US20100120142A1

US20100120142A1 - Culture of human embryonic cells

Info

Publication number: US20100120142A1
Application number: US12/171,866
Authority: US
Inventors: Ulla Impola; Minna Tiittanen; Milla Mikkola; Jukka Partanen; Jari Natunen; Tero Satomaa; Juhani Saarinen
Original assignee: Suomen Punainen Risti Veripalvelu; Glykos Finland Ltd
Current assignee: Suomen Punainen Risti Veripalvelu; Glykos Finland Ltd
Priority date: 2008-07-11
Filing date: 2008-07-11
Publication date: 2010-05-13
Also published as: US8703488B2; US20110306128A1

Abstract

The invention relates to a method for culturing human embryonic stem cells (hESCs) with a lectin. The invention relates also to the use of a lectin in a method for culturing human embryonic stem cells (hESCs) and a culture medium composition containing a lectin.

Description

FIELD OF THE INVENTION

The invention relates to a method for culturing human embryonic stem cells (hESCs) on a lectin. The invention relates also to the use of a lectin in a method for culturing human embryonic stem cells (hESCs) and a culture medium composition containing a lectin attached on the culturing plates.

BACKGROUND OF THE INVENTION

Traditional methods for culturing human embryonic stem cells (hESCs) require the direct use of mouse embryonic fibroblasts (MEFs) as a feeder layer, or feeder-conditioned medium or serum. A medium for a feeder-free culture of hESCs includes an extracellular matrix extracted from a mouse sarcoma and is sold under the trademark Matrigel™ (BD Bioscience, US). Matrigel™ is mostly comprised of laminin and collagen and these compounds in purified form have also been tried in culturing hESCs.
Matrigel™ and the other feeder-free media used currently in cultures suffer from xeno contamination, and in addition are subject to large variability caused by containing growth factors and other undefined molecules.
Mallon B. S. et al. have reviewed the attempts made toward xeno-free culture of hESCs in The International Journal of Biochemistry and Cell Biology 38, 1063-1075, 2006. As can be concluded, the culture of hESCs suffers with respect to both technical and clinical potential by the use of cells or extracts originating from animal sources, such as mouse embryonic fibroblasts and an extract from a mouse sarcoma. The current culture methods are also laborious and difficult to scale. Further, it is often hard to maintain the cells in uniform quality and in an undifferentiated form.
One of the biggest problems of the current methods and media for culturing hESCs arises from the use of animal-derived material in the culture medium.
This problem has now been solved in accordance of the present invention by providing a method for culturing hESCs using a medium containing a lectin as a culturing matrix.

BRIEF DESCRIPTION OF THE INVENTION

The present invention is directed to a method for culturing human embryonic stem cells (hESC) or a population of hESCs with at least one lectin. The invention is also directed to a culture medium composition comprising at least one lectin. Further, the invention is directed to the use of a lectin in a method for culturing hESCs.
In one embodiment, the invention is directed to method for culturing hESCs with a lectin as a matrix and a definitive, serum- and feeder-free medium. The invention is also directed to a culture medium composition comprising at least one lectin and a definitive, serum- and feeder-free medium. Further, the invention is directed to the use of a lectin together with a definitive, serum- and feeder-free media in a method for culturing hESCs.
In one embodiment of the invention the lectin is a natural lectin originating and/or derived from a plant or an animal. In another embodiment, the lectin is a lectin derivative produced by biotechnology methods, such as recombinant technology.
In a further embodiment of the invention, the lectin is ECA (sometimes also called ESL) lectin isolated from Erythrina cristagalli seeds or an essentially similar lectin derivative produced by gene technology means.
The invention is based on the use of at least one lectin, such as a plant lectin, in the culture of hESCs, preferably with a definitive, serum- and feeder-free medium.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative embodiments of the invention and are not meant to limit the scope of the invention as defined in the claims in any way.

FIG. 1 shows the colonies of (A); FES 29 cells cultured on ECA-lectin for 6 passages (original magnification 4×), (B); FES 29 cells during passage 14 on ECA (magnification 10×) and (C); FES 30 cells cultured on ECA for 7 passages (magnification 10×) obtained in Example 1.

