Differentiation of Human Embryonic Stem Cells into Insulin-Producing Clusters - 《stemcells》2004-2005文献干细胞之家



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发表于 2009-3-5 10:38 |只看该作者 |倒序浏览 |打印

a Department of Obstetrics and Gynecology, Rambam Medical Center, Haifa, Israel;
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b The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel

Key Words. ES cells ? Pancreatic differention ? Insulin-secreting cells ? Differentiation) n' a- h3 \5 I! ^- q% @' [6 N
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Joseph Itskovitz-Eldor, M.D., Department of Obstetrics and Gynecology, Rambam Medical Center, P.O.B. 9602, Haifa 31096, Israel. Telephone: 972-4-854-2536; Fax: 972-4-854-2503; e-mail: Itskovitz@rambam.health.gov.il

ABSTRACT
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Human embryonic stem (hES) cells are self-renewing pluripotent cells obtained from the inner cell mass of human blastocysts . Both hES cell lines and clones retain normal karyotypes even after long-term culture. The unique feature of ES cells is their dual ability to be indefinitely cultured in an undifferentiated state and to differentiate into cells representative of all three body lineages: ectoderm, mesoderm, and endoderm . Differentiation of hES cells can be induced by removing the cells from their feeder layer and growing them in suspension. Growth in suspension leads to the formation of embryoid bodies (EBs), which are a necessary step in the process of in vitro differentiation . Such differentiation provides a new perspective for studying the cellular and molecular mechanisms of early development. In addition, their characteristics made hES cells good candidates for transplantation therapies. One of the potential uses may be the cure of diabetes.& p. i  ~; H7 b% E0 v. z2 b) k
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Diabetes mellitus is a major health problem affecting more than 5% of the global population and is the most common metabolic disorder . Diabetes mellitus takes two forms: type I (insulin-dependent), caused by an autoimmune destruction of the insulin-producing ? cells and type II (non-insulin-dependent), resulting from a combination of reduced insulin sensitivity and impaired function of the insulin-secreting ? cells. The present treatments for diabetes do not offer a cure and do not prevent the secondary complications associated with diabetes, such as diabetic retinopathy, nephropathy, and neuropathy . Early treatment of these patients and restoration of the ?-cell function through pancreas or islets transplantation can both free the patients from their dependency on insulin and prevent complications . However, one of the major obstacles in human transplantation is the limited supply of donor tissue ., X; p& `2 R4 ]6 L& ~; g$ ?/ h
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It has previously been shown that hES cells can spontaneously differentiate into insulin-producing cells, can secrete insulin, and can express other ?-cell markers . Research conducted on mouse embryonic stem cells led to insulin-producing cells , which were capable of curing diabetes in streptozotocin-treated mice . Here we show that hES cells can be induced to form islet-like clusters similar to immature pancreatic ? cells and to produce insulin.* Y) q6 Y3 R$ H" F9 U
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MATERIALS AND METHODS
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Previous works using mouse  and hES  cells have shown their differentiation potential toward insulin-secreting cells. To promote the differentiation of hES cells into insulin-secreting cells, the procedure was performed according to a previously described protocol  with some modifications (Fig. 2).2 y: c) o7 x/ T- i; ~5 ]8 ^
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Figure 2. General outline of the differentiation protocol. The protocol consists of several stages. Stage 1: growth of undifferentiated hES cells (bar = 10 μM). Stage 2: formation of EBs (bar = 30 μM). Stage 3: plating EBs in medium I (DMEM/F12 1:1, insulin-transferrin-selenium-fibronectin and 1 mM glutamine) for 1 week (bar = 30 μM). Stage 4: dissociating the cells and plating them in medium II (DMEM/F12 1:1 with N2 and B27 media, 1 mM glutamine, and 10 ng/ml bFGF) for 1 week (bar = 5 μM). Stage 5: change to medium III (removal of bFGF, addition of 10 μM nicotinamide, and reduction of the glucose concentration from 3,151 to 901 mg/l) (bar = 10 μM). Stage 6: dissociation of the cells and growing them in suspension in petri dishes with medium III (bar = 10 μM).

