KR101442783B1 - Composition for Inducing Erythroid Differentiation - Google Patents

Abstract

The present invention relates to a composition comprising poly-L-lysine (PLL) as an active ingredient for inducing differentiation of erythrocyte precursor cells and a method for producing in vitro production of erythrocytes. By introducing an amino group to the surface of a cultivation support without toxicity by using PLL which is a polyamino acid, the present invention provides the optimal conditions for the erythrocyte precursor cells to differentiate into mature erythrocytes and efficiently promotes the differentiation into erythrocytes throughout the process of producing the erythrocytes. Accordingly, the present invention can satisfy the clinical demand of blood for transfusion by presenting a safe mass production method of erythrocytes in vitro.

Description

[Composition for Inducing Erythroid Differentiation]

The present invention relates to a composition for inducing erythrocyte differentiation of erythroid precursor cells containing PLL (Poly-L-lysine) as an active ingredient.

Due to the increased number of surgeries, the demand for blood transfusion continues to increase, but unfortunately the amount of donated blood is scarce as well as the potential risks of immune reactions and viral infection. The most common type of blood cells, red blood cells (RBCs), play a key role in oxygen delivery and are essential for successful blood transfusion. Therefore, a chemical blood substitute has been developed as a substitute for red blood cells in order to meet a blood transfusion requirement. However, these alternatives can cause serious adverse effects such as vasoconstriction, hypertension, free radical generation, and oxidative stress [1-2]. Therefore, mass production of red blood cells derived from the patient's own stem cells may be a promising strategy to overcome obstacles to blood transfusion and blood substitute synthesis.

Hematopoietic stem cells (HSCs) are self-renewing multipotent cells capable of differentiating into all kinds of blood cells (eg, megakaryocytes, leukocyte and erythroid cells) [3]. Generally, hematopoietic stem cells can be obtained from bone marrow, peripheral blood and cord blood. There have been several attempts to obtain hematopoietic stem cells from embryonic stem cells, but such studies have not yet been fully successful [4]. Therefore, transplantation of hematopoietic stem cells is an urgent treatment strategy for leukemia patients with abnormal blood production system.

Production and differentiation of hematopoietic stem cells in the bone marrow are constantly stimulated by physicochemical interactions of specific microenvironment called hematopoietic niche in bone marrow [5].

Hematopoietic stem cells are composed of various types of cells surrounding the extracellular matrix (ECM), chymocaine, cytokines and hematopoietic stem cells. The interaction between the hematopoietic stem cells and their niche compositions plays an important role in the undifferentiated state of stem cells, in vitro proliferation, migration and post-differentiation, and the removal of nuclei of hematopoietic stem cell-derived erythroid cells [6].

Many studies have attempted to promote the in vitro proliferation of hematopoietic stem cells by regulating the microenvironment through co-culture systems [7], cytokines [8] and compounds [9]. In this regard, Rhodes et al. [10] suggested that differentiation of hematopoietic stem cells into erythroid cells is mainly induced in erythroblastic islands containing central macrophages that continue to communicate with erythroblasts during differentiation Respectively. The end result is the maturation of red blood cells through enucleation of primitive erythroid cells called red blood cells.

In vitro proliferation of hematopoietic stem cells and differentiation into erythroid cells can be achieved using physiologically or chemically modified culture substrates. For example, the aminated surface of nanofibers and nanosegment facilitates in vitro proliferation of hematopoietic stem cells [10-12]. In addition, the primary amine-bound surface induced more efficient enucleation during hematopoietic differentiation into erythroid cells [13]. However, a strong toxic APES (cytotoxic 3-aminopropyl trimethoxylsilane) was used for binding of amine groups, and the density of amine groups was not optimized for hematopoietic stem cell expansion and enucleation, and the mechanism of interaction between hematopoietic stem cells and substrate It is not clear yet. Therefore, it will be the most useful means for expanding and differentiating hematopoietic stem cells to allow optimal binding without inducing toxicity of amine groups on the culture plate surface.

Poly-L-lysine (PLL), which is a synthetic polyamino acid having a cationic structure, is advantageous in that it can introduce an amine group into a support by a simple and economical method. However, despite these merits, in vitro proliferation of hematopoietic stem cells, The ability of PLLs to promote differentiation and enucleation has not been studied.

Thus, we hypothesized that the PLL-coated substrate would increase the in vitro proliferation of hematopoietic stem cells and the differentiation into erythroid cells by electrostatic attraction. In the present invention, various concentrations of PLL were introduced to optimize culture conditions for the expansion and differentiation of hematopoietic stem cells. As a result, it was confirmed that PLL was also involved in the final enucleation mechanism of erythrocytes in the differentiation of hematopoietic stem cells into erythroid cells Respectively.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made extensive efforts to develop a method for efficiently mass-producing red blood cells in vitro ( ex vivo ) in order to improve the shortage of blood supply required for surgical operations. As a result, when erythroid precursor cells such as hematopoietic stem cells or erythrocyte-derived cells are cultured in a culture medium containing PLL (poly-L-lysine), expansion, differentiation and denucleation of cells are efficiently performed, and amine groups And the matured red blood cells can be produced with high yield, thus completing the present invention.