FIG. 2 shows the FACS analysis of the surface markers (A); SSEA3 and (B) Tra-1-60 (or Tra-1-81) expressions on FES 29 cells during ECA culture from the beginning of ECA culture (passage 0) to passage 8, and (C) SSEA3 and (1) Tra-1-60 (or Tra-1-81) expressions on FES 30 cells during ECA culture from the beginning of ECA culture (passage 0) to passage 8 obtained in Example 1. The surface marker expressions in the control Matrigel cultures are shown for comparison (p=passage ECA/Matrigel).

FIG. 3 shows the FES 29 cells cultured in suspension for EB formation after 9 passages on ECA described in Example 1.

FIG. 4 shows the hESC colonies on ECA in StemPro® medium obtained in Example 2: (A) FES 29 cells cultured on ECA for 9 passages: first 7 passages in conditioned medium and then 2 passages in StemPro® definitive medium and (8) FES 29 cells during passage 3 in StemPro® medium on ECA passage 10.

FIG. 5 shows the FACS-analysis of the expression of the two surface markers SSEA-3 and Tra-1-60 of undifferentiated hESCs described in Example 2. FES29-cells were cultured on ECA for 10 passages and with StemPro®-medium for the last 3 passages.

FIG. 6 shows the EBs formed from FES 29 cells after 12 ECA passages and 4 StemPro® passages obtained in Example 2.

FIG. 7 shows (A) FiPS1-5 and (B-C) FiPS6′-12 cell colonies after 5 passages on ECA-lectin in conditioned medium obtained in Example 3.

FIG. 8 shows EBs were formed from FiPS6-12 cells after 6 passages on ECA obtained in Example 3.

FIG. 9 shows a list of lectins whose amino acid sequences are highly homologous to that of ECA. Potential N-glycosylation sites have been indicated with highlighting. Lysine residue, which can be used to link the lectin to a surface, have been shown in bolded italics.

