RU2628092C1 - Method for obtaining of msc-associated non-differentiated hemopoietic precursor cells with cd34+/cd133+ phenotype - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to production of cell cultures enriched with hematopoietic precursor cells with CD34⁺/CD133⁺ phenotype. The method includes preparation of a stromal sublayer, addition of an umbilical cord fraction, cultivation and selection. Multipotent stromal cells of adipose tissue (MSC) are obtained from the stromal-vascular fraction of adipose tissue, which are cultured at an O₂ concentration of 5% in the environment. Mononuclear fraction of umbilical cord blood (MNF UB) is added to the MSC monolayer. After cocultivation, the undifferentiated hematopoietic precursors are selected from the mononuclear fraction of UB by adhesion to MSC at 20% O₂, MSC is continued to be cultured with attached MNF UB for 96 hours at O₂ concentration of 20% in the environment.

EFFECT: invention allows to obtain a population enriched with undifferentiated hematopoietic umbilical cord blood precursors with this complex phenotype.

9 dwg, 1 tbl, 1 ex

Description

The invention relates to medicine, namely to biotechnology, and can be used to obtain undifferentiated hematopoietic progenitor cells from umbilical cord blood (GSPK), for clinical use.

Cord blood is currently regarded as a complete source of hematopoietic stem and progenitor cells.

An important advantage, compared with bone marrow and mobilized peripheral blood, is the possibility of non-invasive production of HSPC samples in a sufficiently large number and the use of cells with less correspondence for histocompatibility antigens, sufficient safety of the recipient in connection with more rare cases of the reaction “transplant against the host” [ Broxmeyer, 2005; Gluckman, Rocha, 2009].

However, the absolute number of hematopoietic stem cells in umbilical cord blood is small, so the possibility of their ex vivo expansion for subsequent clinical use is of increasing interest.

Typically, ex vivo expansion employs CD34 ⁺ suspension monocultures in a static mode with the addition of hematopoietic cocktails (containing cytokines that stimulate hematopoiesis) that provide self-renewal and proliferation of primitive hematopoietic precursors [Andrade et al., 2013]. This requires a very accurate selection of cultivation conditions and it is necessary to maintain a constant oxygen concentration and pH of the medium.

To cultivate large volumes of cell suspension, bioreactors are used in which constant mixing of the culture medium is ensured, maintaining a constant concentration of oxygen and its pH. The use of such systems requires large volumes of cultural environments and highly qualified personnel.

Currently, the development of the use of three-dimensional matrices using porous Cytomatrix biomaterials with the aim of ex vivo expansion of HSPC [Ehring et al., 2003] is underway. However, the use of such a support layer is expensive and time consuming.

One of the directions of ex vivo expansion of HSPC is co-cultivation with stromal components, in particular, with multipotent mesenchymal cells (MSCs). The advantage of using feeder-containing systems to maintain poorly differentiated hematopoietic precursors is currently not in doubt.

Even in classical works [Dexter et al., 1977, 1982], a prolonged, for several months, maintenance of hematopoiesis was demonstrated on the sublayer of a mixed population of bone marrow cells. In addition, for successful amplification, direct contact of hematopoietic and stromal cells is necessary, which was convincingly shown by Silva et al., [2010], when the growth of poorly differentiated precursors in transwells was almost 10 times less than with direct interaction. The use of the stromal sublayer allows, to some extent, to imitate the structural and functional features of the hematopoietic niche. In particular, in co-culture with stromal cells, hematopoietic cells can occupy various compartments.

In the so-called "Dexter" cultures from bone marrow stromal cells, which were mentioned above, the localization of hematopoietic cells in 3 regions relative to the stromal sublayer was described.

