Pancreatic islet-like cell structures and a method of preparing thereof

Abstract

The invention relates to a method of preparing pancreatic islet-like cell structures characterized by a unique combination of morphological and functional features which make them particularly suitable for use in both clinical and drug screening application, as well as to the pancreatic islet-like cell structures obtained therefrom.

Claims

1. A method of preparing a spheroid pancreatic islet-like cell structure, comprising culturing a stem cell in a first differentiation liquid cell culture medium comprising a poly-cationic substance selected from the group consisting of poly-lysine and a soluble salt of protamine.

2. The method according to claim 1, wherein said culturing the stem cell in the first differentiation liquid cell culture medium comprising a poly-cationic substance is performed for a period of time of 2 to 4 days.

3. The method according to claim 1, wherein the poly-cationic substance is a soluble salt of protamine.

4. The method according to claim 3, wherein the soluble salt of protamine is protamine sulfate or protamine hydrochloride.

5. The method according to claim 4, wherein the protamine sulfate or protamine hydrochloride is at a concentration comprised within the range of 5 to 20 mM.

6. The method according to claim 1, wherein the poly-cationic substance is poly-lysine.

7. The method according to claim 1, which further comprises replacing the first differentiation liquid cell culture medium comprising a poly-cationic substance with a second differentiation liquid cell culture medium not comprising a poly-cationic substance and culturing until completely differentiated cells are obtained.

8. The method according to claim 7, wherein the culturing in the second differentiation liquid cell culture medium not comprising a poly-cationic substance is performed for a period of time of at least 2 days.

9. The method according to claim 1, wherein the stem cell is an adult stem cell.

10. The method according to claim 9, wherein the adult stem cell is a human liver stem cell.

Description

EXAMPLES

1. HLSC Culture and Expansion

(1) 1.1 Isolation, Characterization and Culture of HLSCs

(2) HLSCs lines are isolated from healthy liver tissues of patients undergoing hepatectomies and characterized as previously described (Mohamad Buang M L et al. Arch Med Res. 2012; 43(1): 83-8). Specifically, HLSCs are seeded in 75 cm.sup.3 culture flasks and cultured in a medium (see Table 1) containing a 3 to 1 proportion of -minimum essential medium and endothelial cell basal medium-1, supplemented with L-glutamine 2 mM, penicillin 100 UI/ml/streptomycin 100 g/ml and 10% Fetal Bovine Serum. Cells are maintained in a humidified 5% CO.sub.2 incubator at 37 C.

(3) 1.2 Detachment of HLSCs

(4) Once up to a 80% confluence, cells are washed twice with PBS and incubated with trypsin-EDTA 1 for about 5 minutes at 37 C., in order to induce cell detachment. Trypsin activity is subsequently neutralized by adding RPMI supplemented with L-glutamine 2 mM, penicillin 100 UI/ml/streptomycin 100 g/ml and 10% Fetal Bovine Serum. Then, cells are harvested by centrifugation at 1200 rpm for 5 minutes, the supernatant is removed and the pellet re-suspended in culture medium and split among five 75 cm.sup.3 culture flasks.

(5) 1.3 Cryopreservation of Cells

(6) Cells up to a 80% confluence are detached and harvested by centrifugation as described in paragraph 1.2. The cells are counted and 10.sup.6 cells per vial are cryopreserved. The cell pellet is resuspended in a 1 ml solution containing 90% FBS and 10% dimethyl sulphoxide (DMSO) and placed into pre-cooled cryovial/s. The cryovials are frozen at 80 C. overnight before being placed into the liquid nitrogen container at 196 C.

(7) 1.4 Thawing of Cryopreserved Cells

(8) A vial of frozen HLSCs is removed from the liquid nitrogen tank and placed in a beaker of water pre-warmed to 37 C. Once the cell suspension has completely thawed, it is placed into a sterile 50 ml falcon tube with 10 ml of sterile media and centrifuged at 1200 rpm for 5 minutes. The cell pellet is then resuspended in culture medium and split in three 75 cm.sub.3 culture flask and left to attach overnight at 37 C. in a humidified 5% CO.sub.2 incubator. The medium is changed the following day.

2. HLSC Differentiation into Islets-Like Structures

(9) HLSCs at a density of 1210.sup.3/cm.sup.2 are seeded in 25 cm.sup.3 culture flasks or in a Petri dish 10020 mm in the differentiation culture medium 1 (see Table 1) consisting in RPMI 1640 or DMEM supplemented with 10% Fetal Bovine Serum, glucose 11.6 mM, protamin chloride 10 g/ml, L-glutamine 2 mM and penicillin 100 UI/ml/streptomycin 100 g/ml. Cells are placed, for a period of 4 days, without changing the medium, in a humidified 5% CO.sub.2 incubator at 37 C. On day 5, the medium is replaced with differentiation culture medium 2 (see Table 3) consisting in RPMI 1640 or DMEM supplemented with 10% FBS, glucose 11.6 mM, L-glutamine 2 mM and penicillin 100 UI/ml/streptomycin 100 g/ml. Medium is subsequently changed every other day. Within 2 to 4 days cells are expected to start organizing in islets-like structures that reach a maximum number after 14-18 days (FIG. 1).

