-induced diabetic mice administered with the diff. cocktail

Immunohistochemistry analysis revealed that islet integrity was disrupted, with uneven boundaries, and insulin-, PDX1-, or NKX6.1-positive cells were rarely observed in the pancreatic tissues of STZ-induced diabetic mice. However, diff. cocktail administration partially restored β-cell destruction caused by STZ induction, as indicated by higher pancreatic levels of insulin-, PDX1-, or NKX6.1-positive cells in mice administered with diff. cocktail than in STZ-treated controls (Fig. 3A). Hence, the pancreatic insulin content in these mice was significantly higher than that in the matched controls (Fig. 3B).

Determination of endogenous BMNC fate in the pancreas

Chimeric mice systemically administered with diff. cocktail, as indicated in Fig. 2A, started recovering the body weight from day 28 after oral administration (Supplementary Fig. 2A). Random blood glucose levels were significantly lower starting from day 7 following oral administration to the end of the study in the STZ-only-treated group (Supplementary Fig. 2B). Consistent with the results shown in Fig. 1D, the chimeric mice administered with the diff. cocktail under hyperglycemic conditions showed improved glucose tolerance, and fasting blood glucose levels on day 36 after oral administration were reduced in the diff. cocktail-administered diabetic mice (Supplementary Fig. 2C). Furthermore, fasting plasma insulin levels were significantly increased with the administration of the diff. cocktail (Supplementary Fig. 2D).

Immunofluorescence staining of pancreatic tissues from chimeric mice confirmed the presence of increased insulin- and GFP-double-positive cell levels in the presence of diff. cocktail (Fig. 2B). These observations were determined by quantification of immunofluorescence images using the Metamorph Image Analysis tool, which revealed a significant increase in insulin-positive and GFP-positive cell areas in the group administered with diff. cocktail. The area of insulin- and GFP-double-positive cells in the pancreatic tissues was also increased by 5.1-fold in diff. cocktail-administered mice, thereby suggesting that diff. cocktail effectively differentiated endogenous BMNCs into IPCs (Fig. 2C). Consistent with these findings, qPCR analysis of the pancreatic tissues from diff. cocktail-administered mice showed an increase in the mRNA levels of Gfp, Ins1, and Ins2 (Fig. 2D), and the pancreatic insulin content in these mice were increased by two-fold (Fig. 2E).

DISCUSSION

The specific contribution of endogenous BMNC-mediated regeneration to the anti-diabetic efficacy of four extrinsic soluble factors has not been elucidated to date. In the current study, oral administration of the four factors (diff. cocktail) effectively lowered the blood glucose levels, improved glucose tolerance, and enhanced glucose-stimulated insulin release in STZ-induced diabetic mice. Direct lineage tracing of BMNCs from MIP-Luc/GFP mice with the diff. cocktail in diabetic mice confirmed that the bone marrow pluripotent cells were capable of engrafting to the pancreas and being programmed in vivo to become functional IPCs. The number and function of β-cells were improved in STZ-treated mice administered with the diff. cocktail. Collectively, these results support that the diff. cocktail exerts an anti-diabetic effect by improving insulin secretion and partially restoring β-cell mass in diabetic mice. These findings will be helpful in developing anti-diabetic modalities that improve insulin secretion and restore β-cell mass.

Bone marrow cells contribute to pancreatic β-cell regeneration at a low frequency, ranging from 1% to 3% [10,23-25]. Ianus et al. [10] provided in vivo evidence that mouse bone marrow-derived cells could differentiate into a glucose-competent pancreatic endocrine β-cell phenotype and demonstrated that this process was unlikely due to in vivo cell fusion. Previous studies, including those of ours, affirmed that allogeneic bone marrow transplantation with as low as 1% chimerism in the pancreatic islets can reverse the diabetogenic process in diabetic mice [20,24]. Hence, we believe that oral supplementation of diff. cocktail in this study more efficiently facilitated the differentiation of endogenous BMNCs into functional IPCs in diabetic mice. The efficiency of GFP-positive cells derived from the bone marrow to differentiate into IPCs in response to oral supplementation of diff. cocktail was 5.1-fold higher in the diff. cocktail-treated mice than in the vehicle-treated control. The pancreatic islets [26,27], pancreatic duct tissue [28,29], and liver [30] cells have the potential to differentiate into cells with a pancreatic endocrine phenotype. Thus, we cannot rule out the possibility that the diff. cocktail also contributed to the differentiation of these cells into IPCs. However, this hypothesis needs to be confirmed in further studies.