Firstly, these are cells that adhere to the stroma upon inoculation of a hematopoietic suspension, secondly, cells that become suspension upon division of attached cells, and thirdly, cells transmigrating under the stromal sublayer [Dexter et al., 1977; Wagner et al., 2007; Jing et al., 2010; Andreeva et al., 2015]. CD34 ⁺ hematopoietic precursors in the three described compartments on the sublayer of mesenchymal stromal cells (MSCs) of the bone marrow were phenotyped, and it was found that the most proliferating cells were found in suspension, while the precursors associated with the stroma divide more slowly and are enriched in undifferentiated hematopoietic cells with phenotype CD34 ⁺ / CD38 ^- [Wagner et al., 2007; Jing et al., 2010].

Thus, the use of the stromal sublayer from mesochemical stromal cells of the bone marrow makes it possible to obtain populations of hematopoietic cells with varying degrees of commitment.

Unfortunately, in various protocols using the MSC / HSPC co-culture, only newly formed suspension HSPCs are considered as target cells for expansion, although, as mentioned above, this population contains more committed precursors than stroma-associated cells.

The routine step in isolating a population and expanding HSPCs is to sort them by CD34 antigen [Brandt et al., 1990]. The CD34 ⁺ HSPC population contains cells that differ in their potentials for differentiation: some of them can be committed to several hematopoietic sprouts, while in others this ability is limited [Perey et al., 1998; Dykstra et al., 2007]. Unfortunately, the immunological selection of CD34 ⁺ cells may lose the earliest HSPCs with the CD34 phenotype ^- [Zanjani et al., 2003]. Such cells express the CD133 antigen, possessing the properties of hematopoietic stem cells [Gallacher et al., 2000; Rutella et al., 2003]. By the presence / absence of CD34 and CD133 antigens

undifferentiated hematopoietic precursors are divided into early (CD34 ^- / CD133 ⁺ ), medium (CD34 ⁺ / CD133 ⁺ ) and late CD34 ⁺ / CD133 ^- ) [McGuckin et al., 2003].

Regardless of which cells were taken for expansion - mononuclear cells from bone marrow, umbilical cord or mobilized peripheral blood, or a CD34 ⁺ population, during ex vivo expansion, newly formed HSPCs lose an undifferentiated phenotype in favor of committed predecessors. This reduces their value as cellular material for the restoration of blood formation.

Of particular interest to researchers are cells capable of long-term restoration of hematopoiesis during transplantation, in contrast to more committed precursors that provide only short-term effects.

It is known that among HSPCs there are cells capable of forming “cobblestone bridge areas” (COOB or CAFC (Cobblestone Area Forming Cell)) under MSC in co-culture. In vitro tests are used to identify such cells, based on the detection of the frequency of COOB or their committed offspring (cells initiating long-term cultures (CIDC) or LTC-IC (from the words Long-term Culture Initiating Cell)) by the method of limiting dilutions [van Os et al ., 2008].

It has been shown that CD34 ⁺ / CD133 ⁺ PS cells are significantly enriched in KIDK, while among CD34 ⁺ / CD133 ^- KIDK is significantly less [De Wynter et al., 1998]. Moreover, it is known that the majority (up to 80%) of hematopoietic cells of pcMNAs are represented by late predecessors with the CD34 ⁺ / CD133 phenotype ^- and, to a lesser extent, cells with the expression of early markers.

In this regard, the task of developing approaches for more efficient and physiologically sound expansion of hematopoietic cells remains one of the most significant in the field of cell

therapy and regenerative medicine.

The closest analogue of the invention is a method of expansion of mononuclear cells of umbilical cord blood (MNC PK) ex vivo in the presence of multipotent mesenchymal stromal cells (MSCs) of a person (RU 2525143, 08/10/2014). This method includes the cultivation of MSCs from the stromal-vascular fraction of adipose tissue to achieve a monolayer at a concentration of O ₂ in a medium of 5%, the addition of a suspension of MNCs to the monolayer of MSCs, cultivation for 72 hours at a concentration of O ₂ in a medium of 5%, selection of loose MSCs and medium replacement, continued cultivation of MSCs with PC MNCs attached to them for 7 days at a concentration of O ₂ in the medium of 5%.

