Umbilical cord extracts improve diabetic abnormalities in bone marrow-derived mesenchymal stem cells and increase their therapeutic effects on diabetic nephropathy

Bone marrow-derived mesenchymal stem cells (BM-MSC) has been applied as the most valuable source of autologous cell transplantation for various diseases including diabetic complications. However, hyperglycemia may cause abnormalities in intrinsic BM-MSC which might lose sufficient therapeutic effects in diabetic patients. We demonstrated the functional abnormalities in BM-MSC derived from both type 1 and type 2 diabetes models in vitro, which resulted in loss of therapeutic effects in vivo in diabetic nephropathy (DN). Then, we developed a novel method to improve abnormalities in BM-MSC using human umbilical cord extracts, namely Wharton’s jelly extract supernatant (WJs). WJs is a cocktail of growth factors, extracellular matrixes and exosomes, which ameliorates proliferative capacity, motility, mitochondrial degeneration, endoplasmic reticular functions and exosome secretions in both type 1 and type 2 diabetes-derived BM-MSC (DM-MSC). Exosomes contained in WJs were a key factor for this activation, which exerted similar effects to complete WJs. DM-MSC activated by WJs ameliorated renal injury in both type 1 and type 2 DN. In this study, we developed a novel activating method using WJs to significantly increase the therapeutic effect of BM-MSC, which may allow effective autologous cell transplantation.


OLETF-MSC activated by WJs ameliorated renal injury in OLETF rats 5
We also examined the therapeutic effects of BM-MSCs using OLETF rats, a

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Abnormal STZ-MSC were detected in the kidney at day 5 after MSC injection 20 and quickly disappeared after a few more days. Conversely, activated STZ-MSC (STZ-21 MSC-WJ) were detected in the kidney earlier, at day 2 after injection, and still 1 detectable 15 days after injection ( Supplementary Fig. S10a). Larger numbers of STZ-2 MSC-WJ were detected also in the interstitium of the lung, liver and spleen, compared  To investigate the mechanism of WJs reducing ER stress in DM-MSC, STZ-7 MSC and OLETF-MSC were cultured with 4-phenylbutyrate (PBA) 1 , which inhibited 8 ER stress by promoting chemical chaperone activity, or WJs. The expression of HRD-9 1 2 , which is ER-associated degradation (ERAD)-associated E3 ubiquitin-protein ligase, 10 PERK  , eIF2 and phospho-IRE-1   which are ER transmembrane proteins and its 11 downstream signaling molecule, were up-regulated by the stimulation with PBA and 12 the addition of WJs in STZ-MSC and OLETF-MSC. Because WJs suppressed the ER 13 stress as similar as PBA, it was thought that inhibition of ER stress is one of mechanism 14 of WJs. On the other hand, to investigate whether HG induces ER stress in normal 15 MSC, Control-MSC were stimulated with Tunicamycin (TM), which is an ER stress 16 inducer by inhibiting N-glycosylation of protein 4 , or Thapsigargin (TG), which is an 17 inhibitor of endoplasmic reticulum Ca 2+ -ATPase and decrease in ER calcium levels 3 , or 18 HG culture media . The expression of XBP-1 was increased by the stimulation with 19 TM, TG and HG in Control-MSC. Because HG suppressed the expression of PERK, it 20 was thought that hyperglycemia mainly induces ER stress via PERK pathway.

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The osteogenic differentiation potentials of BM-MSC in both STZ rats and 1 OLETF rats were significantly decreased. Previous studies have shown that 2 hyperglycemia down-regulated the osteogenic differentiation of BM-MSC via the 3 suppression of heme oxygenase-1 5,6 , down-regulation of bone morphogenetic protein 2 7 , 4 or induction of apoptosis and senescence via an increase of the receptor for AGEs 8 .

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Several studies reported the relationship between diabetes and osteoporosis based on 6 MSC abnormality and endocrine roles for both bone and adipose tissue 9,10 . The  On the other hand, WJs up-regulated adipogenic differentiation potentials and down-11 regulated osteogenic differentiation potentials in both STZ-MSC and OLETF-MSC.

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These results suggested that the differentiation potentials of DM-MSC did not affect the 13 therapeutic effects on DN directly. 14 STZ is commonly used to induce diabetes in experimental animal models and 15 frequently to investigate the pathogenesis of human diabetic nephropathy. However, the 16 cytotoxicity of STZ reportedly affects the pathology of nephropathy in STZ-induced 17 diabetic models 11,12 . Tay YC and Tesch GH reported that focal area of acute tubular 18 necrosis (ATN) with tubular vacuolisation is the primary tissue disorders occurring in 19 the early phase (at 1, 2 and 6 weeks after the administration of STZ) of high-dose ( > 20 200 mg/kg) STZ administered diabetic nephropathy (DN) in model mice. Tay YC also 21 suggested the optimal dose of STZ to induce DN in mice to include two lower doses of 22 at the first time and 150 mg/kg at the second time intravenously injected in 5 days apart. 1 We administered 150 mg/kg of STZ only once, which induced lower irritation than 2 occurred with the above protocol. Although mild interstitial hypercellularity and tubular 3 dilatation were observed, ATN and/or interstitial fibrosis were not observed 4 histologically in STZ mice during the early phase (5 weeks after the injection of STZ).

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Furthermore, to distinguish the drug toxicity of STZ and the influence of hyperglycemia 6 in the histological findings of the kidney, we regulated blood glucose levels by the 7 transplantation of insulin pellets in STZ mice. Although excessive tubular dilatation 8 improved with glycaemic control, interstitial hypercellularity and U-alb / Cr levels were 9 not improved by its treatment alone. These results suggested that glycaemic control 10 alone was insufficient for cellular infiltration and subsequent fibrotic changes. It was 11 also reflected in the result that U-alb / Cr did not improve.

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In addition, we also examined the therapeutic effects of BM-MSCs using 13 OLETF rats to avoid the effects of STZ toxicity on nephropathy. As DM-MSC-WJ 14 inhibited albumin secretion into the urine in OLETF rats similar to STZ rats.

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Histologically, DM-MSC-WJ ameliorated the renal tubular epithelial damage, 16 interstitial inflammatory cell infiltration and the fibrotic change common to type 1 and 17 type 2 DN despite differences in the pathology of diabetes and the age of each model.         Table S1). It 21 was then incubated with horseradish peroxidase-conjugated secondary antibodies (Supplementary Table S2). Immuno-reactivity was developed using the enhanced 1 chemiluminescence kit (Amersham Biosciences). Thapsigargin (TG, Wako Pure Chemical Industries, Ltd., Osaka, Japan) and high-glucose 8 culture media for 24 hours, respectively. The expression of HRD-1, PERK, phospho-9 IRE-1 and translation initiation factor (eIF) 2 were analyzed by immunoblotting.

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Primary and secondary antibodies were listed in Supplementary Table S1 and S2.    Supplementary Tables S1 and S2. Systolic blood pressure was measured in STZ rats using non-invasive blood 8 pressure monitor (MK-2000ST, Muromachi Kikai Co., Ltd. Tokyo, Japan). The blood 9 pressure was measured 5 to 8 times in each rat, and the average value was taken as the 10 value of each rat.                           Immunofluorescence staining of proinsulin (red) and glucagon (green) in the islet tissues of rats. DAPI was used for counterstaining nuclei (blue). Bar: 50 µm.