The gut is a potential source of stem cells that differentiate into endogenous IPCs because it contains enteroendocrine cells that express molecules involved in glucose responsiveness and processing of prohormones; it also shares many differentiation pathways with the pancreas [31]. A previous lineage-tracing study using BMNCs from MIP-Luc/GFP mice showed the presence of GFP- and insulin-double-positive cells scattered in the lamina propria of the intestinal villi [20]; however, further studies are needed to determine the extent to which the intestinal GFP-positive cells secrete insulin to alleviate hyperglycemia.

However, the specific mechanism by which the combination of these four factors effectively differentiates BMNCs into IPCs remains unknown. Putrescine, glucosamine, nicotinamide, and BP-1-102 are beneficial to the pancreas as they maintain the optimal quality and function of β-cells via the upregulation of β-cell marker genes [32-35]. A previous study reported that cotreatment with putrescine and glucosamine improved the differentiation of BMNCs into IPCs, and BMNCs treated with the four extrinsic factors had the most effective mRNA induction of β-cell markers. This indicated a synergistic effect of the four extrinsic factors in BMNC-derived IPCs [22]. However, further research is needed to understand the detailed mechanisms by which the combined treatment with the four soluble factors affects pancreatic β-cell production and maturation.

In conclusion, oral administration of the diff. cocktail in STZ-induced diabetic mice caused the effective differentiation of endogenous BMNCs into IPCs, ultimately affecting β-cell regeneration. The anti-diabetic effects of the diff. cocktail were confirmed by the reversal of hyperglycemia and in vivo insulin release in a glucose-responsive manner and by the presence of an increased insulin-positive cell level in the pancreas. Direct lineage tracing of BMNCs from MIP-Luc/GFP mice in combination with the diff. cocktail confirmed that the bone marrow pluripotent cells were capable of engrafting into the pancreas and being programmed in vivo to become functional IPCs. Collectively, our results indicated that diff. cocktail treatment promoted β-cell regeneration with restored islet function and long-term hypoglycemic effects in an STZ-induced diabetic mouse model. Hence, the diff. cocktail exerts beneficial effects on the pancreas during the metabolic stress state, ultimately leading to efficient and optimal insulin synthesis and secretion. These findings provide new insights into therapeutic options for diabetes.

SUPPLEMENTARY MATERIALS

Supplementary materials related to this article can be found online at https://doi.org/10.4093/dmj.2024.0174.

Supplementary Table 1.

List of antibodies used for immunofluorescence staining

dmj-2024-0174-Supplementary-Table-1.pdf

Supplementary Table 2.

Primer sequences for polymerase chain reaction

dmj-2024-0174-Supplementary-Table-2.pdf

Supplementary Fig. 1.

Effect of differentiation-inducing factors orally supplemented in mice. Water (A) and food (B) intake is measured every 3 to 4 days. All data are presented as the mean±standard error of the mean, obtained from 4–5 mice per group. STZ, streptozotocin; Diff. cocktail, combination of putrescine, glucosamine, nicotinamide, and BP-1-102. ^aP<0.001.

dmj-2024-0174-Supplementary-Fig-1.pdf

Supplementary Fig. 2.

Characterization of streptozotocin (STZ)-induced diabetic chimeric mice harboring bone marrow mononucleated cells (BMNCs) from MIP-Luc/green fluorescent protein (GFP) (Jackson Laboratories) systemically administered with differentiation (diff.) cocktail. Body weight (A) and changes in random feeding blood glucose levels (B) of mice administered with diff. cocktail (filled circle, straight line) or comparable vehicle (empty circle, dotted line) are measured on the indicated days. (C) Blood glucose responses from the intraperitoneal glucose tolerance test (ipGTT). Glucose (1 g/kg, body weight) is injected after overnight fasting at 36 days after systemic infusion with diff. cocktail. (D) Plasma insulin levels after overnight fasting at 46 days following oral administration are measured using ultrasensitive mouse insulin enzyme-linked immunosorbent assay (ELISA). All data are presented as the mean±standard error of the mean, obtained from 5–8 mice per group. Diff. cocktail, combination of putrescine, glucosamine, nicotinamide, and BP-1-102. ^aP<0.05, ^bP<0.01.

dmj-2024-0174-Supplementary-Fig-2.pdf

Notes

CONFLICTS OF INTEREST

Kyong Soo Park has been honorary editors of the Diabetes & Metabolism Journal since 2020. He was not involved in the review process of this article. Otherwise, there was no conflict of interest.

