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symptomes DT1
fonction exo/endocrine
gènes en italique et ce que les gènes codent (xpl : myc marqueur de cancer et FT)
expliquer origine peptide C

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Paufiner le diapo

Date soutenance : jeudi 2 mai 13h30

Ne pas oublier de mettre le nom de behnamou

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Pour article 2 : regarder quel type de gène et implication dans le cancer

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Pour article 2 : regarder quel type de gène et implication dans le cancer

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Individuellement : Avancée sur le diaporama

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Lien CANVA :

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Check out this Presentation designed by Mondoudou.

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francois.patou@gmail.com

Cristina.Nostro@uhn.ca --> Out of office, REP 8 avril

yasaman.aghazadeh@ircm.qc.ca --> Out of office, REP 8 avril

maxime@treefrog.fr

maxime.feyeux@gmail.com

andrea.leonard@treefrog.fr

kevin@treefrog.fn

kevin.alessandri@gmail.com

frederic.desdouits@treefrog.fr

jens.schroder@bayer.com --> mail non fonctionnel

contact@treefrog.fr

Miriam@MiriamETucker.com

b.rajaei@lumc.nl

massumi.mohammad@gmail.com

PYBenhamou@chu-grenoble.fr

charles.thivolet@chu-lyon.fr

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Amélie & Jeanne : Relance Mail

Blanche & Chloé : Diaporama

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Amélie : envoie et préparation des mails

Jeanne : préparation question et expériences

Blanche : Diaporama et expériences

Chloé : Diaporama

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https://docs.google.com/document/d/1NLczC4Si4uMDci2BLMCyrWPJgECZzs2kFBCSoJrI5wY/edit?usp=drivesdk

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Dans expérience 1 : expérience de dedifférenciation

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Dans expérience 1 : expérience de dedifférenciation

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Expérience 2 : différenciation et reinjection

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D'où viennent les fibroblastes prélevés dans la première expérience ?

Pourquoi on s'intéresse spécifiquement au marqueurs de lendothelium ? (Premier acticle)

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mail fr :
remplacer
selon vos préférences
mettre les noms au début (format lettre)
donner une durée/nombre de questions
temps pour réponse ou jusqu'à interview
possibilité de demander aux chercheurs sur l'xp qu'on a imaginé
contacts :
c'est bon
questions :
préciser quels expériences
reformulation de question sur utilisation des stems cells
faut un diagramme de Gantt
organisation temps outil
répartition des tâches
contacts profs/prof
qui fait quoi ?
approfondir les xps
semaine pro (21/03) : initiaition diaporama/ébauche diapo
nombre de diapos par partie et répartition
mettre que on travaille en commun
arrivé avec diapo presque fini
mettre l'expérience/ y réfléchir
à quel moment de l'exposé
2/3 minutes par diapo
plan à mettre ou pas
plan : articles => a quoi ça fait référence
1 : différenciation en cellules souches
2 : différenciation en îlots
diapo intro
qu'est ce que c'est la thérapie cellulaire et qu'est ce que ça permet de faire et en quoi c'est pertinent pour le DT1
=> recontextualisé
contrainte/ tenant et abouttisant
article 1
trouver schéma
comment on fait, comment vérifier que ça marche puis validation
faire les liens
article 2 :
passage cellules souches à animales
expliquer les expériences
encore du travail sur la 2ème partie

faut mettre les outils

FAIRE LES LIENS ENTRE LES ARTICLES
PRECISER DANS L'XP IN VIVO
mettre les articles au bon endroit.

c'est flou dans sa tête

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Hello, Thank you for the time you are offering us. This our list of questions concerning your research and your work in general. As we realise you may not have much time to answer, do not hesitate to skip the ones you do find relevant. Respectfully Blanche Hevry, Jeanne Martial, Chloé Romero and Amélie Siaud.

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résumé séance 14/03 :
mail : ajouter durée interview/nbr de questions/temps jusqu'a interview
question sur notre xp (pdt l'interview)
questions : en ajouter/changer la formulation

pour la séance du 21/03 :
diapo final à travailler
- nombre de diapo par parties
- clarifier les liens entre parties
- dans plan, articles => titre de partie à laquelle l'article répond
1 -> différenciation de cellules en cellules souches (cours 602 de corsi)
2 -> différenciation CS en îlots fonctionnels et immuno-évasif
- qu'est-ce que la thérapie cellulaire, ce que ça permet et       pernitence dans traitement DT1 (contextualisation)
- préciser les outils des articles

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https://docs.google.com/document/d/1p7wlSBGJAD0yYvL5Zbhv1XNwgMZqMjsHUVa-DzsKgNo/edit?usp=drivesdk

lien au google docs pour rédiger le mail.
faudra l'envoyer au profs

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le gars qui a écrit les articles que Jeanne a trouvé
travaille dans une entreprise pharmatieutique sur des traitements pour le diabète

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I-introduction : présentation succincte de notre sujet (diabète, contexte et importance de trouver traitements, modèle) +  énoncer le plan

II. Qu’est ce que le diabète de type I et une maladie auto-immune (Pancréas, mécanisme régulation )

III. Utilisation de SC pour traiter le diabète… (Qu'est-ce que les SC, capacité à se différencier, se régénérer et se multiplier)

IV. controverses/limites de l’application/Risques/critiques + conclure sur les perspectives futures

VIII- conclusion générale

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https://1drv.ms/p/s!AlXvpMq3wTXjhuQfSLKpI-QgoTMFDA

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chercheuse Maria Cristina Nostro, PhD (McEwen Stem Cell Institute, Toronto) pour Medscape Medical News.

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chercheuse Maria Cristina Nostro, PhD (McEwen Stem Cell Institute, Toronto) pour Medscape Medical News.

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En quoi les cellules souches sont un espoir pour le traitement du diabète de type I chez la souris?

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En quoi les cellules souches sont un espoir pour le traitement du diabète de type I chez la souris?

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J’ai ajouté les 5 figures de l’article 1 sur le PowerPoint parce qu’elles étaient toutes pertinentes et que c’était pas si conséquent mais vous pouvez tout supprimer si vous voulez.

:)

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Jeanne et blanche : article 1 trouver les figures

Amélie et Chloé : article 2 trouver les figures

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Pr Trevor W. Reichman, directeur chirurgical de la transplantation de pancréas et de cellules d'îlots de Langerhans à l'Université de Toronto, Canada.

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Pr Trevor W. Reichman, directeur chirurgical de la transplantation de pancréas et de cellules d'îlots de Langerhans à l'Université de Toronto, Canada.

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types de cellules utilisés (quels cellules souches)
si prelèvement de cellules β, modif en cellules souches puis différentiation
où sont les SC de cellules β ?

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-préparer les sous parties
-pourquoi tels articles (préciser quelles figures) à quel endroit on introduit les articles dans le plan ?

- présentation du plan en cours (10 min)

-réfléchir à quelle experience « imaginer », où placer cette experience.

Après:
-quel scientifique contacter + les questions à lui poser.

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STEM CELL
PATHOLOGY

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Tout le monde : questions, recherche de chercheurs et contact

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Nat Commun. 2016; 7: 11463.

Abstract

We recently reported the scalable in vitro production of functional stem cell-derived β-cells (SC-β cells). Here we extend this approach to generate the first SC-β cells from type 1 diabetic patients (T1D). β-cells are destroyed during T1D disease progression, making it difficult to extensively study them in the past. These T1D SC-β cells express β-cell markers, respond to glucose both in vitro and in vivo, prevent alloxan-induced diabetes in mice and respond to anti-diabetic drugs. Furthermore, we use an in vitro disease model to demonstrate the cells respond to different forms of β-cell stress. Using these assays, we find no major differences in T1D SC-β cells compared with SC-β cells derived from non-diabetic patients. These results show that T1D SC-β cells could potentially be used for the treatment of diabetes, drug screening and the study of β-cell biology.

Pancreatic β cells can be generated from pluripotent stem cells. Here, the authors show that human induced pluripotent stem cells from patients with type 1 diabetes can be differentiated into β-like cells that have no detectable differences compared with cells from non-diabetic individuals.

Patient-derived human induced pluripotent stem cells (hiPSCs), differentiated to disease-relevant cells, are becoming quite important due to their potential for cell replacement therapy and drug screening, as well as improving our understanding of the pathophysiology of disease. Type 1 diabetes (T1D) occurs by autoimmune-mediated destruction of pancreatic β-cells, and genome-wide association studies have revealed that most genetic loci associated with T1D are affiliated with the immune system. However, several loci and related networks are expressed in the β-cells or are otherwise non-immune1,2,3. The role intrinsic defects in β-cells from patients, such as reduced mass and function or susceptibility and response to stress, may play in initiating the disease remains unclear1,2,3,4,5,6,7. Furthermore, what T1D patient-specific barriers, if there are any, may impede the use of autologous hiPSC technology for cell replacement therapy are unknown. As β-cells are destroyed during disease progression, procurement of β-cells from T1D patients that have not undergone disease-related environmental stress for study has not been possible.

Transplantation of exogenous β-cells to replace dead or dysfunctional endogenous β-cells is a potential strategy for controlling blood glucose levels in diabetic patients. Allogeneic transplantation of cadaveric islets has already been performed on patients with positive clinical results, but this approach suffers from a limited islet supply and the requirement that patients remain on immunosupressants8. Human pluripotent stem cells9, including both human embryonic stem cells (hESCs)10,11,12,13 and hiPSC13,14,15,16, provide the basis for potentially unlimited numbers of replacement cells. Several groups have detailed the generation of early and intermediate cell types from human pluripotent stem cells, such as definitive endoderm and pancreatic progenitors10,11,12,13. Cells that express low levels of insulin, but few other β-cell markers, have been generated from T1D hiPSC previously. However, these cells have been of limited utility, as they do not resemble bona fide β-cells, lack function in vitro and in vivo, mis-express β-cell and other islet genes, and lack correct granular ultrastructure15,16. In recent times, we reported the generation of SC-β cells from both hESC- and hiPSC-derived from non-diabetic (ND) donors that have many of the defining features of β-cells, including its function both in vitro and in vivo, β-cell marker expression and the ability to control glucose levels in diabetic mice17,18. Other groups have reported similar protocols and findings19,20.

Here we use this novel technology to generate SC-β cells from T1D hiPSC for the first time. These cells, referred to as T1D SC-β cells, express markers found in β-cells, are functionally indistinguishable from ND SC-β cells in vitro and in vivo, and prevent alloxan-induced diabetes in mice. Insulin secretion is increased in these T1D SC-β cells in response to several categories of anti-diabetic drugs. Furthermore, we show that these cells respond to different types of chemically induced stress, develop an in vitro disease model of T1D SC-β cell stress and demonstrate a partial rescue of this stress phenotype with treatment of a small molecule (an Alk5 inhibitor). T1D SC-β cells can be used to better study diabetes and as a potential autologous source for cell replacement therapy.