DETAILED DESCRIPTION OF THE INVENTION

Human embryonic stem cells (hESCs) are derived from the inner cell mass of 3-5 day-old blastocysts. hESCs pose telomerase activity and express surface markers SSEA-3, SSEA-4, Tra-1-60 and Tra-1-81. They proliferate on continuous basis when maintained in an appropriate culture environment and differentiate both in vivo and in vitro into endo-, meso- and ectoderm. The differentiation is detected by formation of embryoid bodies in vitro and teratoma in vivo. hESCs are considered to be the building blocks for all types of cells in humans and thus have huge potential in applications of cell therapy and regenerative medicine. With regard to the safety of the transplantation applications of hESCs and the derivatives thereof, it is important to reduce or even eliminate the xenogenic contamination of these cells.
Induced pluripotent stem (IFS) cells are a type of pluripotent stem cell artificially derived from a non-pluripotent cell, typically an adult somatic cell by inducing a “forced” expression of certain genes. IPS cells are considered to be identical to natural pluripotent stem cells, such as embryonic stem cells in many respects.
In the present invention the term human embryonic stem cell (hESC) refers to natural human embryonic stem cells and IPS cells.
Lectins are sugar-binding proteins. They typically play a role in biological recognition phenomena involving cells and proteins. Most of the lectins are basically non-enzymatic in action and non-immune in origin. Lectins occur ubiquitously in nature. They may bind to a soluble carbohydrate or to a carbohydrate moiety which is a part of a more complex carbohydrate structure, such as a glycoprotein or glycolipid. They typically agglutinate certain animal cells and precipitate glycoconjugates. Lectins serve many different biological functions from the regulation of cell adhesion to glycoprotein synthesis and the control of protein levels in the blood. Lectins are also known to play important roles in the immune system by recognizing carbohydrates that are found exclusively on pathogens or that are inaccessible on host cells. Lectins could be derived from plants, such as legume plants like beans, grains and seeds. In addition, lectins having an animal origin are known. Legume lectins are one of the largest lectin families with more than 70 lectin family members.
Known lectins isolated from plants are, for example, Con A, LCA, PSA, PCA, GNA, HPA, WGA, PWM, WA, ECA, DSA, UEA-1, PNA, SNA and MAA. Galectins are a family of lectins having mammalian origin. Lectins recognizing the “terminal N-acetyllactosamine” structure (Fucα2)_nGalβ4GlcNAc, wherein n is 0 or 1, are a group of preferred lectins of the present invention. These lectins include, in particular, ECA (Erythrina cristagalli lectin) and UEA-1 (Ulex europeaus agglutinin-I), as well as galectin lectins. In addition, a number of other natural lectins may have the specificity of recognizing and/or binding to the “terminal N-acetyllactosamine” structure. Furthermore, natural lectins can be mutagenized to improve their binding or to obtain binding specificity to the “terminal N-acetyllactosamine”. A list of lectins, whose amino acid sequences are highly homologous to ECA is shown in FIG. 9. These lectins potentially have or may readily be modified by e.g. mutagenesis to have the same activity as ECA.
In one embodiment of the invention, the lectin is an animal-free galectin, that is, a recombinant lectin protein produced in cell culture system, preferably in a non-animal cell culture system.
In one embodiment of the invention, lectins include also oligosaccharide-binding protein domains and peptides derived from lectins. Preferably the lectins do not contain a non-lectin domain, such as an enzyme domain or toxic domain found, for example, in ricin agglutinin (RCA). The lectins of the present invention further include any polypeptide or equivalent being functionally a lectin. Antibodies and oligosaccharide-binding enzymes are examples of the proteins being functional lectins. Preferred enzymes include fucosidases and galactosidases modified to remove the catalytic activity. The antibodies include all types of natural and genetically engineered variants of immunoglobulin proteins. Preferred antibodies include blood group H type II and terminal N-acetyllactosamine binding antibodies.
In the present invention the term “terminal N-acetyllactosamine” refers to a neutral N-acetyllactosamine with a non-reducing terminal end; the neutral means that the structure is not modified by sialic acid or other acidic residues. Preferably terminal N-acetyllactosamine is non-substituted type II N-acetyllactosamine or its α2′-fucosylated variant structure (H-type II structure) according to formula (Fucα2)_nGalβ4GlcNAc, wherein n is 0 or 1.
The amount of lectin used in a solution is about 0.1-500 μg/ml, preferably about 5-200 μg/ml or about 10-150 μg/ml. The amount of lectin for immobilization of the cell culture surface is about 0.001-50 μg/cm², preferably from about 0.01-50 μg/cm²to about 0.1-30 μg/cm², more preferably about 0.3-10 μg/cm²for a lectin with Mw of about 50 kDa, or corresponding molar density per surface area used. In one embodiment, about 1-50 μg/cm², or about 5-40 μg/cm², preferably about 1040 μg/cm²of lectin is used in a solution to coat a plastic cell culture surface. In one embodiment, the concentration in the coating solution is between about 50-200 μg/ml for a lectin with Mw of about 50 kDa or corresponding molar density per surface area used. In a specific embodiment, a plastic cell culture well with polystyre surface is coated by passive adsorbtion using about 140 μg/ml solution in amount of about 30 μg/cm²for a lectin with Mw of about 50 kDa.
The present invention relates to a method for culturing human embryonic stem cells (hESC) or a hESC polulation with a lectin. The invention is also directed to a culture medium composition comprising a lectin as a matrix. Further, the invention is directed to the use of a lectin in a method for culturing hESCs.
In one embodiment, the invention is directed to a method for culturing hESCs with at least one lectin and to a culture medium composition comprising at least one lectin. Further, the invention is directed to the use of at least one lectin in a method for culturing hESCs.
In one embodiment of the invention the lectin is a natural plant lectin such as ECA lectin and in another embodiment of the invention at feast one of the lectins is ECA lectin.
In one embodiment, the invention is directed to method for culturing hESCs with a lectin as a culturing matrix and a definitive, serum- and feeder-free medium. The invention is also directed to a culture medium composition comprising a lectin and a definitive, serum- and feeder-free medium. Further, the invention is directed to the use of a lectin together with a definitive, serum- and feeder-free media in a method for culturing hESCs.
In another embodiment, the invention is directed to method for culturing hESCs with at least one lectin and a definitive, serum- and feeder-free medium and to a culture medium composition comprising at least one lectin and a definitive, serum- and feeder-free medium. Further, the invention is directed to the use of at least one lectin together with a definitive, serum- and feeder-free media in a method for culturing hESCs.
A definitive or fully-defined, serum- and feeder-free medium is a medium that is specifically formulated for the uniform growth of hESCs and contains ingredients required for maintaining normal morphology, pluripotency and differentiation capability of hESCs. StemPro® hESC SFM, developed and sold by Invitrogen Corporation, US, is an example of this kind of a definitive, serum- and feeder-free medium developed for culturing of hESCs without feeder cells.
In a further embodiment of the invention, the definitive, serum- and feeder-free medium is StemPro®hESC SFM.
According to the present invention, a lectin is used as a sole culture matrix ingredient or it is added to a culture media applicable to the growth of hESCs or used with such a medium. The culture media can also be supplemented, for example, with a single or a plurality of growth factors selected from, for example, a WNT signaling agonist, TGF-b, bFGF, IL-6, SCF, BMP-2, thrombopoietin, EPO, IGF-1, IL-11, IL-5, Flt-3/Flk-2 ligand, fibronectin, LIF, I-IGF, NFG, angiopoietin-like 2 and 3, G-CSF, GM-CSF, Tpo, Shh, Wnt-3a, Kirre, or a mixture thereof.
In one embodiment of the invention, the hESCs are grown on a lectin, such as a plant lectin or galectin coated plate or vessel.
The hESCs cultured according to the present invention are not exposed to animal-derived material during their cultivation, at least not in such an extent than cells cultured according to the known methods using feeder cells, Matrigel™ and/or other animal-derived material.
The hESCs cultured according to the present invention have shown to have the typical characteristics of human embryonal stern cells, posing telomerase activity and expressing surface markers SSEA-3, Tra-1-60 and Tra-1-81. In addition, the cells have been shown to be able to differentiate by forming embryoid bodies and/or teratomas.
The method and the culture medium composition of the present invention provide means for culturing hESCs substantially free of xenogenic contamination. The human embryonic stem cell(s) and/or cell population(s) cultured according to the present invention are thus safe for the current and future transplantation applications.
The following examples represent illustrative embodiments of the invention without limiting the invention any way.