The clusters formed at the end of the process were examined for their insulin-secretion potential (Fig. 1A). Decreasing the glucose concentration in the growth medium from 3,151 to 901 mg/l during stage V (as described in Materials and Methods) resulted in an increase in the insulin secretion in response to glucose, from 2.13 ± 1.32 to 8.64 ± 3.42 μu/ml per 105 cells/hour. A more dramatic increase in insulin secretion to 225.8 ± 78 μu/ml per 105 cells/hour was obtained by the formation of clusters at step VI (Fig. 1A). The time course of insulin secretion in response to glucose was further examined by incubating the cells for 5–60 min, which revealed that the amount of secreted insulin accumulated in the medium increased with time, reaching maximum effect after 60 min. To determine whether these cells use physiological signaling pathways to regulate insulin release, we examined the effect of several agonists and antagonists on insulin secretion. Incubating the cells with high glucose (16.6 mM) and 100 μM IBMX (3-isobutyl-1-methylxanthine; an inhibitor of cyclic-AMP phosphodiesterase) resulted in a small 40% increase of insulin secretion as compared with 3.3 mM glucose (Fig. 1B).

Figure 1. A) Insulin release in response to 3.3 mM glucose in different growth conditions. Left bar: insulin release from stage V-H cells (high glucose DMEM/F12 supplemented with N2, B27, and nicotinamide). Middle bar: stage V-L cells (low glucose DMEM/F12 supplemented with N2, B27, and nicotinamide). Right bar: clusters at stage VI (with the same medium as stage V-L). B) The effect of IBMX on insulin secretion. Cells were incubated with either 3 mM glucose (left bar) or 16.7 mM glucose (right bar) for 60 min with or without IBMX (100 μM). C) The effect of agonists and antagonists on insulin secretion in response to 16.7 mM glucose. The effect of IBMX, tolbutamide, carbachol, and nifedipine on the insulin secretion was determined and expressed as the percentage of the basal insulin secretion. The data of all experiments are expressed as the mean ± SE of three experiments performed in triplicates.
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Some other agonists and antagonists also had an effect on insulin secretion. Tolbutamide (10 μM), an inhibitor of the KATP±-channel, and Carbachol (100 μM), an agonist of muscarinic cholinergic receptors, increased insulin secretion by 59.6% ± 5% and 50.7% ± 3%, respectively. Nifedipine (50 μM), a blocker of Ca2  channels present in ? cells, inhibited insulin secretion by 37.5% ± 8% (Fig. 1C). These results indicate that the pancreatic machinery is used for glucose-mediated insulin release.7 [( x: T( \2 |  F

RT-PCR reaction, as shown in Figure 3, demonstrated an enhanced expression of pancreatic genes in the differentiating hES cells. The transcription factor, pancreatic duodenal homeobox 1 (PDX1), appeared mainly in stage III mRNA, decreased in stage IV, and reappeared in stage VI. Glucagon and neurogenine 3 (Ngn3) were highly expressed in stage III and then in stage VI. Somatostatin expression was higher at stage IV than at stage III and then disappeared. Pax4 expression increased from stage III to IV and then decreased in stage V-H (high glucose in the medium), but when the glucose concentration was reduced, Pax4 was increased (stage V-L, low glucose in the medium and stage VI). Pax6 was noticed in all stages of differentiation. The KATP-channel genes Kir6.2 and SUR1 were expressed in stages IV and in stage VI, while SUR1 was detected in stage V-L as well. Islet amyloid polypeptide (IAPP) and prohormone convertase 1/3 (PC1/3) expressions increased in stages V-L and VI. Glucokinase (GCK) was detected only in stage V-L. Insulin and other pancreatic ?-cell-specific genes, such as Nkx6.1, Is11, Glut2, and prohormone convertase 2 (PC2), were only noticed in mRNA from stage VI cells. These results indicate that differentiated cells have the ability to transcribe and process insulin.