Results

Derivation and in vitro assessment of T1D SC-β cells

To generate T1D and ND SC-β cells, we derived and characterized hiPSC from skin fibroblasts of patient donors ( Fig. 1a,b ). As described previously15, we found both T1D and ND hiPSC to express pluripotent stem cell markers, differentiate to express markers of all three germ layers and, after undergoing planar differentiation to pancreatic progenitors, produce PDX1+/NKX6-1+ cells that can be transplanted into mice to spontaneously generate glucose-responsive cells in vivo ( Supplementary Figs 1 and 2).

After T1D and ND hiPSC derivation and characterization, we adapted the cells to suspension culture to undergo differentiation to produce SC-β cells ( Fig. 1c )17. We generated numerous batches of cells from all six cell lines. A batch of cells is a single flask of cells that has undergone the entire differentiation protocol and is considered a single biological replicate. Both T1D and ND hiPSC are capable of differentiating to SC-β cells that co-express C-peptide+/NKX6-1+ and C-peptide+/PDX1+, with few cells expressing the α-cell hormone glucagon ( Fig. 1d ). When quantified by flow cytometry, we found on average 24±2% and 27±2% of cells co-expressed C-peptide+/NKX6-1+ for T1D and ND cells, respectively ( Fig. 1e ), similar to what we previously reported with hESCs and ND hiPSCs17. Furthermore, when analysed with electron microscopy, both T1D and ND SC-β cells contained developing and mature insulin granules that are similar to β-cell granules ( Supplementary Fig. 3)17.

T1D SC-β cells function and respond to anti-diabetic drugs

We next tested the cells with an in vitro glucose-stimulated insulin secretion assay, to assess their function. We found that both T1D and ND SC-β cells can respond to sequential glucose challenges ( Supplementary Fig. 4). On average for 18 biological batches (9 for T1D and 9 for ND), T1D and ND SC-β cells secrete 2.0±0.4 and 1.9±0.3 μIU of human insulin per 10 ³ cells in response to 20 mM glucose and have stimulation indexes (ratio of insulin released at 20-2 mM glucose) of 1.9 and 2.2, respectively ( Fig. 1f ). On average, T1D and ND cells responded to 88% and 78% of the challenges, respectively. Insulin content was similar between the two groups, 210±40 μIU per 10 ³ cells and 220±20 μIU per 10 ³ for T1D ( n=3 batches, 1 from each donor) and ND ( n=3 batches, 1 from each donor), respectively. Overall, these T1D SC-β cells function similar to ND SC-β cells generated in this study and to our previous report of SC-β cells and adult primary islets17.

As a proof-of-concept for their possible use in drug screening, a subset of T1D and ND SC-β cell lines were treated with three anti-diabetic compounds that affect insulin secretion by different mechanisms: Sulfonylurea (tolbutamide), GLP-1R agonist (Liraglutide) and GCK activator (LY2608204) ( Fig. 1g ). Treatment with each of the three compounds increases insulin release at both low- and high-glucose challenges compared with control by a factor of 2.0 on average.

T1D SC-β cells function after transplantation

To evaluate their potential use in cell replacement therapy and in vivo physiological tests and further confirm their identity as SC-β cells, T1D and ND SC-β cells were transplanted underneath the kidney capsule of ND immunocompromised mice ( Fig. 2a ). After 2 weeks, graft function was evaluated by measuring serum human insulin before and 30 min after an injection of glucose ( Fig. 2b and Supplementary Table 1). At this early time point, human insulin is detected and the grafts were glucose responsive in most, but not all, mice. Overall, 81% (26/32) and 77% (37/48) secreted more human insulin after glucose injection, for T1D and ND SC-β cells, respectively. The ratio of insulin secretion after glucose challenge compared with before challenge averaged 1.4 and 1.5, for T1D and ND SC-β cells, respectively. Again, no major differences between these T1D and ND SC-β cells were observed17. Immunostaining of recovered grafts revealed clusters of C-peptide+ cells with some glucagon+ cells ( Fig. 2c ).

The presence of both T1D and ND SC-β cells were observed for several months after transplantation, with the grafts continuing to respond to glucose injections and high amounts of human insulin being detected in the serum ( Fig. 2d,e ). After 3-4 months, a subset of mice were treated with alloxan to selectively kill mouse but not human β-cells21, to evaluate the ability of T1D and ND SC-β cells to maintain euglycemia ( Fig. 2a,c ). Destruction of endogenous mouse β-cells was confirmed by demonstrating the absence of serum mouse C-peptide. Mice that did not receive a SC-β cell graft but were treated with alloxan died within 29 days of alloxan treatment. Mice were observed for up to 80 days after alloxan treatment, for a total of 164-192 days since SC-β cell transplantation, with mice that received either T1D or ND SC-β cells maintaining on average <200 mg dl −1 fasting blood glucose ( Fig. 2f ), secreting human insulin in response to a glucose injection ( Fig. 2g ) and rapidly clearing glucose after a glucose injection ( Fig. 2h,i ). This data shows that grafts continue to function for >5 months in vivo.

Development of a disease model of T1D SC-β cell stress

The results summarized above support the conclusion that T1D SC-β cell function is similar to that of ND SC-β cells both in vitro and in vivo. To further compare the two types of SC-β cells, we focused on a subset of cell lines and employed a method we recently reported to analyse global gene expression from fixed, immunostained and sorted cell populations. ( Fig. 3a , Supplementary Fig. 5 and Supplementary Data 1)17,22. There is little, if any, major difference between these T1D and ND SC-β cells as indicated by the clustering of the samples. Furthermore, these cells clustered with our previously reported hESC SC-β cells and adult cadaveric β-cells, but are distinct from fetal β-cells and from T1D and ND hiPSC that had undergone a control planar protocol that produces dysfunctional insulin-expressing cells.

Increased cellular susceptibility to exogenous stress has been reported in patient-derived hiPSC from some diseases, including ALS and Wolfram syndrome23,24. Similarly, β-cells are known to be susceptible to stress, which may play a role in initiating diabetes3,4,5,25,26. To study this in T1D, the in vitro-derived SC-β cells were acutely subjected to several chemical stressors and the persistence of SC-β cells was evaluated through assessment of the co-expression of β-cell markers C-peptide+/NKX6-1+ ( Fig. 3b and Supplementary Fig. 6). Many of the stress treatments reduced the fraction of cells co-expressing C-peptide+/NKX6-1+, with the cytokine stress cocktail (a combination of interleukin-1β (IL), tumour necrosis factor-α (TNF) and interferon-γ (IFN)) resulting in the largest reduction.

Given the role that cytokines and inflammation are thought to play in T1D25, we chose to further investigate the effects of interleukin-1β, tumour necrosis factor-α and interferon-γ on T1D and ND SC-β cells. Cytokine treatment results in the rapid loss of not only C-peptide+ cells that co-express NKX6-1+ but also cells that co-express MAFA+ ( Supplementary Fig. 7) and PDX1+. The reduction of MAFA and NKX6-1 precedes PDX1, as has been reported for primary human β-cells27 ( Fig. 3c ). We hypothesized that the T1D SC-β cells would be more sensitive to cytokine-induced stress than ND SC-β cells, as has been reported in other patient hiPSC disease systems23,24. However, no detectable difference in the T1D and ND SC-β response to cytokine-induced stress was observed ( Fig. 3d ).

The stress conditions identified in Fig. 3 provide the potential basis for a human disease model drug-discovery platform. To demonstrate its utility for drug screening, cells in this cytokine stress assay were treated with Alk5 inhibitor type II (Alk5i), a compound that was previously identified to prevent stress-induced phenotypic loss in mouse β-cells28. With human SC-β cells, treatment with Alk5i partially blocks loss of C-peptide+/NKX6-1+ cells, improving retention by 32% ( Fig. 3e,f ).

Discussion

In summary, we demonstrate that functional SC-β cells can be generated from hiPSC derived from T1D patients in vitro ( Fig. 1a-c ). These cells are very similar to those we previously reported for hESC and ND hiPSC SC-β cell and are similar but not identical to adult β-cells17. T1D SC-β cells express markers found in β-cells, including NKX6-1 and PDX1, and have global gene expression patterns similar to adult β-cells ( Figs 1d,e and ​and3a). 3a ). T1D SC-β cells are functional in vitro and in vivo, responding to glucose challenges by increasing their human insulin release and controlling glucose levels in diabetic mice after transplantation ( Fig. 2 ). After exposure to chemical-induced stress, T1D SC-β cells lose expression of β-cell markers ( Fig. 3b,e ). These cells also respond to cytokine stress, which can be partially prevented by treatment with Alk5i ( Fig. 3b-f )28. Finally, these cells also respond to known anti-diabetic drugs that cause increased insulin secretion ( Fig. 1g ).

These experimental approaches could directly be used for discovery of novel compounds that increase function and prevent stress and death in β-cells. The usefulness of these cells could be further enhanced by a combination with recently reported genetic modification strategies, for example, by the use of TALEN or CRISPR-Cas9 to introduce or correct known diabetes-associated mutations29,30 or engineering reporter constructs to improve functional readout31.

Overall, we do not observe major differences in SC-β cells derived from T1D and ND patients with the assays and cell lines used in this report. It is important to note that this does not mean differences do not exist in T1D β-cells compared with ND β-cells. One possibility is that defects or differences would appear after extended culture or many years after transplantation. Diabetes generally develops over a long period, and we did not test for the effects of ageing. In addition, investigating how T1D SC-β cells interact with the immune system would be quite informative. Indeed, the gene with the largest differential expression in T1D and ND SC-β cells is TAP1, a peptide transporter associated with the major histocompatibility complex that has been implicated in with T1D32. In addition, it is important to note that the three T1D donors used in this study do not represent the whole T1D population; differences among patients are likely to exist.

The lack of observable gross deficiencies in T1D SC-β cells combined with their functional phenotype indicates the potential utility of these autologous cells in cell replacement therapy for diabetes for at least a proportion of the T1D population. We caution that additional studies need to be conducted with a larger number of patient-specific cell lines before clinical use. In combination with a strategy to prevent autoimmune destruction33, these cells may provide an alternative to current approaches in transplantation that have to address the additional hurdle of allo- or xenogeneic rejection.