Example 1

Human Embryonic Stem Cell (hESC) Lines Cultured on ECA-Lectin Coated Plastic Generation and Maintenance of hESC Lines

Processes for generation of hESC lines from blastocyst stage of in vitro fertilized human embryos have been described previously in Thomson et al. (Science, 282:1145-1147, 1998). Cell lines FES 22, FES 29 and FES 30 were initially derived and cultured either on mouse embryonic fibroblasts feeders (MEFs; 12-13 pc fetuses of the ICR strain), or on human foreskin fibroblast feeder cells (HFFs; CRL-2429 ATCC, Mananas, USA) (Mikkola et al. BMG Dew Biol, 6:40-51, 2006). All the lines were cultured in serum-free medium (KnockOut™ D-MEM; Gibco® Cell culture systems, Invitrogen, Paisley, UK) supplemented with 2 mM L-Glutamin/Penicillin streptomycin (Sigma-Aldrich), 20% KnockOut Serum Replacement (Gibco), 1× non-essential amino acids (Gibco), 0.1 mM 3-mercaptoethanol (Gibco), 1×ITS (Sigma-Aldrich) and 4 ng/ml bFGE (Sigma/Invitrogen) on feeder cells, or on Matrigel™ (BD Biosciences) in the same medium (supplemented with additional 4 ng/ml bFGF) conditioned over night on MEFs. Passaging was done either mechanically or enzymatically using collagenase IV (Gibco).