Accordingly, an object of the present invention is to provide a composition for producing red blood cells from erythroid precursor cells.

It is another object of the present invention to provide a method of in vitro production of red blood cells.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a composition for producing red blood cells from erythroid precursor cells comprising PLL (poly-L-lysine) as an active ingredient.

The present inventors have made extensive efforts to develop a method for efficiently mass-producing red blood cells in vitro ( ex vivo ) in order to improve the shortage of blood supply required for surgical operations. As a result, when the erythroid precursor cells such as hematopoietic stem cells or erythrocyte-derived cells are cultured in a culture component containing PLL (poly-L-lysine), the cells are well adsorbed to the substrate and not only the expansion, differentiation and denucleation occur efficiently It is possible to introduce amine groups without toxicity, and it has been found that matured red blood cells can be produced with high yield.

As used herein, the term " red blood cell generation " means the differentiation of undifferentiated cells (e. G., Hematopoietic or erythroid cells) prior to final differentiation into mature red blood cells into erythrocytes, "Has the same meaning as" red blood cell differentiation ".

As used herein, the term " erythroid precursor cell " refers to all cells involved in erythropoiesis, except for matured erythrocytes.

Erythrocyte precursor cells differentiate into mature erythrocyte via the following erythropoiesis: (a) proerythroblast in unipotent stem cells (erythroblast) ≪ / RTI > (b) differentiating whole cells into basophilic erythroblasts; (c) differentiating into a polychromatophilic erythroblast in an alkaline basic cell; (d) differentiating into polyclonal erythroblasts from polybasic cells; (e) differentiating into reticulocytes in the cells of the purifying cells; And (f) differentiating the reticulocytes into erythrocytes.

According to a specific embodiment of the present invention, the erythroid precursor cells of the present invention are hematopoietic stem cells or erythroid cells.

The hematopoietic stem cell (HSC) used in the present invention means a multipotential stem cell capable of differentiating into all kinds of blood cells such as erythrocytes, megakaryocytes, neutrophils and erythrocyte cells, and includes bone marrow, peripheral blood, ≪ / RTI > Hematopoietic stem cells are differentiated into red blood cells. Erythroid cells are differentiated into enucleated erythrocytes through proerythroblast and basophilic erythroblast. According to the present invention, the composition of the present invention promotes not only the in vitro proliferation of hematopoietic stem cells but also the differentiation into erythroid cells, as well as the enucleation of erythroid cells. Therefore, red blood cells can be efficiently obtained by using the composition of the present invention not only in the case of hematopoietic stem cells for erythrocyte differentiation but also in erythroid cells.

According to a specific embodiment of the present invention, the composition of the present invention promotes the expansion of hematopoietic stem cells in vitro.

According to the present invention, the composition of the present invention significantly increases the number in vitro by promoting the S-phase entry of hematopoietic stem cells. The increase in the number of hematopoietic stem cells is a very important feature for ensuring a large amount of blood for blood transfusion required in clinical practice.

According to a specific embodiment of the present invention, the composition of the present invention increases the expression of erythropoiesis-related genes selected from the group consisting of GATA-1, ALAS, alpha-globin and gamma-globin.

The present inventors have observed that the composition of the present invention increases the expression of various erythropoietic-related genes expressed throughout the differentiation process from undifferentiated cells to red blood cells. Thus, the composition of the present invention can be used not only as a step-specific but also as an entire process of erythrocyte differentiation. Specifically, it can promote early expansion and differentiation of hematopoietic stem cells, But also induce final enucleation.

According to a specific embodiment of the present invention, the composition of the present invention promotes enucleation of erythroid cells.

Unlike other blood cells, erythrocytes are free of nuclei, and nuclei present in primitive progenitor cells are removed through differentiation and finally differentiated into mature red blood cells that carry oxygen transfer function. According to the present invention, the composition of the present invention induces enucleation of erythroid cells differentiated from hematopoietic cells with high efficiency, thereby promoting the generation of erythrocytes from the early stage to the last stage of differentiation.