Methods

Cell line derivation and characterization

ND-1 and ND-2 were previously published17. Other hiPSC lines were derived from fibroblasts grown out from skin biopsies received from Columbia University using Sendai viral reprogramming on mouse fibroblasts (Life Technologies; A1378001). Cell lines were characterized by karyotype analysis, expression of pluripotency genes and differentiation genes in a spontaneous differentiation assay. G-banded karyotype analysis was performed by Cell Line Genetics (Madison, WI). In brief for the other assays, colonies were stained with antibodies for TRA-1-60 (Millipore; MAB4360), SSEA-3 (Millipore; MAB4303), SSEA-4 (Millipore; MAB4304), NANOG (Abcam; ab21624) and OCT4 (Abcam; ab19857). Colonies were also stained for alkaline phosphatase (Millipore; SCR004) and reverse transcriptase-PCR performed for DNMT3B, HTERT, NANOG, OCT4, REX1 and SOX2. Differentiation was assessed by a spontaneous embryoid body differentiation and assessment of markers for the three germ layers, using reverse transcriptase-PCR to look for NCAM, PAX6, AFP, GATA4, FLK1 and GATA2. RNA was extracted using RNeasy Mini-Kit (Qiagen; 74106) and complementary DNA synthesized using qScript cDNA SuperMix (Quanta; 101,414-106). The primer sequences used are (gene, forward primer, reverse primer, annealing temperature and cycle number): ACTIN, 5′-ggacttcgagcaagagatgg-3′, 5′-agcactgtgttggcgtacagnnealing-3′, 60 °C, 25; DNMT3B, 5′-ataagtcgaaggtgcgtcgt-3′, 5′-ggcaacatctgaagccattt-3′, 56 °C, 30; HTERT, 5′-tgtgcaccaacatctacaag-3′, 5′-gcgttcttggctttcaggat-3′, 57 °C, 33; NANOG, 5′-tccaacatcctgaacctcag-3′, 5′-gactggatgttctgggtctg-3′, 58 °C, 30; OCT4 5′-gtggaggaagctgacaacaa-3′, 5′-caggttttctttccctagct-3′, 56 °C, 30; REX1, 5′-tggacacgtctgtgctcttc-3′, 5′-gtcttggcgtcttctcgaac-3′, 60 °C, 30; SOX2, 5′-ttgtcggagacggagaagcg-3′, 5′-tgaccaccgaacccatggag-3′, 64 °C, 33; NCAM, 5′-atggaaactctattaaagtgaacctg-3′, 5′-tagacctcatactcagcattccagt-3′, 68 °C, 33; PAX6, 5′-tctaatcgaagggccaaatg-3′, 5′-tgtgagggctgtgtctgttc-3′, 57 °C, 35; AFP, 5′-agcttggtggtggatgaaac-3′, 5′-ccctcttcagcaaagcagac-3′, 58 °C, 30; GATA4, 5′-ctagaccgtgggttttgcat-3′, 5′-tgggttaagtgcccctgtag-3′, 61 °C, 30; FLK1, 5′-agtgatcggaaatgacactgga-3′, 5′-gcacaaagtgacacgttgagat-3′, 63 °C, 32 and GATA2, 5′-gcaacccctactatgccaacc-3′, 5′-cagtggcgtcttggagaag-3′, 58 °C, 35.

Cell culture

After adapting the undifferentiated cells to feeder-free culture on Matrigel (BD Biosciences; 08774552) in mTeSR1 (StemCell Technologies Inc.; 05850), undifferentiated cells were adapted and maintained in mTeSR1 in 500-ml spinner flasks (Corning; 89,089-814) on a stir plate (Chemglass) rotating at 70 r.p.m. in a humidified 37 °C incubator set at 5% CO ₂. Undifferentiated cells were maintained by passaging clusters dispersed with Accutase (StemCell Technologies; 07920) and seeded at 0.5 × 10 ⁶ cells per ml in mTeSR1 supplemented with 10 μM Y27632 (Abcam; ab120129). Cultures were passaged every 72-96 h (ref. 17).

To initiate differentiation to SC-β cells, clusters were dispersed as above and seeded between 0.25 and 1 × 10 ⁶ cells per ml in mTeSR1 supplemented with 10 μM Y27632 to achieve cluster diameters of 150-220 μm after 48-72 h. Differentiation was initiated by changing the media to differentiation media and following feeding schedule seen in Supplementary Tables 2 and 3. S6 basal media used in stage 6 was made by supplementing CMRL 1,066 Supplemented (Mediatech; 99-603-CV) with 10% fetal bovine serum (HyClone; 16,777) and 1% Penicillin/Streptomycin. For this study, we had three batches of differentiation that failed to produce at least 40% PDX1+/NKX6-1+ by the end of stage 4 and were not included in the analysis. SC-β cell samples were acquired and experiments on SC-β cells were performed after 10-17 days in Stage 6.

In a subset of experiments, PDX1+/NKX6-1+ pancreatic progenitors and dysfunctional cells that express C-peptide were generated with a planar differentiation protocol for initial evaluation of hiPSC differentiation capacity. To initiate planar differentiation, undifferentiated cells were seeded at 5.3 × 10 ⁶ cells per cm ² on plates treated with growth factor-reduced Matrigel (BD Biosciences; 356,231) in mTeSR1 supplemented with 10 μM Y27632 and after 24 h the media were changed to differentiation media. To generate PDX1+/NKX6-1+ pancreatic progenitors, the media were changed as seen in Supplementary Table 4. To generate PH cells, the media were changed as seen in Supplementary Table 5.

Immunohistochemistry

Cell clusters or excised kidneys were fixed with 4% paraformaldehyde (PFA; Electron Microscopy Science; 15,714) for 1 h at room temperature (RT) or overnight at 4 °C, respectively, before being paraffin embedded and sectioned at 10 μm. Paraffin was removed with Histoclear (Thermo Scientific; C78-2-G) and rehydrated, and antigens retrieved by 2 h 0.1 M EDTA (Ambion; AM9261) treatment in a pressure cooker (Proteogenix; 2,100 Retreiver). Samples were blocked with 0.1% Triton X-100 (VWR; EM-9400) and 5% donkey serum (Jackson Immunoresearch; 017-000-121) in PBS (staining solution) for 1 h at RT, incubated with primary antibodies diluted in staining solution overnight at 4 °C, washed for 5 min in staining solution, incubated covered with appropriate Alexa Fluor-488 or -594 secondary antibodies diluted 1:300 in staining solution 2 h at RT, washed for 5 min in staining solution, mounted with Vectashield (Vector Laboratories; H-1200) and covered with a coverslip. Images were taken with an Olympus IX51 Microscope. The primary antibodies used to assess differentiation throughout this study are given in Supplementary Table 6 (ref. 17).

Flow cytometry

Clusters were dispersed by treatment with TrypLE Express (Life Technologies; 12,604,013) at 37 °C, fixed with 30 min treatment with 4% PFA on ice, blocked by 30 min incubation in staining buffer, stained with primary antibodies diluted in staining buffer overnight at 4 °C, washed twice with staining buffer, stained with appropriate AlexaFluor-488 and -647 secondary antibody diluted 1:300 in staining buffer and washed twice with staining buffer. Stained cells were measured using an LSR II flow cytometer (BD Biosciences) and analysed using FlowJo17.

Electron microscopy

Cellular ultrastructure was assessed with electron microscopy17. Clusters were fixed with 1.25% PFA, 2.5 glutaraldehyde and 0.03% picric acid in pH 7.4, 0.1 M sodium cacodylate for 2 h at RT, washed in 0.1 M cacodylate buffer, incubated at least 2 h at RT in 1% osmium tetroxide and 1.5% potassium ferrocyanide (OsO ₄/KFeCN ₆), washed in 0.1 M cacodylate buffer, incubated for 1 h in OsO ₄/KFeCN ₆, washed thrice with water, stained for 1 h in 1% aqueous uranyl acetate, washed twice in water and dehydrated with alcohol. After a 1-h incubation in propyleneoxide, samples were infiltrated overnight in 1:1 propyleneoxide and TAAB Epon (Marivac Canada), embedded, sectioned (60 nm thickness), placed onto copper grids, stained with 0.2% lead citrate and analysed using a JEOL 1200EX transmission electron microscope. Assessment of ultrastructure was performed by a blinded observer.

In vitro glucose-stimulated insulin secretion

In vitro function was assessed by measuring in vitro glucose-stimulated insulin secretion17. Clusters were washed twice in Krebs buffer (krb), preincubated for 2 h in krb containing 2 mM glucose (low glucose) at 37  °C and washed once. Clusters were then challenged with three sequential treatments of alternating low-glucose krb and high-glucose krb containing 20 mM glucose (sx total glucose treatments), followed by treatment with low-glucose krb containing 30 mM KCl. Each treatment lasted 30 min, after which 100 μl of supernatant was collected and human insulin quantified using the Human Ultrasensitive Insulin ELISA (ALPCO Diagnostics; 80-INSHUU-E01.1. Human insulin measurements were normalized by viable cell counts that were acquired by dispersing clusters with TrypLE Expression and counted using a ViCell (Beckman Coulter). The formulation for krb buffer is seen in Supplementary Table 7. In a subset of experiments, cells were pre-cultured with an anti-diabetic drug for 24 h in stage 6 media and that same anti-diabetic drug was also included in the krb buffer: Tolbutamide (100 μM; Sigma; T0891), LY2608204 (1 μM; Selleck; S2155) and Liraglutide (1 μM; Bachem; H-6724.0001).

Kidney capsule transplantation

All performed animal work was done in accordance to Harvard University International Animal Care and Use Committee regulations. The number of mice chosen was sufficient to statistically show function previously17. No randomization was used. All procedures were done by blinded individuals. ND male immunodeficient SCID/Beige mice (Taconic) aged 8-10 weeks were anaesthetized with 250 mg kg −1 avertin, an incision made to expose the kidney, a catheter needle inserted underneath the capsule and 5 × 10 ⁶ cells injected. Cells generated with planar protocols were aggregated overnight to form clusters before injection. After the incision site was closed, mice were given 5 mg kg −1 carprofen and another dose 24 h later. Mice were monitored twice a week after transplantation.

Two weeks after transplantation, the function of transplanted cells was assessed by performing in vivo glucose-stimulated insulin secretion. Mice were fasted 16 h and the blood collected before and 30 min after an intraperitoneal injection of 2 g kg −1 glucose by facial bleed with a lancet (Feather; 2,017-01). Serum was separated from blood using microvettes (Sarstedt; 16.443.100) and human insulin quantified using the Human Ultrasensitive Insulin ELISA.

A subset of mice were treated with alloxan to destroy endogenous mouse β-cells 12-16 weeks after transplantation. Alloxan (90 mg kg −1) was delivered by tail vein injection21. Loss of endogenous mouse β-cells was confirmed by measuring serum mouse C-peptide using a Mouse C-peptide ELISA (ALPCO Diagnostics; 80-CPTMS-E01). Blood glucose levels was periodically measured for mice fasted 16 h and for mice injected with 2 g kg −1 glucose.

Global gene expression analysis

Clusters from several independent batches made from a subset of cell lines were dispersed with TrypLE, fixed in 4% PFA supplemented with RNasin (VWR; PAN2615) for 30 min on ice, stained with NKX6-1 and INS primary antibody in RNasin-containing buffer for 30 min on ice and stained with appropriate Alexa Fluor-488 and -647 secondary antibodies diluted in RNasin-containing buffer for 30 min on ice. INS+/NKX6-1+ cells were sorted using an FACSAria (BD Biosciences) and RNA extracted by first incubation sorted cells in digestion buffer (RecoverAll Total Nucleic Acid Isolation Kit; Ambion; AM1975) at 50 °C for 3 h then by following the instructions from the manufacturer. cDNA and cRNA was generated with Illumina TotalPrep RNA Amplifcation Kit (Life Technologies; AMIL1791). cRNA was hybridized to Human HT-12 Expression BeadChips (Illumina) with the Whole-Genome Expression Direct Hybridization kit (Illumina) and chips read with Illumina Beadstation 500. Data were analysed in GenomeStudio (Illumina) with background subtraction and rank-invariant normalization. Previous published data for HUES8 SC-β cells, undifferentiated HUES8, fetal β-cells and adult β-cells was also included in data analysis17,22. Hierarchical clustering was performed using Pearson's correlation and Ward linkage.