(10) TABLE-US-00001 TABLE 1 HLSC culture medium Final concentration Volume (ml) Final volume 250 -MEM 165 EBM 55 FBS 10% 25 Penicillin 10000 UI/ml/ 100 UI/ml/100 ug/ml 2.5 streptomycin 10 mg/ml Glutamine 200 mM 2 mM 2.5

(11) TABLE-US-00002 TABLE 2 HLSC differentiation medium 1 Final concentration Volume (ml) Final volume 250 RPMI 1640 or DMEM 216.85 FBS 10% 25 Penicillin/streptomycin 100 UI/ml/100 ug/ml 2.5 Glutamine 200 mM 2 mM 2.5 Glucose 1M 11.6 mM 2.9 Protamine 10 mg/ml 10 ug/ml 0.25

(12) TABLE-US-00003 TABLE 3 HLSC differentiation medium 2 Final concentration Volume (ml) Final volume 250 RPMI or DMEM 217.1 FBS 10% 25 Penicillin/streptomycin 100 UI/ml/100 ug/ml 2.5 Glutamine 200 mM 2 mM 2.5 Glucose 1M 11.6 mM 2.9

(13) The results obtained are illustrated in the appended drawings, the content of which is briefly illustrated herein below.

(14) FIG. 1: representative 20 micrographs showing HLSCs in basal culture medium (A) and following culture in differentiation medium with protamine (B-D). After 24 h, the cells changed in morphology and started forming small clusters (B) that progressively increased in both size and number, reaching a maximum number after a culture period of 18 days (C). (D) 40 micrograph showing islets-like structures after a culture period of 18 days. (E) Graph showing the total number of islets-like structures per 25 cm.sup.3 culture flask after 18 days of culture. (F) Graph representing the meanSD number of islets-like structures after 18 days of culture (n=10).

(15) FIGS. 2-8: representative pictures showing islet-like structures characterization by immunofluorescence after 14 days of culture: islets-like structures become positively stained for both PDX-1 and NgN3 (FIG. 2), insulin and glucagon (FIG. 3), C-peptide and GLUT-2 (FIG. 4), somatostatin (FIG. 5), ghrelin and PP (FIG. 6), collagen IV (FIG. 7) and von Willebrand Factor (FIG. 8).

(16) FIG. 9: representative pictures showing immunofluorescence characterization of islet-like structures cells following islet-like structures disaggregation (trypsin 1) after 14 days of culture.

(17) FIG. 10: graph showing both blood glucose and human C-peptide levels in non-diabetic SCID mice (solid line, CTRL), streptozotocin-induced (55 mg/kg/day for 5 days) diabetic SCID mice (dotted line, DM) and streptozotocin-induced diabetic SCID mice that received HLSC derived islets-like structures implant (5000 IEQ/kg) under the renal capsule (dashed line, DM+ILS), before and 13 days following the implant. Compared to diabetic mice, diabetic mice that received islets-like structures implant had a significant decrease in blood glucose levels. This was paralleled by a concomitant increase in human C-peptide, that remained undetectable in both non diabetic SCID mice and in diabetic SCID mice that did not receive the implant.

(18) FIG. 11: graph showing islets-like structure (ILS) formation following HLSCs culture in RPMI or DMEM based medium supplemented with FBS 10% (F), FBS 10%+ glucose 11.6 mM (FG) or FBS 10%+ glucose 11.6 mM+ protamine chloride 10 g/ml (FGP) for 48 hours.

(19) FIG. 12: graphs showing the size distribution (A), the meanSD diameter (B) and IEQ/100 ILS (C) of islets-like structures derived from HLSCs following 14 days of culture in RPMI based medium supplemented with FBS 10%+ glucose 11.6 mM+ protamine chloride 10 g/ml.

(20) FIG. 13: Glucose does not affect islets-like structures (ILS) formation (A), but plays a key role in inducing endocrine specification and insulin/glucagon expression (B). A. Representative picture and graph showing islets-like structure formation following culture (4 days) in RPMI/DMEM supplemented with protamine chloride 10 ug/ml (P) without or with different glucose concentrations (6, 11.6 and 28 mM). B. Representative picture showing PDX-1, NgN3, insulin and glucagon expression in RPMI-based medium with glucose 1 or 25 mM.

(21) FIG. 14: representative pictures showing the expression of PDX-1, NgN3, GLUT-2, C-peptide, glucagon and somatostatin in cells cultivated in a poly-lysine differentiation medium.