According to a specific embodiment of the present invention, the erythroid cells of the present invention are CD34 ⁺ cells. (This entry should be deleted because it is not an implementation example, but starting from here)

As used herein, the term " CD34 ⁺ cell " means a cell expressing CD34. CD34 is a cell-cell adhesion factor, which is the name of a human gene that encodes the cell surface glycoprotein that mediates the contact of stem cells to extracellular matrix or stromal cells of the bone marrow. Cells expressing CD34 are found in umbilical cord and bone marrow such as, but not limited to, normal blood vessel hematopoietic cells, endothelial progenitor cells and endothelial cells. Specifically, the CD34 ⁺ cells of the present invention are cells derived from cord blood.

According to a specific embodiment of the present invention, the PLL of the present invention is contained in a form coated on the surface of the culture supporter.

As used herein, the term " culture support " means a culture container, a culture substrate or any supporting material which physically and chemically interacts with the cells to be cultured while simultaneously providing a surface for adsorption. According to the present invention, the PLL, which is an active ingredient of the present invention, is coated on a culture supporter such as a culture plate, thereby introducing an amine group to the surface of the supporter, thereby efficiently adsorbing, expanding, differentiating and enucleating the cultured cells. The culture supporter need not necessarily have a planar surface and may have spherical or other various three dimensional structures to the extent that it can provide a significant surface for cell adsorption. Therefore, the PLL-coated culture supporter is not only a general culture container such as a culture plate, but also a three-dimensional network nanofiber having a nano unit fiber shape similar to an extracellular matrix (ECM) A variety of synthetic polymers commonly used as artificial substrates for cell culture, micropatterned solid substrates or porous substrates, including, but not limited to, any support material that provides a support surface.

According to a specific embodiment of the present invention, the PLL of the present invention is 0.001-0.1 wt%.

The composition of the present invention may contain components commonly used for culturing animal cells in addition to PLL to improve the survival rate of erythroid precursor cells. Examples of animal cell culture components that may be included in the composition of the present invention include culture media customary in the art such as Eagle's MEM [Eagle's minimum essence medium, Eagle, H. Science 130: 142 (1959)], α-MEM [Stanner, et al., NAT. New Biol. 230: 52 (1971)], Iscove's MEM [Iscove, N. et al., J. Exp. Med. 147: 923 (1978)], 199 medium [Morgan et al., Proc. Soc. Exp. BioMed., 73: 1 (1950)], CMRL 1066, RPMI 1640 [Moore et al., J. Amer. Med. Assoc. 199: 519 (1967)], F12 [Ham, Pro. Natl. Acad. Sci. USA 53: 288 (1965)], F10 [Ham, R.G. Exp. Cell Res. 29: 515 (1963)], DMEM [Dulbecco's modification of Eagle's medium, Dulbecco, R. et al., Virology 8: 396 (1959)], DMEM and F12 [Barnes, D. et al., Anal. Biochem. 102: 225 (1980)], Way-mouth's MB752 / 1 [Waymouth, C. J. Natl. Cancer Inst. Iscove's modified Dulbecco's medium, Iscove's modified Fisher's medium or Iscove's modified Eagle's medium, McCoy's 5A [McCoy, T. A., et al, Pro. soc. Exp. Bio. Med. 100: 115 (1959)], the series of MCDB [Ham, R. G et al., In Vitro 14:11 (1978)], AIM-V medium and modified medium thereof. A detailed description of the medium is given in R. Ian Freshney, Culture of Animal Cells, A Manual of Basic Technique, Alan R. Liss, Inc., New York.

According to another aspect of the present invention, there is provided an in vitro production of a red blood cell comprising culturing erythroid precursor cells in a culture medium containing PLL (poly-L-lysine) ≪ / RTI >

Since erythroid precursor cells, PLL, and other culture components used in the present invention have already been described above, the description thereof is omitted in order to avoid excessive redundancy.

According to a specific embodiment of the present invention, the PLL of the present invention is coated on the surface of a culture support.

The PLL used in the present invention increases the expression of various erythropoietic-related genes expressed throughout the differentiation process from undifferentiated cells to red blood cells, promotes early expansion and differentiation of hematopoietic stem cells, Lt; RTI ID = 0.0 > enucleation. ≪ / RTI > Thus, PLL may be used initially in the culture of erythrocyte precursor cells following the erythrocyte formation process, or may be used for further culture at an intermediate stage (e. G., A denucleation stage, 17 days of culture). Erythrocyte precursor cells can be obtained from a variety of sources, such as peripheral blood, cord blood, or bone marrow. CD34 ⁺ cells as erythroid precursor cells can be isolated according to various cell separation methods known in the art, such as immuno magnetic-bead separation methods using CD34 ⁺ antibodies.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides an optimal condition for erythrocyte precursor cells to differentiate into mature erythrocytes by introducing an amine group on the surface of a culture supporter without toxicity using a polyamino acid PLL.

(b) The present invention efficiently promotes erythrocyte differentiation throughout the process of producing erythrocytes.