Chemically induced stress

Cell clusters were dispersed by TrypLE Express treatment and plated onto growth factor reduced Matrigel-coated 96-well plates overnight in S6 media supplemented with 10 μM Alk5i, 1 μM T3 and 10 μM Y27632. Plated cells were then washed twice with S6 then cultured with S6 media alone or S6 media with combinations of chemical stressors ( Supplementary Table 8) for up to 48 h. In a subset of experiments, cells were also cultured with 10 μM Alk5i. After chemical treatment, cells were fixed with 4% PFA for 30 min at RT, blocked with staining buffer for 30 min at RT, stained with primary antibody diluted in staining buffer overnight at 4 °C, stained with appropriate Alexa Fluor-488 and -594 secondary antibody diluted in staining buffer for 2 h at RT and stained with 4,6-diamidino-2-phenylindole. Stained cells were quantified by automated cell counting using Cellomics ArrayScanVTI, counting at least 1,000 cells per batch.

Statistical analysis

Statistical significance was determined by the use of two-sided unpaired and paired t-tests, as appropriate. Data shown as mean±s.e.m. n indicates the total number of batches (biological replicates) represented by the data. The box and whisker plot was generated with defaulting statistical settings with BoxPlorR ( http://boxplot.tyerslab.com/).

Additional information

Accession codes: Global Gene Expression Analysis has been deposited in the NCBI Gene Expression Omnibus database under accession code GSE70901.

How to cite this article: Millman, J. R. et al. Generation of stem cell-derived β-cells from patients with type 1 diabetes. Nat. Commun. 7:11463 doi: 10.1038/ncomms11463 (2016).

Supplementary Material

Supplementary Information:
Supplementary Figures 1-7 and Supplementary Tables 1-8

Supplementary Data 1:
Microarray Data Set

Acknowledgments

We thank the following people and organizations for assistance in this study: Jennifer Kenty, Isaac Alter, Pauline Gabrieli, Alexandrine Corneloup, Priya Makhijani and George Kenty for technical assistance; HSCI iPS Core Facility for assistance with the derivation and characterization of the hiPSC lines; HSCI Histology Core and Flow Cytometry Core facilities for technical assistance; Maria Ericsson, Elizabeth Beneochi and Louise Trakmias from HMS Conventional Electron Microscopy Facility for technical assistance; Professor Pamela Itkin-Ansari for helpful discussions related to alloxan treatment; all members of the Melton laboratory for helpful discussions. J.R.M. was supported by an HSCI fellowship. D.A.M. is a Howard Hughes Medical Institute investigator. Grants from HSCI, NIH, Helmsley Charitable Trust and the JPB Foundation along with the generosity of M. and A. Barry supported this research.

Footnotes

J.R.M., M.G., F.W.P. and D.A.M. are inventors on patent applications for content described in this manuscript. F.W.P. is an employee at Semma Therapeutics.

Author contributions J.R.M., M.G., F.W.P. and D.A.M. conceived the experimental design. J.R.M. and C.X. performed all of the in vitro experiments, except electron microscopy, which was performed by A.V.D. J.R.M. and C.X. performed all the in vivo experiments. J.R.M. and D.A.M. wrote the manuscript. All authors edited and reviewed the manuscript.

References

Kroon E. et al. Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells [in vivo. Nat. Biotechnol. 26, 443-452 (2008). [PubMed] Google Scholar]

Pagliuca F. W. et al. Generation of functional human pancreatic β cells [[in vitro. Cell 159, 428-439 (2014).PubMed] PMC free article] [Google Scholar]

Rezania A. et al. Reversal of diabetes with insulin-producing cells derived [in vitro from human pluripotent stem cells. Nat. Biotechnol. 32, 1121-1133 (2014). [PubMed] Google Scholar]

Russ H. A. et al. Controlled induction of human pancreatic progenitors produces functional beta-like cells [[in vitro. EMBO J. 34, 1759-1772 (2015).PubMed] PMC free article] [Google Scholar]

Hua H. et al. iPSC-derived beta cells model diabetes due to glucokinase deficiency. J. Clin. Invest. 123, 3146-3153 (2013). [[PubMed] PMC free article] [Retracted Google Scholar]

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# data[].attributes.attachment_link.title
Generation of stem cell-derived β-cells from patients with type 1 diabetes

# data[].attributes.attachment_link.description
We recently reported the scalable in vitro production of functional stem cell-derived β-cells (SC-β cells). Here we extend this approach to generate the first SC-β cells from type 1 diabetic patients (T1D). β-cells are ...

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Blanche : Violet
Jeanne : Rouge
Amélie : Vert
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Commun : Jaune

# data[].attributes.headline
Cell Therapy for Type 1 Diabetes: From 
Islet Transplantation to Stem Cells

# data[].attributes.subject
Cell Therapy for Type 1 Diabetes: From 
Islet Transplantation to Stem Cells

# data[].attributes.body
La transplantation des îlots n'est pas un traitement universel mais elle montre qu'il est possible de remplacer les cellules β par thérapie cellulaire. De plus, la transplantation permet l'étude de nouvelles thérapies cellulaires pour le diabète de type 1.

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Affiliations

¹ Department of Stem Cell and Regenerative Biology, Harvard University, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Boston, MA, USA.

² Department of Stem Cell and Regenerative Biology, Harvard University, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Boston, MA, USA. Electronic address: dmelton@harvard.edu.

Free PMC article

Free PMC article

Affiliations

¹ Department of Stem Cell and Regenerative Biology, Harvard University, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Boston, MA, USA.

² Department of Stem Cell and Regenerative Biology, Harvard University, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Boston, MA, USA. Electronic address: dmelton@harvard.edu.

Abstract

Immunological protection of transplanted stem cell-derived islet (SC-islet) cells is yet to be achieved without chronic immunosuppression or encapsulation. Existing genetic engineering approaches to produce immune-evasive SC-islet cells have so far shown variable results. Here, we show that targeting human leukocyte antigens (HLAs) and PD-L1 alone does not sufficiently protect SC-islet cells from xenograft (xeno)- or allograft (allo)-rejection. As an addition to these approaches, we genetically engineer SC-islet cells to secrete the cytokines interleukin-10 (IL-10), transforming growth factor β (TGF-β), and modified IL-2 such that they promote a tolerogenic local microenvironment by recruiting regulatory T cells (T regs) to the islet grafts. Cytokine-secreting human SC-β cells resist xeno-rejection and correct diabetes for up to 8 weeks post-transplantation in non-obese diabetic (NOD) mice. Thus, genetically engineering human embryonic SCs (hESCs) to induce a tolerogenic local microenvironment represents a promising approach to provide SC-islet cells as a cell replacement therapy for diabetes without the requirement for encapsulation or immunosuppression.

Keywords: cell engineering; cell replacement; diabetes; immune evasion; immune tolerance; stem cells.

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Conflict of interest statement

Declaration of interests D.G., Q.Z., and D.A.M. are named on US provisional patent application serial no. 63/340,453, which is related to this work. D.G. is an employee of Inventia Life Sciences. D.A.M. and Q.Z. are employees of Vertex Pharmaceuticals. These companies were not involved in any aspect of the work.

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national center for biotechnology information

# data[].attributes.attachment_link.title
Engineering human stem cell-derived islets to evade immune rejection and promote localized immune tolerance - PubMed

# data[].attributes.attachment_link.description
Immunological protection of transplanted stem cell-derived islet (SC-islet) cells is yet to be achieved without chronic immunosuppression or encapsulation. Existing genetic engineering approaches to produce immune-evasive SC-islet cells have so far shown variable results. Here, we show that targetin ...

# data[].attributes.attachment_link.title
Cell Therapy for Type 1 Diabetes: From Islet Transplantation to Stem Cells

# data[].attributes.attachment_link.description
Abstract. The field of cell therapy of type 1 diabetes is a particularly interesting example in the scenario of regenerative medicine. In fact, β-cell replacement has its roots in the experience of islet transplantation, which began 40 years ago and is currently a rapidly accelerating field, with several ongoing clinical trials using β cells derived from stem cells.

# data[].attributes.headline
Long-term glycemic control using polymer-encapsulated human stem cell–derived beta cells in immune-competent mice

# data[].attributes.subject
Long-term glycemic control using polymer-encapsulated human stem cell–derived beta cells in immune-competent mice

# data[].attributes.body
Correction à long terme le diabète chez des souris en utilisant des cellules humaines (SC-β) dérivées de cellules souches embryonnaires. Ces cellules sont encapsulées dans de l'alginate pour atténuer les réponses immunitaires et ont été implantées chez des souris diabétiques (sans nécessiter l'utilisation d'immunosuppression). Les résultats ont montré un contrôle glycémique efficace jusqu'à 174 jours après l'implantation, soulignant le potentiel de cette approche pour le traitement du diabète sans les limitations actuelles.

# data[].attributes.headline
Immune response after autologous hematopoietic stem cell transplantation in type 1 diabetes mellitus

# data[].attributes.subject
Immune response after autologous hematopoietic stem cell transplantation in type 1 diabetes mellitus

# data[].attributes.body
L'article examine la réponse immunitaire après greffe de cellules souches hématopoïétique. Les patients traités ont montré une meilleure sécrétion d'insuline et une réduction de la dose d'insuline (entre autre). Il y a aussi une diminution de cellules impliquées dans la réponse immunitaire (th1 et th17) et dans la réaction inflammatoire (certaines cytokines). Tout cela pourrait expliquer l'efficacité thérapeutique supérieure du traitement par AHSCT par rapport à l'insulinothérapie traditionnelle.

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ça vous va si on se donne chacun une couleur ? comme ça quand on fait qlqc sur le padlet on voit qui ?

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Stem Cell Research & Therapy 8, Article number: 90 (2017) Cite this article

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Abstract

Background

This study explored the details of the immune response after autologous hematopoietic stem cell transplantation (AHSCT) treatment in type 1 diabetes mellitus.

Methods

Peripheral blood mononuclear cells (PBMCs) from 18 patients with type 1 diabetes mellitus were taken at baseline and 12 months after AHSCT or insulin-only therapy. The lymphocyte proliferation, mRNA expression and secretion of pro-inflammatory and anti-inflammatory cytokines belonging to T-helper type 1 (Th1), T-helper type 17 (Th17) and regulatory T (Treg) cells in PBMC culture supernatants were assessed.