AUTHOR CONTRIBUTIONS

Conception or design: S.A.L., H.S.J., K.S.P.

Acquisition, analysis, or interpretation of data: all authors.

Drafting the work or revising: S.A.L., H.S.J., S.S.C., K.S.P.

Final approval of the manuscript: K.S.P.

FUNDING

This study was supported by grants from the Korea Health Technology R&D Project “Strategic Center of Cell and Bio Therapy for Heart, Diabetes & Cancer” through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare (MHW) (HI17C2085) and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2020R1A6A1A03047972), Republic of Korea. This research was also supported by the BK21FOUR Program of the National Research Foundation of Korea (NRF), funded by the Ministry of Education (5120200513755) and by grants from the Seoul National University Hospital Research Fund (No. 0320223020) and the Yangyoung Foundation, Republic of Korea.

ACKNOWLEDGMENTS

None

REFERENCES

1. Sun H, Saeedi P, Karuranga S, Pinkepank M, Ogurtsova K, Duncan BB, et al. IDF Diabetes Atlas: global, regional and country-level diabetes prevalence estimates for 2021 and projections for 2045. Diabetes Res Clin Pract. 2022; 183:109119.

2. Olokoba AB, Obateru OA, Olokoba LB. Type 2 diabetes mellitus: a review of current trends. Oman Med J. 2012; 27:269–73.

3. Reed J, Bain S, Kanamarlapudi V. A review of current trends with type 2 diabetes epidemiology, aetiology, pathogenesis, treatments and future perspectives. Diabetes Metab Syndr Obes. 2021; 14:3567–602.

4. Chiang JL, Kirkman MS, Laffel LM, Peters AL; Type 1 Diabetes Sourcebook Authors. Type 1 diabetes through the life span: a position statement of the American Diabetes Association. Diabetes Care. 2014; 37:2034–54.

5. ElSayed NA, Aleppo G, Aroda VR, Bannuru RR, Brown FM, Bruemmer D, et al. 2. Classification and diagnosis of diabetes: standards of care in diabetes-2023. Diabetes Care. 2023; 46(Suppl 1):S19–40.

6. Rother KI, Harlan DM. Challenges facing islet transplantation for the treatment of type 1 diabetes mellitus. J Clin Invest. 2004; 114:877–83.

7. Croon AC, Karlsson R, Bergstrom C, Bjorklund E, Moller C, Tyden L, et al. Lack of donors limits the use of islet transplantation as treatment for diabetes. Transplant Proc. 2003; 35:764.

8. Ciceri F, Piemonti L. Bone marrow and pancreatic islets: an old story with new perspectives. Cell Transplant. 2010; 19:1511–22.

9. Kassem M, Abdallah BM. Human bone-marrow-derived mesenchymal stem cells: biological characteristics and potential role in therapy of degenerative diseases. Cell Tissue Res. 2008; 331:157–63.

10. Ianus A, Holz GG, Theise ND, Hussain MA. In vivo derivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion. J Clin Invest. 2003; 111:843–50.

11. Hess DC, Hill WD, Martin-Studdard A, Carroll J, Brailer J, Carothers J. Bone marrow as a source of endothelial cells and NeuN-expressing cells after stroke. Stroke. 2002; 33:1362–8.

12. Watanabe T, Masamune A, Kikuta K, Hirota M, Kume K, Satoh K, et al. Bone marrow contributes to the population of pancreatic stellate cells in mice. Am J Physiol Gastrointest Liver Physiol. 2009; 297:G1138–46.

13. Feng G, Mao D, Che Y, Su W, Wang Y, Xu Y, et al. The phenotypic fate of bone marrow-derived stem cells in acute kidney injury. Cell Physiol Biochem. 2013; 32:1517–27.

14. Lagasse E, Connors H, Al-Dhalimy M, Reitsma M, Dohse M, Osborne L, et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med. 2000; 6:1229–34.

15. Tanaka R, Komine-Kobayashi M, Mochizuki H, Yamada M, Furuya T, Migita M, et al. Migration of enhanced green fluorescent protein expressing bone marrow-derived microglia/macrophage into the mouse brain following permanent focal ischemia. Neuroscience. 2003; 117:531–9.

16. Rennert RC, Sorkin M, Garg RK, Gurtner GC. Stem cell recruitment after injury: lessons for regenerative medicine. Regen Med. 2012; 7:833–50.