(c) The present invention can meet the clinical demand for transfusion blood by presenting a safe in vitro mass production method of erythrocytes.

Figure 1 shows that PLL substrates (0.1, 0.01 and 0.001%) promote the in vitro proliferation of hematopoietic stem cells. In vitro proliferation of CD34 ⁺ cells on the PLL substrate was increased at 3 days compared to the control, but not at 1 day (Fig. 1a). CD34 ⁺ cells showed a higher rate of cell division in the PLL matrix than in the control (Fig. 1b). The percentage of CD34 ⁺ Lin ^- cells was similar in all of the substrates on day 1 and day 3 (FIG. 1c).
FIG. 2 is a graph showing that the PLL substrate (0.01%) does not affect the pluripotency of hematopoietic stem cells. The total number of colonies (Figure 2a) and the percentage of colonies of various types (Figure 2b) were determined through colony formation analysis. CFU-E, CFU-G and CFU-M: CFU (colony forming unit) of erythroid cells, granulocytes, red blood cells and megakaryocytic colonies, respectively.
Figure 3 is a graph showing that the PLL substrate (0.1, 0.01 and 0.001%) promotes the differentiation of CD34 ⁺ cells into erythroid cells. FIG. 3A shows the increase in the number of erythroid cells cultured on the PPL substrate for 21 days in erythroid cell differentiation medium, and FIG. 3B shows the ratio of CD71 ⁺ GPA ⁺ cells, respectively. CD71 and GPA (glycophorin A) were used as surface markers to detect erythrocyte differentiation of erythroid cells. FIG. 3c is a graph showing the results of the enucleation analysis by flow cytometry analysis using anti-GPA antibody and SYTO64. GPA ⁺ SYTO64 Cells were considered to be denucleated erythrocytes.
Figure 4 shows the effect of PLL substrate (0.01%) on erythroid cell differentiation through benzidine staining and RT-PCR. FIG. 4A is a graph showing the results of measurement of the number of erythrocytes completely differentiated on the PLL substrate through hematoxylin staining after benzidine staining in step IV. FIG. These cells are benzidine ⁺ / hematoxylin and do not contain nuclei. FIG. 4B is a graph showing the results of RT-PCR measurement of the expression of erythropoiesis-related genes at the end of each step. FIG.
FIG. 5 is a graph showing that denucleation in the downward direction is promoted by the interaction between the hematopoietic stem cells and the PLL substrate (0.01%). Light in order to detect the suspensions in erythroid cells perform gimja dyeing and, to detect the departure nucleus of the bottom culture plate hematoxylin and was performed for DAPI staining (Fig. 5a), mm determination of the nuclear attached per ^second Respectively. Figure 5b shows the results of observing the enucleated cells using the anti-GPA antibody, paloidin and DAPI through the confocal image of the Z-section (top panel). A model of the enucleation mechanism is shown in the panel below. Enucleation appears to be mediated by selective interactions between the anion of the cell membrane of the PLL molecule and the cell membrane of the erythrocyte cell, thereby promoting downward enucleation.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Method

Cord blood CD34 ⁺  Isolation of cells

The cord blood samples from normal pregnant women were collected in bags (Green Cross Corp., Yong-in, Korea) containing 45 ml of CPDA-1 (citrate phosphate dextrose A-1) This study was approved by the Institutional Review Board of the Korean Medical School. Mononuclear cell (MNC) fractions were separated using Ficoll paque PLUS (GE Healthcare Biosciences, Pittsburgh, Pa.) According to manufacturer's instructions. CD34 ⁺ cells were isolated according to the manufacturer's instructions using an immuno-magnetic microbead separator (MACS CD34 isolation kit, Miltenyi Biotech, Auburn, Calif.) [15].

To prepare the PLL substrate, the PLL was coated onto the tissue culture plate at room temperature for 2 hours and washed twice with distilled water. The isolated CD34 ⁺ cells seeded 1 x 10 ⁴ cells per cm ² onto PLL-treated plates, respectively.

CD34 ⁺  In vitro proliferation of cells

In order to induce the in vitro proliferation of isolated CD34 ⁺ cells, cells were cultured in the presence of 20% FBS, 1% penicillin / streptomycin, 50 ng / ml stem cell factor (SCF; Peprotech, Rehovot, Israel) 20 ng / ml IL-3 (Peprotech) and 50 ng / ml IL-6 (Peprotech). For labeling CFSE (carboxy fluorescein succinimidyl ester), 1 × 10 ⁶ cells / ml in DMEM was treated with 5 μM CFSE, incubated for 10 min at 37 ° C, and washed 4 times in cold DMEM.

The same number of cells were plated three times in a methylcellulose-based culture medium containing human cytokines and colony assays were performed according to the manufacturer's instructions (R & D systems, Minneapolis, MN). Colonies containing 30 or more cells were sorted and classified using an optical microscope 14 days later.

