Jeoung Eun Lee

Establishment and Maintenance of Human Embryonic Stem Cell Lines on Human Feeder Cells Derived from Uterine Endometrium under Serum-Free Condition

Abstract Human embryonic stem (hES) cells are usually established and maintained on mouse embryonic fibroblast (MEFs) feeder layers. However, it is desirable to develop human feeder cells because animal feeder cells are associated with risks such as viral infection and/or pathogen transmission. In this study, we attempted to establish new hES cell lines using human uterine endometrial cells (hUECs) to prevent the risks associated with animal feeder cells and for their eventual application in cell-replacement therapy. Inner cell masses (ICMs) of cultured blastocysts were isolated by immunosurgery and then cultured on mitotically inactivated hUEC feeder layers. Cultured ICMs formed colonies by continuous proliferation and were allowed to proliferate continuously for 40, 50, and 55 passages. The established hES cell lines (Miz-hES-14, -15, and -9, respectively) exhibited typical hES cells characteristics, including continuous growth, expression of specific markers, normal karyotypes, and differentiation capacity. The hUEC feeders have the advantage that they can be used for many passages, whereas MEF feeder cells can only be used as feeder cells for a limited number of passages. The hUECs are available to establish and maintain hES cells, and the high expression of embryotrophic factors and extracellular matrices by hUECs may be important to the efficient growth of hES cells. Clinical applications require the establishment and expansion of hES cells under stable xeno-free culture systems.

Human Somatic Cell Nuclear Transfer Using Adult Cells

Derivation of patient-specific human pluripotent stem cells via somatic cell nuclear transfer (SCNT) has the potential for applications in a range of therapeutic contexts. However, successful SCNT with human cells has proved challenging to achieve, and thus far has only been reported with fetal or infant somatic cells. In this study, we describe the application of a recently developed methodology for the generation of human ESCs via SCNT using dermal fibroblasts from 35- and 75-year-old males. Our study therefore demonstrates the applicability of SCNT for adult human cells and supports further investigation of SCNT as a strategy for regenerative medicine.

Heat Shock 70‐kDa Protein 8 Isoform 1 Is Expressed on the Surface of Human Embryonic Stem Cells and Downregulated upon Differentiation

The cell‐surface markers used routinely to define the undifferentiated state and pluripotency of human embryonic stem cells (hESCs) are those used in mouse embryonic stem cells (mESCs) because of a lack of markers directly originated from hESC itself. To identify more hESC‐specific cell‐surface markers, we generated a panel of monoclonal antibodies (MAbs) by immunizing the irradiated cell clumps of hESC line Miz‐hES1, and selected 26 MAbs that were able to bind to Miz‐hES1 cells but not to mESCs, mouse embryonic fibroblast cells, and STO cells. Most antibodies did not bind to human neural progenitor cells derived from the Miz‐hES1 cells, either. Of these, MAb 20‐202S (IgG1, κ) immunoprecipitated a cell‐surface protein of 72‐kDa from the lysate of biotin‐labeled Miz‐hES1 cells, which was identified to be heat shock 70‐kDa protein 8 isoform 1 (HSPA8) by quadrupole time‐of‐flight tandem mass spectrometry. Immunocytochemical analyses proved that the HSPA8 protein was also present on the surface of hESC lines Miz‐hES4, Miz‐hES6, and HSF6. Two‐color flow cytometric analysis of Miz‐hES1 and HSF6 showed the coexpression of the HSPA8 protein with other hESC markers such as stage‐specific embryonic antigen 3 (SSEA3), SSEA4, TRA‐1‐60, and TRA‐1‐81. Flow cytometric and Western blot analyses using various cells showed that MAb 20‐202S specifically bound to the HSPA8 protein on the surface of Miz‐hES1, contrary to other anti‐HSP70 antibodies examined. Furthermore, the surface expression of the HSPA8 protein on Miz‐hES1 was markedly downregulated upon differentiation. These data indicate that a novel MAb 20‐202S recognizes the HSPA8 protein on the surface of hESCs and suggest that the HSPA8 protein is a putative cell‐surface marker for undifferentiated hESCs.

Evaluation of 28 human embryonic stem cell lines for use as unrelated donors in stem cell therapy: implications of HLA and ABO genotypes.