17. Arany EJ, Waseem M, Strutt BJ, Chamson-Reig A, Bernardo A, Eng E, et al. Direct comparison of the abilities of bone marrow mesenchymal versus hematopoietic stem cells to reverse hyperglycemia in diabetic NOD.SCID mice. Islets. 2018; 10:137–50.

18. Lee RH, Seo MJ, Reger RL, Spees JL, Pulin AA, Olson SD, et al. Multipotent stromal cells from human marrow home to and promote repair of pancreatic islets and renal glomeruli in diabetic NOD/scid mice. Proc Natl Acad Sci U S A. 2006; 103:17438–43.

19. Iskovich S, Goldenberg-Cohen N, Stein J, Yaniv I, Fabian I, Askenasy N. Elutriated stem cells derived from the adult bone marrow differentiate into insulin-producing cells in vivo and reverse chemical diabetes. Stem Cells Dev. 2012; 21:86–96.

20. Oh JE, Choi OK, Park HS, Jung HS, Ryu SJ, Lee YD, et al. Direct differentiation of bone marrow mononucleated cells into insulin producing cells using pancreatic β-cell-derived components. Sci Rep. 2019; 9:5343.

21. Oh SH, Muzzonigro TM, Bae SH, LaPlante JM, Hatch HM, Petersen BE. Adult bone marrow-derived cells trans-differentiating into insulin-producing cells for the treatment of type I diabetes. Lab Invest. 2004; 84:607–17.

22. Lee SA, Kim S, Kim SY, Park JY, Nan J, Park HS, et al. Direct differentiation of bone marrow mononucleated cells into insulin-producing cells using 4 specific soluble factors. Stem Cells Transl Med. 2023; 12:485–95.

23. Trucco M. Regeneration of the pancreatic beta cell. J Clin Invest. 2005; 115:5–12.

24. Zorina TD, Subbotin VM, Bertera S, Alexander AM, Haluszczak C, Gambrell B, et al. Recovery of the endogenous beta cell function in the NOD model of autoimmune diabetes. Stem Cells. 2003; 21:377–88.

25. Hess D, Li L, Martin M, Sakano S, Hill D, Strutt B, et al. Bone marrow-derived stem cells initiate pancreatic regeneration. Nat Biotechnol. 2003; 21:763–70.

26. Zulewski H, Abraham EJ, Gerlach MJ, Daniel PB, Moritz W, Muller B, et al. Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes. Diabetes. 2001; 50:521–33.

27. Abraham EJ, Leech CA, Lin JC, Zulewski H, Habener JF. Insulinotropic hormone glucagon-like peptide-1 differentiation of human pancreatic islet-derived progenitor cells into insulin-producing cells. Endocrinology. 2002; 143:3152–61.

28. Bonner-Weir S, Taneja M, Weir GC, Tatarkiewicz K, Song KH, Sharma A, et al. In vitro cultivation of human islets from expanded ductal tissue. Proc Natl Acad Sci U S A. 2000; 97:7999–8004.

29. Ramiya VK, Maraist M, Arfors KE, Schatz DA, Peck AB, Cornelius JG. Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells. Nat Med. 2000; 6:278–82.

30. Yang L, Li S, Hatch H, Ahrens K, Cornelius JG, Petersen BE, et al. In vitro trans-differentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells. Proc Natl Acad Sci U S A. 2002; 99:8078–83.

31. Mojibian M, Glavas MM, Kieffer TJ. Engineering the gut for insulin replacement to treat diabetes. J Diabetes Investig. 2016; 7(Suppl 1):87–93.

32. Kulkarni A, Anderson CM, Mirmira RG, Tersey SA. Role of polyamines and hypusine in β cells and diabetes pathogenesis. Metabolites. 2022; 12:344.

33. Hong Y, Park EY, Kim D, Lee H, Jung HS, Jun HS. Glucosamine potentiates the differentiation of adipose-derived stem cells into glucose-responsive insulin-producing cells. Ann Transl Med. 2020; 8:561.

34. Wong RS. Extrinsic factors involved in the differentiation of stem cells into insulin-producing cells: an overview. Exp Diabetes Res. 2011; 2011:406182.

35. Miura M, Miyatsuka T, Katahira T, Sasaki S, Suzuki L, Himuro M, et al. Suppression of STAT3 signaling promotes cellular reprogramming into insulin-producing cells induced by defined transcription factors. EBioMedicine. 2018; 36:358–66.

Fig. 1.