CD34 ⁺  Erythroid cell differentiation of cells

To induce the differentiation of isolated CD34 ⁺ cells into erythroid cells, cells were cultured with a slight modification of the method reported by Baek [18]. Based on Iscove's DMEM supplemented with several reagents, several cytokines in serum-free medium were added to the medium for erythrocyte cell differentiation. The basic differentiation method consists of four steps. In the medium supplemented with 1 μM hydrocortisone (HC, Sigma), 100 ng / ml SCF, 10 ng / ml IL-3 and 6 IU / ml EPO (erythropoietin; R & D systems) in step I (0-7 days) CD34 ⁺ cells were cultured. On the fourth day, the supernatant was removed and half of the medium was replaced by adding the same amount of culture medium.

In step II (7-13 days), the expanded erythroid cells were cultured at a density of 1x10 ⁵ / cm ² in 24-well plates under 50 ng / ml SCF, 10 ng / ml IL-3 and 3 IU / ml EPO . Cells were diluted 2-4 times every 3 or 4 days. In step III (13-17 days), 50 ng / ml SCF and 2 IU / ml EPO were added. Finally, in step IV (enucleation phase, 17-21 days), erythroid cells were transferred to fresh medium without cytokines. 0.05% of P188 was added from the 13th day to the last day. At the end of each step, cultured cells were collected and the number of erythroid cells was measured using a hemocytometer. All cultures were maintained in humidified 37 ° C, 5% CO ₂ environment.

Flow cytometry

To analyze the expression of the surface protein markers of the in vitro proliferating stem cells, the cultured cells were cultured on day 1 or day 3 with an anti-lineage specific antigen cocktail (LinFITC; Biolegend) conjugated with FITC (fluorescein isothiocyanate) CD34-Peridin chlorophyll protein (CD34-PerCP; Biolegend). To measure the expression of differentiation surface markers such as CD71 and glycophorin A (GPA), cells cultured on the last day of each step were incubated with anti-CD71-picoerythrin (CD71-PE; Biolegend) and anti- - < / RTI > allophycocyanin (GPAAPC; Biolegend). Specifically, in the enucleation assay, erythroid cells were incubated with anti-GPA-FITC and gently washed to remove unbound antibodies. Cells were resuspended with 0.5 μM SYTO64 (Invitrogen), a cell permeable red fluorescent dye in 10 mM HEPES buffer, pH 7.4, containing 40 mM NaCl and 5 mM MgCl ₂ for enucleation. Cells were washed twice in PBS with 2% FBS and measured with a FACSCalibur System (BD Bioscience, Heidelberg, Germany). Flow cytometry data were analyzed using CellQuest software (BD Bioscience) and WinMDI2.9 software.

Wright-giemsa staining and benzidine staining

To observe the morphology of the cells and the degree of differentiation of erythroid cells into erythrocytes, erythrocyte cells were collected on each slide and cytospin was performed. The slides were dried, stained with a light-gum dye for 5 minutes, and washed three times with distilled water.

For total hemoglobin staining, air-dried cytosine dye samples were fixed in 100% methanol for 4 minutes. After washing for 10 minutes with PBS, cells were stained with 3'3'-diaminobenzidine reagent (Sigma-Aldrich) according to the manufacturer's instructions. Cells such as red blood cells containing hemoglobin were stained brown, and the nuclei of the cells were stained with hematoxylin in purple.

Immune cell chemistry

Hematopoietic stem cells were cultured in the presence or absence of PLL substrate, washed three times with PBS, fixed in 10% formalin, and permeabilized with 0.3% Triton X-100 for 5 minutes. These cells were incubated with 1% bovine serum albumin containing PBS for 1 hour at room temperature to block non-specific protein binding. Cells were then stained with anti-GPA produced in a lightproof box and detected using an anti-mouse FITC-conjugated secondary antibody (Abcam). The nuclei were stained with DAPI (4,6-diamidino-2-phenylindole) and dihydrochloride (Molecular Probes) and mounted on a glass cover slip with Gel / Mount (Biomedia). Samples were examined using a Zeiss LSM 510 Meta laser scanning confocal microscope (Carl Zeiss Microimaging GmbH).

Reverse transcription polymerase chain reaction (PCR) and real-time PCR

The total RNA obtained in the cultured ASC was separated using the TRIzol reagent (Gibco BRL) according to the manufacturer's instructions, and the RNA concentration was determined after resuspension of the RNA precipitate. RNA was reverse transcribed twice for each sample using the TOPscript ™ cDNA synthesis kit (Enzynomics). PCR was performed using AccuPowerGreenStar qPCR premix (Bioneer) and xiCycler 96 Real-time Quantitative Thermal Block (Bioneer). The PCR conditions were as follows: 94 ° C for 10 minutes and 95 ° C for 40 cycles of 10 seconds, 58 ° C for 30 seconds, and 72 ° C for 30 seconds. Fluorescence intensity was calculated using Exicycler3 analysis software. Primer sequences associated with erythroid cell differentiation are shown in Table 1.