Results

Compared with patients receiving insulin-only treatment, the patients receiving AHSCT treatment showed better residual C-peptide secretion, lower anti-GAD titers and less exogenous insulin dosages after 12 months of follow-up. AHSCT treatment was associated with significantly reduced Th1 and Th17 cell proportions as well as decreased IFN-γ, IL-2, IL-12p40 and IL-17A levels in the PBMC culture supernatants (all P  < 0.05). Although there was no significant Treg cell expansion after AHSCT treatment, we observed increased IL-10, TGF-β and Foxp3 mRNA expression and increased TGF-β levels. However, we found no significant changes in the T-cell subpopulations after insulin treatment, except for higher IL-12p40 mRNA expression and a lower proportion of Treg cells.

Conclusions

AHSCT treatment was associated with decreased expansion and function of Th1 and Th17 cells, which may explain the better therapeutic effect of AHSCT compared with the traditional intensive insulin therapy.

Trial registration

Clinicaltrials.gov NCT00807651. Registered 18 December 2008.

Background

Type 1 diabetes mellitus is an autoimmune disease in which autoreactive immune cells attack pancreatic beta cells, eventually causing complete insulin deficiency [ 1]. CD4⁺ T cells play a pivotal role in this pathogenesis. Specifically, they predominate in the early phase of insulitis, causing or accelerating disease onset in young NOD mice [ 2]. CD4⁺ T cells recognize antigenic peptides presented by HLA class II on the surface of antigen-presenting cells, interact directly with these cells via costimulatory and adhesion molecules, produce cytokines and chemokines, and lead to local inflammation [ 3]. The inflammation damage is compensatory until the amount of the beta cell mass is insufficient to maintain glucose hemostasis, which is followed by overt type 1 diabetes mellitus and severe complications. Current therapies mainly include insulin replacement; however, they have failed to diminish immune damage.

The first case that implemented autologous hematopoietic stem cell transplantation (AHSCT) in a type 1 diabetes mellitus subject was executed by Voltarelli's group in 2003 [ 4, 5]. Since then, more studies have been performed to evaluate the therapeutic effect of AHSCTs. These studies consistently demonstrated a well-improved endogenous beta cell function [ 6- 8]. Burt et al. [ 9] speculated that AHSCT may shift the balance between destructive immunity and tolerance through undefined mechanisms. Brinkman DM et al. [ 10] showed that AHSCT led to a predominance of tolerating autoreactive T cells and restoration of the CD4⁺CD25⁺ immunoregulatory network in juvenile idiopathic arthritis (JIA) patients. de Oliveira et al. [ 11] found that AHSCT upregulated fas/fasL and downregulated anti-apoptotic bcl-xL genes expression in peripheral blood mononuclear cells (PBMCs).

Although the clinical and laboratory outcomes are constantly being updated, the exact mechanism of AHSCT is lacking. Here, we explored the immune responses by investigating the change of the peripheral T-cell subsets after AHSCT [ 6, 12] and traditional insulin injection in patients with type 1 diabetes mellitus.

Subjects

Between January 2010 and May 2011, 18 subjects with newly diagnosed type 1 diabetes mellitus (age 12-35 years) were enrolled. Type 1 diabetes mellitus was diagnosed according to the 2011 American Diabetes Association (ADA) criteria [ 13]. Newly diagnosed diabetes was defined as disease duration < 6 months. The protocol for AHSCT was reported in the previous study [ 6, 12]. Briefly, hematopoietic stem cells were mobilized with cyclophosphamide (2.0 g/m ²) and granulocyte colony stimulating factor (10 mg/kg/day), and then collected from peripheral blood by leukapheresis and cryopreserved. The cells were injected intravenously after conditioning with cyclophosphamide (200 mg/kg) and rabbit antithymocyte globulin (4.5 mg/kg). Eight patients received AHSCT treatment (AHSCT group) and 10 patients received traditional insulin injection (Insulin-only group). The age, sex ratio, basal daily insulin dose and blood glucose and C-peptide levels were matched in the two groups. Fifteen normal controls with matched age, gender and BMI were enrolled. The board of medical ethics of Shanghai Ruijin Hospital approved the study, and written informed consent was obtained from all of the subjects and/or their parents prior to enrollment.

Methods

Biochemical assessment

The daily insulin dose and duration of treatment were monitored and recorded. Serum C-peptide levels were measured by radioimmunoassay using a commercially available kit (Roche Diagnostics, Germany). The serum levels of anti-GAD antibodies were also measured by radioimmunoassay using a commercial kit (RSR Limited, Cardiff, UK), and levels > 7.5 U/ml were considered positive. Hemoglobin A1c was measured by high-pressure liquid chromatography.

Flow cytometry analysis

First, a portion of the PBMCs was stained with CD3-FITC, CD4-PeCy5, CD8-PE or TCR-α/β-FITC for 30 min in the dark to evaluate the portion of CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺ and TCR-α/β lymphocytes. Meanwhile, PBMCs were harvested and incubated with CD4-FITC, CD25-APC or CD127-PE for 30 min on ice to identify regulatory T (Treg) cells. For the intracellular cytokine staining of the T-helper type 1 (Th1) and T-helper type 17 (Th17) cells, PBMCs were stimulated with 50 ng/ml PMA and 500 ng/ml ionomycin (Sigma-Aldrich, St. Louis, MO, USA) for 6 h and then 3 μg/ml Golgi Plug/Brefeldin A (eBioscience, CA, USA) was added to the cells 4 h after stimulation. The cells were incubated with CD3-FITC and CD4-PeCy5 for 30 min at 4 °C in the dark, treated with a fixation/permeabilization solution for 30 min and stained by IFN-γ-APC and IL-17A-PE (all from eBioscience) for 20 min at room temperature [ 14]. Isotype controls that were labeled with the same fluorescent probe were included for nonspecific binding normalization. Four-color flow cytometry was performed with a Moflo XDP apparatus (Beckman Coulter, Miami, FL, USA), and the data were analyzed with Summit version 5.2.

Cytokine protein and mRNA measurements

A total of 2 × 10 ⁵ PBMCs were cultured in 200 μl of complete medium for 72 h after they were stimulated with 5 μg/ml phytohemagglutinin (Sigma-Aldrich). The supernatants were then collected and stored at −80 °C until they were analyzed. The cytokine analyses were run in duplicate with a Procarta® Cytokine Human Multiplex Kit (Panomics, Santa Clara, CA, USA) according to the manufacturer's protocols. Total RNA was isolated from the PBMCs with the RNeasy mini kit (Qiagen, Hilden, Germany). Quantitative real-time PCR was performed with a Takara SYBR Green PCR Kit. The reaction was run on a LightCycler480 Detection System (Roche, Switzerland), and the data were evaluated with the 2 -△ △ CT method. Experiments were performed in triplicate for each data point.

PBMC proliferation evaluations

Proliferation was evaluated with a water-soluble tetrazolium salt (WST) method by the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer's instructions. PBMCs were resuspended at a density of 2 × 10 ⁶/ml in a total volume of 100 μl and cultured for 72 h. WST was analyzed by adding the CCK-8 reagent, and the absorbance was measured at 450 nm (OD450) after 3 h with a microplate reader (wavelength 450 nm; Bio-Rad, Hercules, CA, USA). The relative absorbance of the treated group was normalized to the control group. The means ± standard deviation of each triplicate is presented in each graph. All of the experiments were repeated at least three times.

Statistical analysis

The data are presented as the means (±SD) or medium (95% CI range). The difference between the values amongst the different time points and groups was determined with ANOVAs for normally distributed data or Kruskal-Wallis tests for skewed data. Statistical analysis was performed with the SPSS 17.0 system. P  < 0.05 for two tails was considered statistical significance.

Results

Better islet function in the AHSCT group compared with the Insulin-only group

As shown in Table 1, no significant differences were found at baseline between the patients receiving AHSCT (AHSCT-0 M group) and Insulin-only (Insuin-0 M group) treatments. Twelve months later, patients in the Insulin-only group achieved remarkably decreased HbA1c level (Insulin-12 M vs Insulin-0 M, 7.33 ± 1.42 vs 12.20 ± 3.50%, P = 0.002), with no significant changes in the levels of fasting blood glucose, anti-GAD, fasting C peptide, AUCC and insulin dosage (all P > 0.05). Compared with the Insulin-only group, patients in the AHSCT group showed remarkably increased fasting C-peptide levels (AHSCT-12 M vs Insulin-12 M, 1.01 ± 0.23 vs 0.60 ± 0.50 nmol/L, P = 0.031) and C-peptide AUCs (9.59 ± 2.98 vs 4.76 ± 1.42, P = 0.002), and significantly decreased anti-GAD levels (150.4 (31.83-1315.52) vs 1200.0 (725.41-1980.02) units/ml, P = 0.043). Although the two groups displayed similar FBG (5.59 ± 1.40 vs 6.04 ± 1.70 mmol/L, P > 0.05) and HbA1c levels (6.80 ± 0.60 vs 7.33 ± 1.42%, P > 0.05) at baseline, the AHSCT group had less insulin dosages (0.15 ± 0.15 vs 0.52 ± 0.34 U/kg/day, P  = 0.004) at 12 months.

Table 1 Clinical characteristics of type 1 diabetic patients before and after insulin and AHSCT treatments

Decreased Th1 cell profiles in the AHSCT group compared with the Insulin-only group

The proportions of lymphocytes (40.84 ± 6.93 vs 34.6 ± 4.6%, P = 0.019) and monocytes (6.75 ± 1.26 vs 5.50 ± 0.81%, P = 0.04) were significantly higher in the type 1 diabetes mellitus patients compared with the normal controls. At baseline, the AHSCT and Insulin-only groups showed similar proportions of WBCs and lymphocytes. After 12 months of treatment, however, the AHSCT group showed lower proportions of WBCs and lymphocytes than the Insulin-only group (4.16 ± 1.17 vs 5.96 ± 1.38%, P = 0.011 and 33.64 ± 7.01 vs 42.39 ± 5.90%, P = 0.036, respectively). In addition, the AHSCT group showed lower CD3⁺CD4⁺ cell levels (30.59 ± 5.30 vs 43.57 ± 8.70%, P = 0.002), but similar CD3⁺CD8⁺ cell (30.83 ± 11.34 vs 27.65 ± 7.93%, P > 0.05) and monocyte (7.40 ± 0.32 vs 7.12 ± 1.49%, P > 0.05) levels compared with the Insulin-only group. The proportion of TCR(α/β) was significantly decreased in the AHSCT group compared with that in the Insulin-only group after treatment (57.90 ± 10.49 vs 69.94 ± 6.43%, P < 0.05) (Table 2).

Table 2 Changes in the peripheral blood cell proportion before and after insulin and AHSCT treatments

As shown in Fig. 1, R2 represents the CD3⁺CD4⁺ T cells (Fig. 1a) and R20 represents the IFN-r⁺ Th1 cells (Fig. 1b), which were prominently increased in the newly diagnosed patients compared with the normal controls (2.39 ± 0.74 vs 1.52 ± 0.71%, P = 0.005; Fig. 1c). The level was significantly reduced after AHSCT treatment ( P = 0.045); however, no significant changes were found before and after the traditional insulin therapy in the Insulin-only group. At the 12-month follow-up, a much lower proportion of the Th1 subset cells was observed in the AHSCT group compared with the Insulin-only group (1.64 ± 0.14 vs 3.16 ± 0.88%, P = 0.005; Fig. 1c).