Characterization of streptozotocin (STZ)-induced diabetic mice treated with four differentiation-inducing factors (diff. cocktail). (A) Summarized scheme of animal experiments. Male C57BL/6 mice are intraperitoneally administered STZ (diabetic; red lines in the panels) or vehicle (normal; black lines in the panels). After 7 days, the diff. cocktail is administered via oral gavage for 5 consecutive days, twice at 2-week intervals. Body weight (B) and changes in random feeding blood glucose levels (C) of the mice administered diff. cocktail (filled circle, straight line) or comparable vehicle (empty circle, dotted line) are measured at the indicated days. (D) Blood glucose responses from the intraperitoneal glucose tolerance test (ipGTT). Glucose (1 g/kg, body weight) is injected after an overnight fast at 42 days after systemic administration. The area under the curve (AUC) (right panel) of glucose is measured. (E) Plasma insulin levels at 1 hour post-glucose injection (2 g/kg, body weight) following an overnight fast are measured using ultrasensitive mouse insulin enzyme-linked immunosorbent assay (ELISA) at 50 days after oral administration. C57BL/6 normal group (n=4–5); STZ-induced diabetic group (n=3–4). Data are presented as the mean±standard error of the mean. ip, intraperitoneal; diff. cocktail, combination of 200 mg/kg of putrescine and glucosamine, 500 mg/kg of nicotinamide, and 3 mg/kg of BP-1-102; GSIS, glucose-stimulated insulin secretion. ^aP<0.05, ^bP<0.01, ^cP<0.001 for diff. cocktail- vs. vehicle-supplemented mice.

Fig. 2.

Characterization of endogenous bone marrow mononucleated cell (BMNC) fate in the pancreas. (A) Summarized scheme of animal experiments. Chimeric mice are generated by injecting BMNCs (1×10⁶ cells/mouse) derived from MIP-Luc/green fluorescent protein (GFP) (Jackson Laboratories) mice into the lethally irradiated (500×2 cGy) male C57BL/6 mice (8 to 12 weeks old) via the retro-orbital vein. At 4 weeks after BMNC reconstitution, mice are intraperitoneally administered a low dose of streptozotocin (STZ; 50 mg/kg, body weight) daily for 5 consecutive days. After 7 days, 1× differentiation (diff.) cocktail is systemically infused via oral gavage for 5 consecutive days, twice at 2-week intervals as described in the summarized scheme of Fig. 1. (B) Representative immunofluorescent images of the pancreatic sections stained with anti-GFP (green) and anti-insulin (red), followed by 4´,6-diamidino2-phenylindole (DAPI) staining (blue) for nuclei and observed under a confocal fluorescence microscope. Original magnification, 400×; scale bar, 50 μm. (C) Numbers of total insulin- and GFP-positive cells and the GFP- and insulin-double-positive cells from the whole pancreatic section are quantified using the MetaMorph Image Analysis software (Molecular Devices). (D) The mRNA expression of Gfp, Ins1, and Ins2 in the pancreas is measured using quantitative real-time polymerase chain reaction. (E) Pancreatic insulin content in pancreatic tissues harvested 46 days after diff. cocktail supplementation is measured using high-range mouse insulin enzyme-linked immunosorbent assay (ELISA). All data are presented as the mean±standard error of the mean, obtained from 5–8 mice per group. iv, intravenous; ip, intraperitoneal; EGFP, enhanced green fluorescent protein; Diff., differentiation; Ins1, insulin 1; Ins2, insulin 2. ^aP<0.05.

Fig. 3.

Increased insulin- and pancreatic and duodenal homeobox 1 (PDX1)-positive cell expression in the pancreas of diabetic mice supplemented by differentiation (diff.) cocktail. (A) Immunohistochemical images of the sectioned pancreas for insulin (upper), PDX1 (middle), and NK6 homeobox 1 (NKX6.1; lower) harvested 56 days after diff. cocktail supplementation. All images are acquired with an ECLIPSE Ci-L microscope (Nikon Instruments Inc.). Magnification, 100× for insulin staining in the pancreas; 200× for PDX1 and NKX6.1 staining in the pancreas; scale bar, 100 μm. (B) Pancreatic insulin content in mice is also determined from the pancreas harvested 56 days after diff. cocktail supplementation, using high-range of mouse insulin enzymelinked immunosorbent assay (ELISA). Data are presented as the mean±standard error of the mean, obtained from 3–6 mice in each group. STZ, streptozotocin; diff. cocktail, combination of putrescine, glucosamine, nicotinamide, and BP-1-102. ^aP<0.05.

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