The primer sequences used for quantitative PCR for the analysis of erythrocyte differentiation gene order GATA-1 Forward 5'-ATCACACTGAGCTTGCCACA-3 ' Reverse 5'-CAGGCCAGGGAACTCCA-3 ' Globin, alpha 1 Forward 5'-CCGGTCAACTTCAAGCTCCT-3 ' Reverse 5'-AAGAAGCATGGCCACCGAG-3 ' Globin, gamma 1 Forward 5'-GCTGCATGTGGATCCTGAGA-3 ' Reverse 5'-GCCACTGCAGTCACCATCT-3 ' ALAS Forward 5'-TGCAGGGCAACAGGACTTTA-3 ' Reverse 5'-GGGACAGCGTCCAATACCAA-3 ' GAPDH Forward 5'-ACATCGCTCAGACACCATG-3 Reverse 5'-TGTAGTTGAGGTCAATGAAGGG-3 '

Experiment result

  In vitro proliferation and differentiation of hematopoietic stem cells in PLL matrix

Tissue culture plates were coated with various concentrations of PLL (0.0001, 0.01 and 0.1 wt.%) And the oxygen, nitrogen and carbon contents of the coating surface were quantified by X-ray photoelectron spectroscopy (XPS, Table 1). Nitrogen content of the substrate increased with increasing PLL concentration. As the amount of PLL adsorbed increased, the number of amine groups on the culture surface increased.

To investigate the effect of PLL on the self-renewal and extracellular proliferation of hematopoietic stem cells, CD34 ⁺ cells were separated from human umbilical cord blood and cultured in the proliferation medium for 1 or 3 days (Fig. 1). The in vitro proliferation of hematopoietic stem cells on days 1 and 3 was examined using PLL-coated surfaces (0.001, 0.01 and 0.1%) (Fig. 1). As shown in FIG. 1A, on the third day, a larger number of hematopoietic stem cells were observed than on the uncoated control group, but not on the first day. The higher the PLL concentration (0.01 and 0.1%), the more pronounced this trend was, but the lower the concentration (0.001%), the more significant the difference was. Similar dose-dependent results were observed in cell division rates in the fourth cell division (Fig. 1B). However, the proportion of CD34 ⁺ Lin cells was similar at all ratios (Figure 1c).

To determine the differentiation potential of CD34 ⁺ cells, we selected a 0.01% PLL substrate and analyzed the formation of colonies after 14 days in culture on methylcellulose (FIG. 2). The number of total colonies formed from strongly differentiating cells (CFU-E, BFU-E, CFU-G, CFU-M, CFU-GM and CFU-GEMM, 2a) and the ratio of each type of colonies (FIG. 2b) did not show any significant difference between the PLL and control substrates. When synthesis this, 0.01% or in more coating concentrations, PLL is increased by whole without changing the differentiation potential of the proportion of the stem cells with the expression pattern of CD34 ⁺ Lin surface marker or CD34 ⁺ cells, CD34 cell division ⁺ The number of cells is increased.

Erythrocyte cell differentiation and RBC generation on PLL substrate

Next, the differentiation of hematopoietic stem cells into erythroid cells was examined in PLL of 0.001, 0.01 and 0.1% concentration (FIG. 3) and 0.01% concentration (FIG. 4). CD34 ⁺ cells were treated with cytokines and other factors (hydrocortisone (HC), stem cell factor (SCF), IL-3 and EPO (erythropoietin)) in accordance with the desired differentiation stage (Step I- And cultured in four kinds of differentiation medium having different combinations (Fig. 3). The number of erythroid cells was measured by counting cells at the end of each step (Fig. 3A). Measurements of hematopoietic stem cells cultured on a 0.01% PLL substrate and in a control substrate, 0.001 or 0.1% PLL substrate showed the highest increase in stage I, whereas in stage II, III and IV, No specific changes were observed (Fig. 3a). On the other hand, the fluorescence-activated cell sorting (FACS) analysis showed that the ratio of erythroid cells (CD71 ⁺ GPA ⁺ cells) cultured on 0.01% PLL substrate was higher only in step II than in the control group. These results demonstrate that 0.01% PLL substrate promotes the expansion of hematopoietic stem cells that initially differentiate only in the early stages, but not in later stages.