Figure 3. RT-PCR analysis of pancreatic gene expression in several differentiation stages. Total RNA isolated from both undifferentiated hES cells and differentiating cells was subjected to RT-PCR analysis with primers for the indicated genes. Lane 1: undifferentiated hES cells. Lane 2: stage III cells grown in ITSF. Lane 3: stage IV cells grown in DMEM/F12 with N2, B27, and bFGF. Lane 4: stage V-H cells grown in DMEM/F12 medium with N2, B27, and nicotinamide and high glucose. Lane 5: stage V-L cells grown in the same conditions as stage V-H but with low glucose concentrations. Lane 6: stage VI cells grown in suspension in the same media as stage V-L. Lane 7: represents no RTase in the PCR reaction.
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We further examined insulin gene expression by using the in situ hybridization technique and were able to ascertain its distribution in stage VI cells (Fig. 4). Stage VI clusters were examined by immunofluorescence for the expression of insulin, C-peptide, somatostatin, and glucagon (Fig. 5). The results revealed a high percentage of insulin- (70%), somatostatin- (43%), or glucagon (50%)-expressing cells in the clusters. The cells coexpressed both insulin and C-peptide, indicating an undeniable production of insulin. A substantial number of the cells were costained for insulin and glucagon or somatostatin, suggesting that these cells resemble immature pancreatic endocrine cells. This phenomenon has been previously described in the development of the mouse and human pancreas . When staining the cells for the neuron marker nestin, only 10% were stained positive. Likewise, staining for the detection of smooth muscle cells resulted in only a 5% positive staining (data not shown). Similarly, RT-PCR analysis revealed that transcripts for neuronal marker NF-68KD and cardiac actin were detected only in stages IV and V-L and were absent in stage VI cells (Fig. 3).  v2 p; K2 q4 f0 S3 I' [, G: q, g

Figure 4. mRNA detection of insulin using in situ hybridization. Stage VI cells were hybridized with dig-labeled insulin probe, as described in Materials and Methods. Detection was performed using rhodamine-conjugated antidioxigenin antibody. A) Phase contrast image of stage VI cluster. B) Insulin mRNA in the same cells. C) Overlap of A and B showing cells stained in red as part of the whole cluster. Bar = 10 μm.

Figure 5. Confocal microscopy of immunostaining for insulin, C-peptide, somatostatin, and glucagon. Immunofluorescence staining of stage VI clusters. Left panel: A represents C-peptide staining, D represents somatostatin staining, and G represents glucagon staining (all in green). Middle panel: B, E, and H represent insulin staining (red). Right panel: C, F, and I represent costaining of insulin and the second marker (C-peptide, somatostatin, and glucagon, respectively). Overlapping costaining was seen as orange. Slides were analyzed using a confocal microscope. Bar = 10 μm.( B( g7 H7 {6 T7 h( _" ?
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Cell proliferation was further tested using a BrdU staining kit (Zymed). BrdU was incorporated into proliferating cells at the S-phase. At stage VI, 20% of the cells were stained with BrdU, thus confirming that the clusters were still proliferating (Fig. 6A). Furthermore, the aggregation step increased the survivability of the cells, from 1 week at stage V to over 1 month at stage VI., O0 K; f. X  _; Q2 S& P
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Figure 6. A) In order to determine the percent of the proliferating cells, BrdU Streptvidin-Biotin labeling was used followed by anti-mouse IgG Cy3 (red) conjugated antibody. Nuclei were stained with TO-PRO-3 iodide. Slides were analyzed using a confocal microscope. Bar = 10 μm. B) Stage 4b clusters staining for TUNEL  and Insulin. Stage 4b cells were stained for TUNEL  nuclei (green) with In Situ Cell Death Detection Kit followed by Immunofluoresence staining with mouse anti-insulin (red). Slides were analyzed using a confocal microscope. Bar = 10 μm3 b$ t* `9 ^+ [. z; M7 [- i+ ]
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The cells were further tested with TUNEL  combined with insulin staining. Some of the cells were stained positive for both TUNEL and insulin, suggesting apoptosis. However, in contrast to a previous work , 80% of the cells from stage IV that stained positive for insulin were not apoptotic (Fig. 6B). BrdU staining, TUNEL and C-peptide staining of the cells, confirmed that stage VI cells still proliferate and produce de novo insulin.* A# ^) S6 i; Q% n% W

DISCUSSION
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In this work, hES cells were modified to form insulin-producing cells. By improving a protocol that had been previously used in mouse ES cells, an enriched population of insulin-producing cells was obtained. Even though these cells secreted a substantial amount of insulin, their immunostaining and RT-PCR expression revealed a similarity to immature pancreatic cells. Further maturation protocols of the nonmature cells and in vivo studies are needed. Developing hES cells to form mature ?-cell-like structures will enable the use of these cells for future cell therapy of type I diabetes.

ACKNOWLEDGMENT

* These authors contributed equally.% b  S6 u0 P) B% s; Y5 z" R
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