Flow cytometry analysis, cytokine proteins and mRNA measurements of Th1 cells from PBMCs in the AHSCT and Insulin-only groups before and after treatment ( 0 M and 12 M). a, b Representative flow cytometry plots of CD3⁺CD4⁺ (R2) and Th1 cells (R20). c Proportion of IFN-γ⁺ Th1 cells in PBMCs. d, e, f mRNA expression levels of IL-2, IL-12p40 and T-bet respectively. g, h, j Concentrations of IL-2, IL-12p40 and IFN-γ in the cell culture supernatants respectively. * P < 0.05, ** P < 0.01, *** P < 0.001. AHSCT autologous hematopoietic stem cell transplantation, ns no significance, PBMC peripheral blood mononuclear cell

Additionally, the mRNA level of IL-2/IL-12p40 and the key transcription factor, T-bet, were consistently higher in newly diagnosed T1D patients compared with the normal controls (all P < 0.05). They are significantly decreased in the AHSCT group at 12 months compared with the Insulin-only group (IL-2, 1.19 ± 0.37 vs. 2.00 ± 0.74, P = 0.009; IL-12p40, 1.08 ± 0.51 vs 2.38 ± 0.36, P = 0.002; T-bet, 0.84 ± 0.23 vs. 1.60 ± 0.43, P = 0.001, respectively; Fig. 1d- f). The concentrations of IL-2, IL-12p40 and IFN-r in the cell supernatants were all elevated in T1D compared with the healthy group. They were decreased after AHSCT treatment while there was no significant change after insulin therapy in the Insulin-only group. There was a significant decrease in AHSCT-12 M compared with Insulin-12 M (Fig. 1g, h, j).

Increased Th17 cell proportion was reversed by AHSCT treatment

Newly diagnosed T1D patients showed increased Th17 cell proportions compared with the normal controls (0.48 ± 0.08 vs 0.34 ± 0.09%, P = 0.001, Fig. 2b). They were significantly decreased after AHSCT treatment (0.28 ± 0.03 vs 0.46 ± 0.09%, P = 0.000), while no significant change was found after insulin therapy in the Insulin-only group ( P > 0.05). As shown in Fig. 2a, R21 represents the plot area for IL-17A⁺ Th17 cells. Importantly, the Th17 cell proportions were much lower in the AHSCT-12 M group than in the Insulin-12 M group (0.28 ± 0.03 vs 0.51 ± 0.15%, P = 0.004; Fig. 2b). Furthermore, the mRNA level of IL-17A and transcription factor ROR-rt were downregulated after AHSCT treatment (all P < 0.05) and the AHSCT-12 M group was lower than the Insulin-12 M group (IL-17A, 0.83 ± 0.18 vs 1.53 ± 0.36, P = 0.002; ROR-rt, 0.7 ± 0.07 vs 1.37 ± 0.28, P = 0.000) (Fig. 2c, d).

Flow cytometry analysis, cytokine proteins and mRNA measurements of Th17 cells from PBMCs in the AHSCT and Insulin-only groups before and after treatment ( 0 M and 12 M). a Representative flow cytometry plots of Th17 cells (R21). b Proportion of CD3⁺CD4⁺IL-17A⁺ Th17 cells in PBMCs. c, d mRNA expression levels of I IL-17A and ROR-rt respectively. e Concentration of IL-17A in the cell culture supernatants. * P < 0.05, ** P < 0.01, *** P < 0.001. AHSCT autologous hematopoietic stem cell transplantation, ns no significance, PBMC peripheral blood mononuclear cell

Significantly higher IL-17A concentrations in the PBMC supernatants was observed in T1D patients compared with the normal controls (435.26 ± 285.44 vs 122.68 ± 75.64 pg/ml, P = 0.002). Consistent with the mRNA levels, the IL-17A cytokine levels were significantly reduced in the AHSCT-12 M group compared with the Insulin-12 M group (139.54 ± 63.67 vs 286.90 ± 150.68 pg/ml, P = 0.032) and the AHSCT-0 M group ( P = 0.018) (Fig. 2e).

Increased Treg cells and higher defined cytokines in the AHSCT group

As shown in Fig. 3a, R3 represents the plot area of CD25⁺CD127 ⁻ Treg cells. The proportion of CD4⁺CD25⁺CD127 ⁻ Treg cells was remarkably lower in the newly diagnosed group compared with the normal controls ( P = 0.000, Fig. 3b). After treatment, the Treg cells presented a higher proportion in the AHSCT-12 M group compared with the Insulin-12 M group (2.59 ± 0.44 vs 2.02 ± 0.43%, P = 0.040). The proportion of Treg cells was decreased after insulin treatment (2.02 ± 0.43 vs 2.46 ± 0.45%, P = 0.023) but remained at similar levels after AHSCT treatment (2.59 ± 0.44 vs 2.82 ± 0.64%, P > 0.05; Fig. 3b). The IL-10, TGF-β and foxp3 mRNA levels were increased after AHSCT treatment (all P < 0.05) and were higher in the AHSCT-12 M group than in the Insulin-12 M group (0.62 ± 0.16 vs 0.49 ± 0.16, P = 0.017, 1.05 ± 0.29 vs 0.81 ± 0.11, P = 0.033 and 1.12 ± 0.30 vs 0.81 ± 0.21, P = 0.016, respectively; Fig. 3c- e).

Flow cytometry analysis, cytokine proteins and mRNA measurements of Treg cells from PBMCs in the AHSCT and Insulin-only groups before and after treatment ( 0 M and 12 M). a Representative flow cytometry plots of Treg cells (R3). b Proportion of CD4⁺CD25⁺CD127 ⁻ Treg cells in PBMCs. c, d, e mRNA expression levels of IL-10, TGF-β and foxp3 respectively. f Concentration of TGF-β in the cell culture supernatants. * P < 0.05, ** P < 0.01, *** P < 0.001. AHSCT autologous hematopoietic stem cell transplantation, ns no significance, PBMC peripheral blood mononuclear cell

Finally, we analyzed the IL-10 and TGF-β concentrations in the PBMC supernatants. The TGF-β concentrations of the newly diagnosed patients were much lower than the normal controls ( P = 0.017, Fig. 3f). After AHSCT treatment, the TGF-β levels were significantly increased (485.33 ± 190.12 vs 317.29 ± 99.09 pg/ml, P = 0.036); however, no changes were observed after insulin treatment in the Insulin-only group. A marginally higher level was found in the AHSCT-12 M group compared with the Insulin-12 M group (485.33 ± 190.12 vs 328.87 ± 167.10 pg/ml, P = 0.107) (Fig. 3f). The IL-10 levels were undetectable.

The activated PBMC proliferation levels were downregulated in both groups

As shown in Fig. 4, the proliferation of PBMCs was significantly elevated in the newly diagnosed T1D patients compared with the normal controls (1.75 vs 1, P = 0.000). After treatment with either AHSCT or insulin therapy, the proliferation level was significantly downregulated (AHSCT-12 M vs AHSCT-0 M, 1.15 vs 1.66, P = 0.012; Insulin-12 M vs Insulin-0 M, 1.06 vs 1.87, P = 0.010). However, no significant difference was observed between the two groups after 12 months of treatment (1.15 vs 1.06, P  > 0.05).

PBMC proliferation levels detected with CCK-8 in the AHSCT and Insulin-only groups before and after treatment ( 0 M and 12 M). * P < 0.05, *** P < 0.001. AHSCT autologous hematopoietic stem cell transplantation, ns no significance

Discussion

The use of AHSCT to treat severe autoimmune diseases began in the early 1990s [ 15]. The rationale for AHSCT utilization as a treatment for type 1 diabetes mellitus comes with the double-edged effects of immunosuppressive drugs [ 16], which are hypothesized to reconstitute immunotolerance and therefore improve insulitis and increase the islet beta cell recovery capacity; however, AHSCT damages the bone marrow, leading to neutropenia and potentially serious infections. In 2008, Burt's group was the first to apply AHSCT in patients with newly diagnosed type 1 diabetes mellitus [ 17]. We also previously evaluated the efficacy and safety of AHSCT treatment in type 1 diabetes mellitus [ 6]. In the present study, we studied the expansion and function of Th1, Th17 and Treg cells after AHSCT treatment.

The CD4⁺ T-cell subsets have divergent CD4⁺ T-cell responses and may contribute to the chronic autoimmune responses in type 1 diabetes mellitus [ 9, 11, 18]. In our study, although the PBMC distribution was almost the same before treatment, the two groups exhibited an amazing discrepancy 1 year later. The decreased lymphocyte percentages, especially the CD3⁺CD4⁺ T cells, in the AHSCT group dominated the difference. Th17 immunity upregulation has been detected in peripheral blood T cells from children with T1D, and it potentiated both inflammatory and pro-apoptotic responses [ 19]. Circulating IL-17⁺ β-cell specific autoreactive CD4⁺ T cells were a feature of T1D, and the inhibition of Th17 cells reduced the islet-specific inflammatory T-cell infiltration [ 20]. In 2011, Wang et al. [ 21] reported that T-cell vaccination markedly inhibited the retinoic acid-related orphan receptor γt (ROR-γt) mRNA levels as well as the signal transducer and activator of transcription 3 (Stat3) phosphorylation levels, which are the key factors in Th17 cell polarization. At the same time, this treatment decreased blood glucose levels and protected against body weight loss in a diabetic model. Th17 cells may be directly involved in the inflammatory process of the pancreatic islets, causing severe T1D. Th17 cell suppression in the pancreas played a critical role in autoimmune diabetes [ 21]. Diminished Th17 cell responses underlie multiple sclerosis disease abrogation after HSCT. The post-therapy T-cell repertoire exhibited a significantly diminished capacity for Th17 cell responses, with complete abrogation of new clinical relapses. Additionally, a possible mechanism is the restoration of tolerance to self-antigens, which causes the inhibition of inflammatory cytokine production [ 22]. With regards to Th1 cells, increased serum CXCL10 concentrations in children with newly diagnosed type 1 diabetes mellitus sign a predominant Th1-driven autoimmune process, suggesting that a Th1 immune response is involved in the initiation of the insulitis [ 23]; while T-bet-deficient NOD mice were found to have profound defects in diabetogenic CD4⁺ T cells, both in the innate and adaptive immune systems. Moreover, this might lead to the disabilities in initial T-cell priming and proliferation, because T-bet is the critical transcription factor for Th1 polarization in CD4⁺ T cells, which helps to explain the critical role of Th1 cells in insulitis and diabetes development [ 24]. IFN-γ is produced by Th1 cells, which have also been associated with disease activity in multiple sclerosis (MS). No differences have been observed between T cells from HSCT-treated MS patients and healthy controls in the ability to proliferate and produce IFN-γ after polyclonal stimulation. Meanwhile, signs of immunological disease activity were demonstrated in 10 out of 15 control-treated patients, whereas these changes were not observed in the HSCT-treated group or controls. This lends support to the supposition that HSCT causes the removal of autoreactive T-cell clones [ 25]. In our study, we observed that Th1 and Th17 cells were significantly increased and activated in the newly diagnosed patients compared with the normal controls. More importantly, we found markedly decreased proportions of these two cell types after AHSCT, which was associated with the downregulated expression of their cytokines (IL-2/IL-12p40/IFN-γ and IL-17A) as well as their transcription factors (T-bet and ROR-rt). These findings suggest a functional role of these two cells in disease progression. Additionally, they had no changes after insulin therapy when blood glucose was controlled, suggesting that the initial high Th1 and Th17 cell-associated cytokines are not related to a specific inflammatory process, which may accompany the metabolic decompensated phase during T1D onset. Because these two cell types mediate extracellular and intracellular pathogen-induced inflammation as well as autoimmunity, we speculate that AHSCT can weaken autoimmune inflammatory reactions and apoptosis, which likely resulted from elimination of the aggressive destruction by the self-recognized effector T cells due to an "immune reset" by stem cells. This effect of AHSCT led to a halt of any further damage to the pancreas and a slowing down of diabetes progression.