As shown in FIG. 3C, the percentage of enucleated cells (erythrocytes) in erythroid cells in steps III and IV was measured using antibodies against GPA (glycophorin A, erythrocyte surface marker) and SYTO64 (nuclear marker). In step III, 0.01 and 0.1% PLL substrates showed higher erythrocyte (GPA ⁺ SYTO64 cells) ratios relative to the control and 0.001% PLL substrate. However, this difference was not observed in step IV, indicating that the PLL substrate induces enucleation of erythroid cells in step III, rather than step IV. Thus, it can be seen that 0.01% PLL coatings on supports in tissue culture provide the ideal conditions for generating enucleated erythrocytes from hematopoietic cells in vitro.

To compare the degree of erythrocyte cell differentiation in the control and 0.01% PLL substrate, erythrocytes were imaged by benzidine staining to detect hemoglobin (FIG. 4A) and analyzed for erythropoiesis-related gene expression (FIG. 4B). The red blood cells (arrows) of step IV were incubated with benzidine ⁺ / hematoxylin Cells (Fig. 4A). These cells were found more in the PLL substrate than in the control group. According to the results of the quantitative real-time PCR (RT-PCR) analysis (Fig. 4B) in step I-III, the erythrocyte cell differentiation markers (GATA-1, globin transcription factor- Cell ALA synthase (ALAS)) was higher than that of PLL substrate, especially in step III. These analyzes confirm that the 0.01% PLL substrate promotes the enucleation of erythrocyte cells and increases the expression of erythropoiesis-related genes during erythroid cell differentiation.

Efficiency and direction of enucleation on PLL substrate

Figure 5a shows that the degree of enucleation in Stage III erythroid cells (arrows) is higher in the 0.01% PLL substrate (right panel) than in the control (left panel). Interestingly, hematoxylin and DAPI staining also show that many nuclei remain in the bottom of the culture medium, especially the PLL substrate, even after the enucleation process (Fig. 5A). In fact, the number of these nuclei was about three times higher in the PLL substrate when compared to the control group. To examine the direction of enucleation, cells were stained with anti-GPA antibody, paloidin and DAPI during erythrocyte differentiation (Fig. 5b). In the confocal image, green indicates GPA, red indicates actin filament, and blue indicates nuclei. GPA and actin filaments pushing nuclei were co-localized, and the direction of pushing nuclei in most erythroid cells was lateral or downward. However, as a result of quantitation of the adsorbed nuclei, downward enucleation occurred more frequently in the PLL substrate than in the control (Figure 5b). Therefore, the PLL substrate promotes the enucleation of erythrocyte cells by stimulating the nucleus to be downward through selective interaction between the cations of the PLL molecule and the enucleated cells (FIG. 5, schematic diagram).

Further discussion

Interactions between hematopoietic stem cells and cultured matrices are crucial for controlling in vitro proliferation and differentiation of stem cells. Many studies have used various substrates coated with matrix proteins (eg, tropoelastin [14], fibronectin [15] and fibrin [16]) to improve hematopoietic cell expansion and differentiation. Of these, tropoelastin has been found to be particularly efficient. For example, the tropoelastin-coated culture substrate expanded human and mouse hematopoietic cells about 3-fold compared to the control. Other studies have focused on physically and chemically modifying the culture medium for in vitro proliferation and differentiation of hematopoietic stem cells. For example, chemical modification of nanofibers with amine groups increases the in vitro proliferation of hematopoietic stem cells in unmodified controls [11] - [12]. Unfortunately, however, most hematopoietic cells lose more than 70% of the CD34 ⁺ characteristic surface marker during in vitro proliferation. In contrast, the present inventors confirmed that the PLL substrate increased the original number of hematopoietic stem cells by three times, and that more than 80% of them were positive for CD34 (FIG. 6). These results are compared with the results by tropoelastin (Fig. 6).

The present inventors confirmed that the PLL substrate (0.01 and 0.1%) increased the hematopoietic stem cells by about 30% as compared with the control (Fig. 1A). In addition, hematopoietic cells having a faster cell division rate were significantly increased on the PLL substrate as compared with the control group (Fig. 1B). Thus, the PLL substrate increases the in vitro proliferation of hematopoietic stem cells by promoting the entry of cells into the S phase.

During hematopoietic differentiation into hematopoietic cells, cells generated from hematopoietic cells interact with the microenvironment or niche consisting of ECM and various types of cells [6]. These interactions promote the proliferation, differentiation and enucleation of erythroid cells, ultimately producing red blood cells. Lazar-Karsten et al. Have shown that hematopoietic stem cells cultured in isolated ECM proteins containing a mixed protein ECM gel consisting of collagen IV, laminin, fibronectin and laminin, collagen IV, heparan sulfate proteoglycan and entatin are differentiated into erythroid cells . The ECM gel provided the highest cell viability compared to other substrates, whereas the laminin substrate showed a 15% higher denucleation rate. However, the fibronectin substrate showed 40% lower expansion of erythroid cells than the control, and the collagen substrate did not have a definite effect on erythroid cell differentiation.