ECA-Lectin Coating of Cell Culture Plates

ECA-lectin (EY laboratories, USA) was dissolved in phosphate buffered saline 140 μg/ml. Lectin dilution was sterile filtrated using Millex-GV syringe driven filter units (0.22 μm, SLGV 013 SL, Millipore, Ireland) and allowed to passively adsorb on cell culture plate by overnight incubation at +4° C. After incubation the wells were washed three times with phosphate buffered saline and stem cells were plated on them.
hESC Culturing on ECA-Lectin Coated Cell Culture Plates
The hESC lines (FES 22, FES 29, FES 30) were cultured at least three passages on Matrigel™ before transferring them onto ECA coated plates, FES 29 was transferred also directly from MEFs onto ECA coated plates in conditioned medium. All lines were maintained on Matrigel™ as controls. The growing cell aggregates were then passaged to new plates at 3-7 day intervals.
hESC Embryoid Body (EB) Formation
EBs were generated as previously described in Mikkola et al. (BMC Dev Biol, 6:40-51, 2006) with small modifications. Briefly, to induce the formation of EBs the confluent hESC colonies were first treated with 200 U/ml collagenase IV and transferred on non-adherent Petri dishes to form suspension cultures. The formed EBs were cultured in suspension for the next 10 days in standard culture medium (see above) without bFGF.

Teratoma Assay

In order to study teratoma formation about 200 000 morphologically good looking hESCs were injected into the testes of nude mice. The resulting tumors wore harvested 8 weeks later and fixed with formalin for immunohistological examination (Mikkola et al. BMC Dev Biol, 6:40-51, 2006).

Flow Cytometry

hESCs were detached enzymatically and washed in 1% ultra pure BSA in PBS. Monoclonal antibodies against SSEA-3, Tra-1-60 and Tra-1-81 (1:50; gifts kindly provided by ESTOOLS www.estools.org) were used as markers for undifferentiated hESCs. Staining was performed according to manufacturer's instructions, FACS analysis was done with FACS Calibur machinery and CellquestPro software (Becton Dickinson).

Results

Three different hESC-lines, FES 22, FES 29 and FES 30, were cultured on ECA-coated wells in MEF-conditioned medium up to 23 passages. The morphology of hESCs was similar to the control Matrigel cultures and hESCs looked undifferentiated after ECA-lectin passages (FIG. 1). Lines FES 29 and FES 30 were repeatedly successfully transferred from Matrigel to ECA-plates. FES 29 cells were also transferred onto ECA-lectin straight from feeder cells (MEFs).
The expression of surface markers of undifferentiated hESCs (SSEA-3 and Tra-1-60/Tra-1-81) were analyzed every 2 or 3 passages by flow cytometry. The follow-up of the surface marker expression during the culture of FES 29 and FES 30 cells on ECA is shown in FIG. 2.
The pluripotency of hESCs after several ECA passages was verified by their ability to form EBs in suspension culture or teratomas in nude mice. FES 30 cells cultured 23 passages on ECA and FES 29 Cells cultured 4 passages on ECA formed teratoma-containing tissues from all three germ cell layers (data not shown). EBs were successfully formed from FES 29 and FES 30 cells after ECA-culture (FIG. 3).

Example 2

Culturing hESCs on ECA Lectin in Definitive Medium Culturing hESCs

The FES 29 hESC line (see example 1) was cultured 14 passages on Matrigel™ before transferring the cells on ECA-lectin coated plates. The hESCs were cultured on ECA-lectin coated plates for 7 passages in MEF-conditioned medium and then changed to a definitive medium, StemPro® hESC SFM (Gibco, Invitrogen A10007-01). Matrigel™ was used as a control. For enzymatic passaging the cells were exposed to 200 units/ml collagenase IV (Gibco) for 1-2 min at 37° C., washed once in PBS end dissociated by gently pipetting and plated on 2-3 new dishes.
hESC Embryoid Body (EB) Formation
EBs were generated as previously described in Mikkola et al. (2006). Briefly, to induce the formation of EBs the confluent hESC colonies were first treated with 200 U/ml collagenase IV and then transferred on non-adherent Petri dishes to form suspension cultures. The formed EBs were cultured in suspension for the next 10 days in the standard culture medium (see above) without bFGF.

Flow Cytometry

hESCs were detached enzymatically and washed with 1% ultra pure BSA in PBS. Monoclonal antibodies against SSEA-3, Tra-1-60 and Tra-1-81 (1:50; gifts kindly provided by ESTOOLS www.estools.org) were used as markers for undifferentiated hESCs. Staining was performed according to manufacturer's instructions. FACS analysis was done with FACS Calibur machinery and CellQuestPro software (Becton Dickinson).