For human embryonic stem cells (hESCs) to be used clinically, it is imperative that immune responses evoked by hESCs and their derivates after transplantation should be prevented. Human leukocyte antigens (HLA) and ABO blood group antigens are important histocompatibility factors in graft rejection. HLA matching between recipient and unrelated donors, in particular, is important in improving outcomes in hematopoietic cell transplantation (HCT). We have established and successfully maintained 29 hESC lines and analyzed the HLA and ABO genotypes of these lines. HLA-A, -B, -C and -DR (DRB1) genotyping was performed by polymerase chain reaction (PCR) sequence-based typing and ABO genotyping was carried out by PCR restriction fragment length polymorphism methods. To determine what proportion of the Korean population would be covered by these cell lines in organ transplantation, 27 cell lines with HLA-A, -B, and -DR data were evaluated for HCT (cord blood) donors and 28 cell lines with HLA-DR and ABO data were evaluated for solid organ (kidney) transplantation donors, and then compared the data with those from 6,740 donated cord bloods. When 2 HLA mismatches are allowed for HCT, as currently accepted for cord blood transplantation, it was estimated that about 16% and 25% of the possible recipients can find one or more donor cell lines with ≤2 mismatches at A, B, DRB1 allele level and at A, B antigen/DRB1 allele level, respectively. When HLA-DR antigen level matching and ABO compatibility was considered for solid organ (kidney) transplantation, it was estimated that about 29% and 96% of the possible recipients can find one or more ABO-compatible donor cell lines with 0 and 1 DR mismatches, respectively. We provided the first report on the HLA and ABO genotypes of hESC lines, and estimated the degree of HLA and ABO matching in organ transplantation for the Korean population.

Effects of type IV collagen and laminin on the cryopreservation of human embryonic stem cells.

Previous reports have indicated that extracellular matrices (ECMs) affect the developmental fate of human embryonic stem cells (hESCs). Specially, type IV collagen and laminin, which belong to a group of macromolecular proteins with a substantial proportion of ECMs, are known to influence the proliferation and differentiation of hES cells. In this study, we evaluated the effects of type IV collagen and laminin in freezing medium on the survival and differentiation rates of hES cells after slow freezing and rapid thawing. The addition of type IV collagen (1 μg/ml) to the freezing medium significantly increased the survival rate of hES cells after thawing compared with that of a control group. The spontaneous differentiation rates of groups treated with type IV collagen (1 μg/ml) or laminin (1 μg/ml) were significantly lower than those of the control group. Frozen‐thawed hES cells have currently been cultured for more than 70 passages and retain key properties of hES cells such as morphological characteristics, normal karyotype, marker expression (alkaline phosphatase, SSEA‐1, SSEA‐3, SSEA‐4, TRA‐1‐60, TRA‐1‐81, Rex‐1, and Oct‐4), basement membrane–related gene expression, and the potential to differentiate into derivatives of all three germ layers. This new slow freezing method by ECM treatment is a reliable and effective cryopreservation method for pluripotent hES cells.

Induction of acrosome reaction in human spermatozoa accelerates the time of pronucleus formation of hamster oocytes after intracytoplasmic sperm injection

OBJECTIVE To assess the relationship between the incidence of acrosome reaction (AR) and the timing of pronucleus (PN) formation after intracytoplasmic sperm injection (ICSI). DESIGN Prospective study. SETTING Infertility Research Center, Jeil Womens Hospital. MAIN OUTCOME MEASURE(S) Human semen obtained from fertile donors was prepared by one of the following methods: washing only (washed control); Percoll gradient; pentoxifylline; human follicular fluid (FF); pentoxifylline + FF; or platelet-activating factor (PAF) treatment. The AR of each group was assessed by fluorescein isothiocyanate-conjugated Pisum sativum agglutinin or Arachis hypogea agglutinin. Spermatozoa of washed control, pentoxifylline + FF, and PAF treated groups, with significantly higher AR rate than others, were injected into mature hamster oocytes. Spermatozoon-injected oocytes were cultured for 6, 9, 12, or 15 hours. Then they were stained with Toluidine blue for PN formation examination under a light microscope. RESULT(S) Acrosome reaction rates of washed control, Percoll gradient, pentoxifylline, FF, pentoxifylline + FF, and PAF treated groups were 10.5% +/- 2.6%, 10.3% +/- 1.7%, 16.4% +/- 1.8%, 24.8% +/- 5.6%, 28.4% +/- 3.8%, and 33.3% +/- 5.2%, respectively. Pronuclear formation rate in washed control, pentoxifylline + FF, and PAF treated groups were 5.6% (3/54), 19.0% (11/58), and 18.9% (10/53) at 6 hours; 32.7% (18/55), 51.8% (29/56), and 57.4% (31/54) at 9 hours; 36.1% (22/61), 53.6% (30/56), and 50.0% (27/54) at 12 hours; and 47.2% (25/53), 64.8% (35/54), 53.6% (30/56) at 15 hours after ICSI. Pronuclear formation rate was significantly higher in pentoxifylline + FF, and PAF treated groups than that in the washed control group at 6 and 9 hours after ICSI. CONCLUSION(S) Pronuclear formation of oocytes takes place faster on those that were injected with acrosome-reacted spermatozoon than those injected with acrosome-intact spermatozoon. It could be concluded that induction of the AR of spermatozoa accelerates the time of PN formation and early development of the embryo in ICSI.