We also observed significantly decreased Treg cell proportions in the newly diagnosed T1D patients compared with the normal controls. As reported, Treg cells regulate and ensure immune tolerance in healthy individuals, while Treg cell dysfunction might result in the excessive immune attacks and autoimmune diseases [ 26]. Deletion of Treg cells could accelerate T1D onset because spontaneous diabetes was exacerbated in both B7-1/B7-2-deficient and CD28-deficient NOD mice [ 27]. Treg cells can regulate ongoing immune reactions. In NOD mice, the administration of Treg cells could suppress the self-reactive effector T-cell activity and stop the destruction of pancreatic islets, which may potentially benefit T1D [ 28]. Patients with refractory lupus acquired long-term remission after stem cell transplantation [ 29]. Their Treg cells returned to the levels observed in the normal subject, accompanied by an almost complete inhibition of the pathogenic T-cell response to major lupus autoantigen from apoptotic cells [ 29]. In our study, the Treg cell proportion decreased after 1 year of insulin therapy, while it remained stable after the AHSCT therapy. We observed more Treg cells and higher Treg-associated cytokine level in the AHSCT-12 M group than the Insulin-12 M group. Our observations indicated that AHSCT might help Treg cell function and suppress the autoimmune insulitis process, thus leading to a better preservation of β-cell function. However, the absolute amounts of Treg cells in the AHSCT group did not return to normal levels. Cyclophosphamide (CTX) used in the conditioning stage before the stem cell transplantation might impair Treg cells, because Treg cells were more sensitive to CTX than Th cells and CTLs [ 30]. The administration of purified Treg cells might be a new attempt for immune suppressor therapy [ 28].

Regarding the PBMC proliferation levels, we found a remarkable reduction after either ASHCT or insulin treatment. These proliferation levels reflected the capacity of the cells to produce clones in vitro. According to Reinhold et al. [ 31], PBMC proliferation in normal individuals was inversely proportional to the glucose concentration. However, the proliferation indexes were higher in the diabetic patients than in the normal individuals, which could be explained by metabolic defects [ 32]. Meanwhile, insulin insufficiency may be another reason for elevated PBMC proliferation [ 33]. Consistent with our finding, this elevation was reversed after metabolic control in both groups [ 34]. However, there were no significant differences in proliferation between the two groups after 1 year of treatment. This result was unexpected because stem cell transplantation led to decreased proliferation. This discrepancy may be due to the similar metabolic control state, because no significant changes in the blood glucose levels were observed.

There were limitations in this study. The two main limitations of this study included the small number of patients and the duration of the study. A larger sample size and long-term follow-up data are needed in the future. Furthermore, the proliferation capacity of each T-cell subset was not analyzed; the expansion of Treg cells after AHSCT was not consistent with the upregulation of the associated cytokine; and the local pathologic changes in the pancreatic islets were unavailable, which may require further study.

Conclusions

In summary, our results suggest that AHSCT treatment is tightly associated with the inhibition of T-cell proliferation and pro-inflammatory cytokine production. These changes in the decreased expansion and function of Th1 and Th17 cells have demonstrated the novel immunomodulatory function of AHSCT interactions. When T1D progresses with unavoidable immune damage to the islet cells, AHSCT treatment combined with high-dose immunosuppressive therapy and transplantation of autologous hematopoietic stem cells may play an important role in immune resetting, which could potentially be targeted in therapeutic approaches to T1D. Future clinical studies should shed light on the validity of our hypothesis.

Abbreviations

AHSCT:

Autologous hematopoietic stem cell transplantation

IFN-γ:

Interferon gamma

IL:

Interleukin

PBMC:

Peripheral blood mononuclear cell

TGF-β:

Transforming growth factor-β

Th1:

T-helper type 1

Th17:

T-helper type 17

Treg:

Regulatory T

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Acknowledgements

The authors thank Elsevier for help in the English language editing of this manuscript.

Funding

This study was supported by grants from the Chinese National Natural Science Foundation (81370934), the National Basic Research Program of China (2015CB553601), the Chinese Academy of Science (XDA12030102) and Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (20161403).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

WG, LY and BW designed the experiments. LL and MY collected the clinical data. LL, MY, LY and WG performed the experiments, analyzed the data and wrote the manuscript. WG and LY revised the manuscript. JH, WW and GN contributed to discussion. WG is the guarantor of this work and had full access to all data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. LL now works at Department of Endocrinology, Wuhan Central Hospital. Wuhan 430014, Hubei Province, P.R. China. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

The board of medical ethics of Shanghai Ruijin Hospital approved the study, and written informed consent was obtained from all of the subjects and/or their parents prior to enrollment.

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Authors and Affiliations

1. The Department of Endocrinology and Metabolism, Ruijin Hospital, Shanghai Jiao-tong University School of Medicine, Shanghai Institution of Endocrine and Metabolism Diseases, Endocrine and Metabolic E-Institutes of Shanghai Universities and Key Laboratory for Endocrinology and Metabolism of Chinese Health Ministry, No. 197 Ruijin 2nd Road, Shanghai, 200025, People's Republic of China

Lei Ye, Li Li, Minglan Yang, Jie Hong, Weiqiong Gu, Weiqing Wang & Guang Ning

2. The Shanghai Institute of Immunology, Institutes of Medical Sciences, Shanghai Jiao-tong University School of Medicine and Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & SJTUSM, Shanghai, People's Republic of China

Bing Wan

3. The Chinese Academy of Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai Institutes for Biological Sciences, Laboratory of Endocrinology and Metabolism, Institute of Health Sciences, Shanghai, People's Republic of China

Guang Ning

Corresponding author

Correspondence to Weiqiong Gu.

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Ye, L., Li, L., Wan, B. et al. Immune response after autologous hematopoietic stem cell transplantation in type 1 diabetes mellitus. Stem Cell Res Ther 8, 90 (2017). https://doi.org/10.1186/s13287-017-0542-1
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Immune response after autologous hematopoietic stem cell transplantation in type 1 diabetes mellitus - Stem Cell Research & Therapy

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Background This study explored the details of the immune response after autologous hematopoietic stem cell transplantation (AHSCT) treatment in type 1 diabetes mellitus. Methods Peripheral blood mononuclear cells (PBMCs) from 18 patients with type 1 diabetes mellitus were taken at baseline and 12 months after AHSCT or insulin-only therapy.

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Long-term glycemic control using polymer-encapsulated human stem cell-derived beta cells in immune-competent mice - Nature Medicine

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When encapsulated with alginate derivatives that resist the foreign-body response, human embryonic stem cell-derived beta cells restore long-term normoglycemia in immunocompetent mice without the need for immunosuppression. The transplantation of glucose-responsive, insulin-producing cells offers the potential for restoring glycemic control in individuals with diabetes1.

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Affiliations

¹ Department of Medical Genetics, Institute of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.

² National Center for Transgenic Mouse Research, Institute of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.

³ School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

⁴ Department of Physiology, University of Toronto, Toronto, Ontario, Canada.

⁵ Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.

Affiliations

¹ Department of Medical Genetics, Institute of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.

² National Center for Transgenic Mouse Research, Institute of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.

³ School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

⁴ Department of Physiology, University of Toronto, Toronto, Ontario, Canada.

⁵ Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.

Abstract

Human-induced pluripotent stem cells (hiPSCs) can potentially serve as an invaluable source for cell replacement therapy and allow the creation of patient- and disease-specific stem cells without the controversial use of embryos and avoids any immunological incompatibility. The generation of insulin-producing pancreatic β-cells from pluripotent stem cells in vitro provides an unprecedented cell source for personal drug discovery and cell transplantation therapy in diabetes. A new five-step protocol was introduced in this study, effectively induced hiPSCs to differentiate into glucose-responsive insulin-producing cells. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, primitive gut-tube endoderm, posterior foregut, pancreatic endoderm, and endocrine precursor. Each stage of differentiation were characterized by stage-specific markers. The produced cells exhibited many properties of functional β-cells, including expression of critical β-cells transcription factors, the potency to secrete C-peptide in response to high levels of glucose and the presence of mature endocrine secretory granules. This high efficient differentiation protocol, established in this study, yielded 79.18% insulin-secreting cells which were responsive to glucose five times higher than the basal level. These hiPSCs-derived glucose-responsive insulin-secreting cells might provide a promising approach for the treatment of type I diabetes mellitus. J. Cell. Physiol. 232: 2616-2625, 2017. © 2016 Wiley Periodicals, Inc.

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induced pluripotent stem cells / metabolism*

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induced pluripotent stem cells / pathology

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kruppel-like transcription factors / genetics

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kruppel-like transcription factors / metabolism

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octamer transcription factor-3 / genetics

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octamer transcription factor-3 / metabolism

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proto-oncogene proteins c-myc / genetics

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proto-oncogene proteins c-myc / metabolism

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soxb1 transcription factors / metabolism

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national center for biotechnology information

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Pancreatic Endoderm-Derived From Diabetic Patient-Specific Induced Pluripotent Stem Cell Generates Glucose-Responsive Insulin-Secreting Cells - PubMed

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Human-induced pluripotent stem cells (hiPSCs) can potentially serve as an invaluable source for cell replacement therapy and allow the creation of patient- and disease-specific stem cells without the controversial use of embryos and avoids any immunological incompatibility. The generation of insulin ...

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Pancreatic Endoderm-Derived From Diabetic Patient-Specific Induced Pluripotent Stem Cell Generates Glucose-Responsive Insulin-Secreting Cells

# data[].attributes.subject
Pancreatic Endoderm-Derived From Diabetic Patient-Specific Induced Pluripotent Stem Cell Generates Glucose-Responsive Insulin-Secreting Cells

# data[].attributes.body
Des cellules souches pluripotentes induites (hiPSC) dérivées d’un patient diabétique ont été avec succès différenciées en cellules sécrétrices d’insuline sensibles au glucose. Ce processus implique un protocole en cinq étapes imitant l’organogenèse pancréatique in vivo.