Results

hESCs, FES 29 line, were cultured on ECA-lectin for 7 passages with MEF-conditioned culture medium. In the 8^thpassage conditioned medium was changed to the commercial definitive medium, StemPro® hESC SFM. FES 29 cells maintained their undifferentiated state and pluripotency during up to 5 passages in definitive medium on ECA. In FIG. 4 the typical hESC colonies on ECA in the StemPro® medium are shown. FACS-analysis of the expression of surface markers of undifferentiated hESCs (SSEA-3 and Tra-1-60) is presented in FIG. 5. EBs were formed after 12 passages on ECA and 4 passages in the StemPro® medium (FIG. 8).

Example 3

Culturing Induced Pluripotent Stem (IPS) Cells on ECA Lectin Coated Plastic

Culturing IPS Cells

Two lines of IPS cells were originated either from human embryonal lung fibroblasts or human foreskin fibroblasts with protocol modified from Okita et al. (Nature, 448:313-317, 2007) and Wernig et al. (Nature, 448:318-324, 2007). FiPS1-5 and FiPS6-12 lines were cultured for 10 or 8 passages on MEFs before transferring them onto ECA or Matrigel™ coated dishes in MEF-conditioned medium. For enzymatic passaging the cells were subjected to 200 units/ml collagenase IV for 1-2 min at 37° C., washed once in PBS and dissociated by gently pipetting and plated on 2-3 new dishes.
hESC Embryoid Body (EB) Formation
EBs were generated as previously described in Mikkola et al. (2006). Briefly, to induce the formation of EBs the confluent cell colonies were first treated with 200 U/ml collagenase IV and then transferred on non-adherent Petri dishes to form suspension cultures. The formed EBs were cultured in suspension for the next 10 days in standard culture medium (see above) without bFGF.

Results

IPS-cells were cultured in similar in vitro conditions as hESCs. Two IPS cell-lines, FiPS1-5 and FiPS6-12, were transferred from MEFs (after passage 10 or 8, respectively) to Matrigel or to ECA-coated plates in MEF-conditioned medium. IPS cells were morphologically similar to hESCs in all culturing conditions (FIG. 7). EBs were formed from FiPS6-12 cells after 6 passages on ECA (FIG. 8).

Claims

1. A method for culturing human embryonal stem cells (hESCs) wherein a cell or a cell population is contacted with at least one lectin.

2. The method of claim 1 wherein a cell or a cell population is contacted with at least one lectin and with a definitive serum- and feeder-free medium.

3. The method of claim 1 or 2 wherein a cell or a cell population is contacted with one lectin.

4. The method of claim 1 wherein the lectin recognizes the structure (Fucα2)_nGalβ4GlcNAc, wherein n is 0 or 1.

5. The method of claim 4 wherein the lectin is ECA, galectin, or an essentially similar protein biotechnologically produced thereof.

6. The method of claim 2 wherein the definitive, serum- and feeder-free medium is StemPro® hESC SFM.

7. A culture medium composition for culturing human embryonal stem cells (hESCs), wherein the composition comprises at least one lectin as a matrix.

8. The composition of claim 7 wherein it comprises in addition to at least one lectin as a matrix, a definitive serum and feeder-free medium.

9. The composition of claim 7 or 8 wherein it comprises one lectin.

10. The composition of claim 7, wherein the lectin recognizes the structure (Fucα2)_nGalβ4GlcNAc, wherein n is 0 or 1.

11. The composition of claim 10 wherein the lectin is ECA, galectin, or an essentially similar protein biotechnologically produced thereof.

12. The composition of claim 8 wherein the definitive, serum- and feeder-free medium is StemPro® hESC SFM.

13. Use of one or more lectins in a method for culturing hESCs.

14. The use of claim 13 wherein one or more lectins is used as a matrix together with a definitive serum- and feeder-free medium.

15. The use of claim 13 or 14 wherein one or more lectins recognizes the structure (Fucα2)_nGalβ4GlcNAc, wherein n is 0 or 1.

16. The use of claim 15 wherein at least one of the lectins is ECA, galectin, or an essentially similar protein biotechnologically produced thereof.

17. The use of claim 14 wherein the definitive, serum- and feeder-free medium is StemPro® hESC SFM.