High concentration of synthetic serum, stepwise equilibration and slow cooling as an efficient technique for large-scale cryopreservation of human embryonic stem cells

OBJECTIVE To develop an efficient freezing method suitable for large-scale cryopreservation of human embryonic stem cells (hESCs). DESIGN Experimental study. SETTING Research institute. PATIENT(S) None. INTERVENTION(S) Two genetically modified hESC lines, H9-EF1-GFP and CHA-hES3-EF1-GFP, were cryopreserved in cryovials using a combination of two equilibration methods (one-step and stepwise) and two cooling vehicles (cryo-container and program-controlled freezer). After thawing, the survival and differentiation rate were compared among groups. MAIN OUTCOME MEASURE(S) The hESC survival was assessed by alkaline phosphatase staining and differentiation status was determined by flow cytometry using an SSEA-4 antibody. RESULT(S) In both H9-EF1-GFP and CHA-hES3-EF1-GFP cells, the survival rate was highest in the group using stepwise equilibration and program-controlled freezer, and lowest in the group using one-step equilibration and cryo-container. In the groups using cryo-container, the survival and the frequency of undifferentiated cells in both cell lines was highly improved in a stepwise equilibration compared with one-step. Thawed hESCs were positively stained with pluripotent markers SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase. The karyotypes and expression of three germ layer markers in both cell lines were not changed after freezing/thawing. CONCLUSION(S) The stepwise equilibration of Knockout Serum Replacement and cryoprotectant during freezing and thawing resulted in higher survival rates by reducing osmotic damage irrespective of cooling vehicles.

Stem cell factor/c-Kit signaling in in vitro cultures supports early mouse embryonic development by accelerating proliferation via a mechanism involving Akt-downstream genes

PurposeStem cell factor (SCF)/c-Kit regulates the proliferation and survival of germ cells or stem cells; however, little is known about the role of SCF/c-Kit in pre-implantation embryo development.MethodsUsing exogenous SCF supplementation and c-Kit siRNA injection, we investigated the role and mechanism of SCF/c-Kit in pre-implantation mouse embryos.ResultsAddition of soluble SCF to the culture medium improved blastocyst formation. c-Kit gene silencing reduced the rate of blastocyst formation and delayed embryonic development. The number of proliferating cells in c-Kit gene-silenced blastocysts decreased, whereas the number of apoptotic cells in blastocysts obtained from both experimental and the control groups was not affected. RT-PCR, immunostaining and western blotting revealed that proliferation-related Akt downstream targets were substantially affected by c-Kit gene silencing.ConclusionSCF/c-Kit signaling through Akt downstream targets is likely involved in mediating the cleavage and proliferation of blastomeres during mouse pre-implantation embryogenesis.

Three-step method for proliferation and differentiation of human embryonic stem cell (hESC)-derived male germ cells.

The low efficiency of differentiation into male germ cell (GC)-like cells and haploid germ cells from human embryonic stem cells (hESCs) reflects the culture method employed in the two-dimensional (2D)-microenvironment. In this study, we applied a three-step media and calcium alginate-based 3D-culture system for enhancing the differentiation of hESCs into male germ stem cell (GSC)-like cells and haploid germ cells. In the first step, embryoid bodies (EBs) were derived from hESCs cultured in EB medium for 3 days and re-cultured for 4 additional days in EB medium with BMP4 and RA to specify GSC-like cells. In the second step, the resultant cells were cultured in GC-proliferation medium for 7 days. The GSC-like cells were then propagated after selection using GFR-α1 and were further cultured in GC-proliferation medium for 3 weeks. In the final step, a 3D-co-culture system using calcium alginate encapsulation and testicular somatic cells was applied to induce differentiation into haploid germ cells, and a culture containing approximately 3% male haploid germ cells was obtained after 2 weeks of culture. These results demonstrated that this culture system could be used to efficiently induce GSC-like cells in an EB population and to promote the differentiation of ESCs into haploid male germ cells.

An integrated systems biology approach identifies positive cofactor 4 as a factor that increases reprogramming efficiency

Spermatogonial stem cells (SSCs) can spontaneously dedifferentiate into embryonic stem cell (ESC)-like cells, which are designated as multipotent SSCs (mSSCs), without ectopic expression of reprogramming factors. Interestingly, SSCs express key pluripotency genes such as Oct4, Sox2, Klf4 and Myc. Therefore, molecular dissection of mSSC reprogramming may provide clues about novel endogenous reprogramming or pluripotency regulatory factors. Our comparative transcriptome analysis of mSSCs and induced pluripotent stem cells (iPSCs) suggests that they have similar pluripotency states but are reprogrammed via different transcriptional pathways. We identified 53 genes as putative pluripotency regulatory factors using an integrated systems biology approach. We demonstrated a selected candidate, Positive cofactor 4 (Pc4), can enhance the efficiency of somatic cell reprogramming by promoting and maintaining transcriptional activity of the key reprograming factors. These results suggest that Pc4 has an important role in inducing spontaneous somatic cell reprogramming via up-regulation of key pluripotency genes.