In the present invention, the PLL substrate significantly increased the number of total differentiated cells in stage I of erythroid cell differentiation, the number of erythroid cells in stage II, and the number of enucleated cells in stage III respectively (FIG. 3). In addition, PLL increased the expression of erythropoietic-associated genes such as GATA-1, ALAS and gamma-globin in step III. These results show that the PLL substrate can regulate the whole process of erythrocyte cell differentiation, promote the initial expansion and differentiation of hematopoietic stem cells, and induce the final enucleation of hematopoietic stem cell-derived cells. Indeed, PLL increased the expansion of erythroid cells by 28% and their enucleation by 35% compared to the control (Fig. 3).

Denisov et al. Reported that electrostatic reactions can cause rearrangement of the membrane and cause deformation of a specific membrane on the contact surface. The present invention aims to control the density of the cation added by the amine group on the cell culture surface by controlling the coating concentration of the PLL. As a result of XPS analysis, it was confirmed that the highest concentration PLL (0.1%) applied in the present invention not only provides the highest nitrogen content in the culture substrate (Table 2), but also provides the largest number of enucleated cells in step III ).

Oxygen nitrogen and carbon concentration by PLL coating of various concentration density Oxygen nitrogen carbon Non-coated 10.67 Not detected 89.33 0.001% 11.38 3.30 85.31 0.01% 12.86 3.55 83.60 0.1% 10.97 3.97 85.06

As suggested in previous studies, the present inventors assumed that enucleation of many cells would be due to cell membrane deformation in addition to the electrostatic action between the anions of the cell membrane and the cations of the PLL molecules. In the previous report, it was hypothesized that electrons produced more denucleated erythroid cells when compared to substrates rich in amines and electrons, resulting in electrostatic attraction between cell membranes and cationic substrates . In addition, we anticipated that the direction of enucleation would be upward, and there was no direct evidence of this. Meanwhile, in the present invention, it was confirmed that three times as many nuclei as the control group remained on the PLL substrate after erythrocyte enucleation (Fig. 5A). Furthermore, observations of confocal microscopy revealed that the nuclei of the enucleated cells were removed downward rather than upwards, and then adsorbed to the PLL substrate (FIG. 5b).

During the enucleation of erythrocyte cells, the removed nuclei are located around Emp (erythroblast macrophage protein), a cell surface protein with cations and lysine- and arginine-rich N-terminal extracellular domains [18-19]. Thus, the positive PLL amine moiety acts as Emp and appears to promote red cell nuclei to move down the cell surface. However, in order to fully understand the mechanism of interaction between hematopoietic stem cells / hematopoietic stem cell-derived cells and PLL, membrane proteins and related signaling pathways must be identified.

conclusion

The present invention shows that PLL substrates increase the number of total hematopoietic cells without altering the expression of surface markers associated with stem cell characteristics. In addition, the higher the concentration of PLL coating, the greater the in vitro proliferation of CD34 ⁺ cells without affecting the proportion of CD34 ⁺ Lin cells. Furthermore, the number of cells undergoing enucleation after erythrocyte differentiation was increased over PLL compared to the control group. Taken together, our studies show that PLL can be used as an effective composition for promoting in vitro proliferation of hematopoietic stem cells, differentiation into erythroid cells, and enucleation of erythrocyte cells to produce erythrocytes.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

references

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Claims (12)

A composition for inducing the differentiation of erythroid precursor cells into erythrocytes (red blood cells) containing PLL (poly-L-lysine) as an active ingredient.
The composition of claim 1, wherein the erythroid precursor cells are hematopoietic stem cells or erythroid cells.
The composition of claim 1, wherein the composition promotes the expansion of hematopoietic stem cells in vitro.
2. The composition of claim 1, wherein the composition increases expression of an erythropoietic-related gene selected from the group consisting of GATA-1, ALAS, alpha globin and gamma globin.
The composition of claim 1, wherein the composition promotes enucleation of erythroid cells.
2. The composition of claim 1, wherein the erythroid precursor cells are CD34 ⁺ cells.
2. The composition of claim 1, wherein the PLL is coated on the surface of the culture support.
The composition of claim 1, wherein the PLL is 0.001-0.1 wt%.
A method of inducing in vitro differentiation of erythroid precursor cells into red blood cells comprising culturing erythroid precursor cells in a medium containing poly-L-lysine (PLL).
10. The method according to claim 9, wherein the erythroid precursor cells are hematopoietic stem cells or erythroid cells.
10. The method of claim 9, wherein the PLL is coated on a culture support surface.
10. The method of claim 9, wherein the PLL is coated on the surface of the culture substrate.

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