Les cellules différenciées ont démontré des propriétés de cellules β fonctionnelles.

L’étude a rapporté une efficacité élevée, avec 79,18% des cellules sécrétant de l’insuline et réagissant à des niveaux de glucose cinq fois supérieurs au niveau basal.

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Generation of stem cell-derived β-cells from patients with type 1 diabetes

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Generation of stem cell-derived β-cells from patients with type 1 diabetes

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Les auteurs ont générés des cellules β pancréatiques à partir de cellules souches pluripotentes. Les cellules souches pluripotentes induites de patients atteints de diabète de type 1 peuvent être différenciées en β-like cellules qui n’ont pas de différence détectable par rapport aux cellules des individus non diabétiques. Les cellules souches pluripotentes induites de patients atteints de diabète de type 1 sont fonctionnelles in vitro et in vivo et permettent de réguler la glycémie quand elles sont implantés chez des souris diabétiques.

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Engineering human stem cell-derived islets to evade immune rejection and promote localized immune tolerance

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Engineering human stem cell-derived islets to evade immune rejection and promote localized immune tolerance

# data[].attributes.body
Des cellules souches d’îlots pour transplantation sont crées pour sécréter des protéines empêchant le rejet et favorisant la croissance cellulaire, les îlots permettent de réguler le diabète pendant 8 semaines après transplantation chez des souris diabètiques

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faut mettre la définition de diabète de type 1
les types de thérapies qui existe jusqu'à présent (transplants, injection insuline produite par animales,...) et celle sur laquelle on se concentre : thérapie cellulaire

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I-introduction : présentation succincte de notre sujet (diabète, contexte et importance de trouver traitements, modèle)

II- énoncer le plan

III-Qu’est ce que le diabète de type I et une maladie autoimmune

IV- Qu’est ce que les cellules souches ?

V- utilisation de SC pour traiter le diabète….

VI- controverses/limites de l’application/Risques/critiques

VII-conclure sur les perspectives futures

VIII- conclusion générale

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Intro : Mise en situation : Nb de personne ayant le diabète, en quoi c'est un problème qui augmente (en population) estimation de vie, coût du traitement de la pompe insuline et tout(je crois c'est 💰 : Insee "Une enquête estime à plus de 5 000 € le coût moyen annuel du traitement d'un diabétique.")

Partie I. : le diabète de type I (+détaillé d'un pdv cellulaire, qui fait quoi...)

Partie II : Les cellules souches et les traitements actuels

Partie III : les cellules souches pour le diabète de type I. avantage et limites

Conclusion : ?? idk yet

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Faut faire un powerpoint non? Vous voulez qu'on fasse ça sur quoi ?

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C’est pas pour maintenant mais je voulais essayer 

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Doodle is the simplest way to schedule meetings with clients, colleagues, or friends. Find the best time for one-to-ones and team meetings with our user-friendly calendar tool. Get started today!

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Question : qu'est-ce qui cause la réponse immunitaire (cmh ?) ?

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Question : qu'est-ce qui cause la réponse immunitaire (cmh ?) ?

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Diabète de type 1 : une maladie auto-immune, vraiment ? 

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Diabète de type 1 : une maladie auto-immune, vraiment ? 

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Diabete de type 1 : une maladie auto-immune, vraiment ?

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Diabete de type 1 : une maladie auto-immune, vraiment ?

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La destruction des cellules β est du à l'induction de la mort cellulaire par les LTCD4 et LTCD8

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San Diego, Etats-Unis - La greffe de cellules d'îlots de Langerhans pancréatiques obtenus à partir de cellules souches (VX-880, Vertex Pharmaceuticals) continue d'être une piste prometteuse dans le traitement du diabète de type 1, selon les dernières données obtenues chez six patients et présentées au congrès de l' American Diabetes Association 2023.

Deux des six patients ayant reçu les perfusions de VX-880 n'ont plus besoin d'insuline depuis plus d'un an et trois autres, qui les ont reçues plus récemment, suivent une trajectoire similaire. L'un d'entre eux a abandonné l'étude pour des raisons indépendantes de la thérapie.

Les cinq autres continuent de recevoir un traitement immunosuppresseur pour prévenir le rejet des cellules.

Les six patients avaient tous une sécrétion d'insuline indétectable et souffraient d'hypoglycémies sévères comme critère d'entrée dans l'étude de phase 1/2.

" Ces nouveaux résultats démontrent le potentiel des cellules d'îlots de Langerhans dérivés de cellules souches en tant que futur traitement pour les patients atteints de diabète de type 1, une nouvelle ère qui pourrait potentiellement supprimer la nécessité d'administrer de l'insuline exogène pour obtenir un contrôle glycémique ", a indiqué l'investigateur principal, le Pr Trevor W. Reichman, directeur chirurgical de la transplantation de pancréas et de cellules d'îlots de Langerhans à l'Université de Toronto, Canada.

Le Pr Reichman a présenté ces données lors des sessions scientifiques annuelles de l'ADA 2023. Il s'agit d'une actualisation du rapport présenté lors de l'ADA 2022 sur les deux premiers patients ayant reçu la thérapie. " Nous espérons que cette recherche inédite changera la donne pour le traitement du diabète de type 1 ", a-t-il souligné.

" Les données de l'essai clinique sont extrêmement intéressantes... La sécrétion d'insuline était presque la même que chez une personne ne souffrant pas de diabète de type 1. Cela fait si longtemps que nous essayons de générer ces cellules. C'est fantastique de voir ce résultat ", a commenté la chercheuse Maria Cristina Nostro, PhD (McEwen Stem Cell Institute, Toronto) pour Medscape Medical News.

Deux patients ont atteint le critère d'évaluation primaire, trois autres sur la bonne voie

Les six patients (3 hommes, 3 femmes) avaient un âge moyen de 44 ans et un diabète depuis 23 ans en moyenne. Leur taux moyen d'A1c à l'entrée dans l'étude était de 8,1 % et le peptide C à jeun (évaluation de la sécrétion d'insuline endogène) était indétectable. Ils avaient connu en moyenne 3,3 épisodes d'hypoglycémie sévère au cours de l'année précédant la perfusion, qui a été administrée dans la veine porte de manière similaire à la procédure utilisée avec les îlots de Langerhans issus de donneurs décédés.

Les deux premiers patients et celui qui a abandonné l'étude, ont reçu des demi-doses cibles de VX-880 (phase A de l'essai), tandis que les autres, entrés dans l'étude de manière séquentielle (phase B), ont reçu chacun la dose cible complète de VX-880 administrée en une seule perfusion.

Un sérum anti-lymphocytaire (GAT) et des immunosuppresseurs d'entretien, tacrolimus/sirolimus, ont été administrés aux receveurs.

Après la perfusion, les six participants ont présenté une production de peptide C (insuline endogène), une réduction de l'A1c malgré une utilisation réduite d'insuline exogène, et aucun épisode d'hypoglycémie sévère n'a été rapporté à partir du 90e jour.

Les deux participants ayant au moins un an de suivi ont rempli les critères du critère principal, à savoir un taux d'A1c inférieur à 7 % et l'absence d'épisodes d'hypoglycémie sévère. Le premier participant avait un taux d'A1c de 5,3 % au 21e mois et le second de 6,0 % à 12 mois. Tous deux ont présenté une production d'insuline répondant au glucose lors d'un test de tolérance à un repas mixte et ont dépassé l'objectif de l'ADA de plus de 70 % de temps dans l'intervalle de glycémie évalué par la surveillance continue de la glycémie.

Sécurité : pas de problèmes majeurs jusqu'à présent

Parmi les six patients, les effets indésirables comprenaient des élévations de l'enzyme hépatique transaminase, survenues peu après la perfusion de VX-880, qui ont été transitoires et se sont normalisées. Aucun événement indésirable grave n'a été considéré comme lié au traitement.

" Je n'ai aucune inquiétude. Parce qu'ils utilisent des immunosuppresseurs, si quelque chose se passait mal, il suffirait de supprimer l'immunosuppression et les cellules seraient détruites par le système immunitaire. C'est donc un essai parfait d'une certaine manière ", a commenté Maria Cristina Nostro avant d'ajouter " à l'avenir, lorsque nous développerons un système qui sera génétiquement modifié pour éliminer l'immunosuppression. Je pense que la préoccupation sera de s'assurer que les cellules génétiquement modifiées sont sans danger ".

Dans un essai en cours avec des cellules génétiquement modifiées, " ils placent le produit à l'intérieur d'un dispositif de sorte que les cellules puissent être récupérées. Ce n'est peut-être pas parfait, mais au moins cela nous permettra de savoir si le produit génétiquement modifié est sûr ", a-t-elle expliqué en introduction de la session.

Lors de son intervention, la chercheuse a également résumé les travaux en cours dans le domaine, notamment les efforts visant à améliorer la génération d'îlots dérivés de cellules souches sans cellules non-bêta " hors cibles " afin de garantir la cohérence, l'optimisation de la prise de greffe et l'élimination de l'immunosuppression.

Elle a précisé à Medscape : " [VX-880] est le début. C'est le premier produit qui sera utilisé en clinique, mais je peux imaginer que dans cinq ou dix ans, nous aurons des solutions différentes et plus performantes pour le diabète de type 1 et, qui sait, peut-être même pour le diabète de type 2 ".

Sur la base des données obtenues jusqu'à présent, l'essai VX-880 passe maintenant à la phase C, dans laquelle 10 participants recrutés simultanément recevront la dose cible complète du produit. L'essai, qui se déroulait auparavant exclusivement aux États-Unis, s'est maintenant étendu à des sites supplémentaires en Norvège, en Suisse et aux Pays-Bas.

L'étude a été financée par Vertex. Le Pr Reichman fait partie des conseils consultatifs de Vertex et de Sernova Inc. Maria Cristina Nostro a été consultante pour Sigilon Therapeutics de 2018 à 2022, reçoit actuellement un financement de recherche de Universal Cells et possède un brevet sous licence pour Sernova.

Cet article a été publié initialement sur Medscape.com sous l'intitulé 33% of Type 1 Diabetes Patients Insulin-Free With Stem Cells Traduit et adapté par Aude Lecrubier.

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DT1 : des patients deviennent insulino-indépendants grâce à une greffe de cellules souches

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Une greffe d'îlots de Langerhans dérivés de cellules souches VX-880 de Vertex Pharmaceutical a été réalisée chez six patients diabétiques de type 1, dont deux sont insulino-indépendants à un an.

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Amélie et Chloé : Diaporama et article 1 (graphique + explication)

Jeanne et Blanche : Diaporama et article 2 (graphique + explication)

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définitions :

thérapie cellulaire

cellules souches

cellules β des îlots de Langerhans

diabète de type 1

modèle de test : souris (pourquoi on a choisi?)