It was shown that MSCs from adipose tissue can effectively support the viability of hematopoietic precursors and ex vivo co-cultivation of MNCs and MSCs, enriching the population of umbilical cord blood mononuclear cells with hematopoietic precursors.

The disadvantage of this method is the production of HSPC cells of varying degrees of commitment. In addition, this method of expansion shows enrichment of the population with progenitor cells positive for each individual marker CD34 ⁺ and CD133 ⁺ , while such a positive sorting of CD34 ⁺ and CD133 ⁺ cells includes a population of cells with phenotypes of later differentiation.

Therefore, the objective of the invention was to develop a technology that allows not only to increase the number of hematopoietic precursors in an in vitro experiment, but also to obtain a population enriched in undifferentiated hematopoietic umbilical cord blood precursors with a complex phenotype CD34 ⁺ / CD133 ⁺ , which corresponds specifically to early hematopoietic precursors.

To obtain MSC-associated undifferentiated

the hematopoietic progenitor cells with the CD34 ⁺ / CD133 ⁺ phenotype carried out the following steps:

- preparing the stromal sublayer from MSCs from the stromal-vascular fraction of adipose tissue, constantly cultivating at a concentration of O ₂ in the medium of 5%,

- added the mononuclear fraction of umbilical cord blood (MNC PK) to the MSC monolayer and co-cultured for 72 hours,

- selection of undifferentiated hematopoietic precursors from MNC PC was performed due to adhesion to MSCs at 20% O ₂ ,

- MSCs were cultured with PC MNCs attached to them for 96 hours at an O ₂ concentration of 20% with the formation of a stroma-associated population of HSPCs with the CD34 ⁺ / CD133 ⁺ phenotype.

The technical result of the proposed method is to obtain a population of HSPCs that is more than 90% represented by progenitor cells with the complex CD34 ⁺ / CD133 ⁺ phenotype associated with the stromal sublayer of multipotent stromal cells (MSCs) of adipose tissue and forming cobblestone areas (COOB).

In addition, cell preparations obtained after co-cultivation do not need to separate MSCs, since allogeneic MSCs contribute to the engraftment of hematopoietic grafts.

This technical result is achieved in that in a method for producing a population of undifferentiated hematopoietic umbilical cord blood precursors (HSPCs) containing HSPCs with the CD34 ⁺ / CD133 ⁺ phenotype associated with the stromal sublayer from multipotent stromal adipose tissue cells (MSCs) including the preparation of a stromal sublayer from MSCs from stromal-vascular fraction of adipose tissue, constantly culturing at a concentration of O ₂ in a medium of 5%, the addition of a mononuclear fraction of umbilical cord blood (MNC

PC) to the MSC monolayer, their co-cultivation for 72 hours with further selection of undifferentiated hematopoietic precursors from MNC PCs due to adhesion to MSCs at 20% O ₂ , continued cultivation of MSCs with PC MNCs attached to them for 96 hours at a concentration of O ₂ in the medium 20% with the formation of a population of HSPCs, including HSPCs with the phenotype CD34 ⁺ / CD133 ⁺ associated with the stromal sublayer from MSCs of adipose tissue.

A brief description of the drawings.

Figure 1 - Scheme for the production of HSPC after co-cultivation of MNC PC with MSC.

Figure 2 - Suspension of HSPC in co-culture with MSCs from adipose tissue after 7 days of ex vivo expansion.

Figure 3 - MSC-associated HSPCs on the surface of MSCs and under the stromal sublayer - "cells forming the cobblestone bridge area - (COOB)" in co-culture with MSCs from adipose tissue after 7 days of ex vivo expansion.

Figure 4 - Suspension GSPK when analyzing on a flow cytometer, the distribution of size and granularity.

Figure 5 - Distribution of suspension HSPCs when analyzed on a flow cytometer — gating of the HSPC population by CD45 antigen.

Figure 6 - Distribution of suspension HSPCs when analyzed on a flow cytometer - CD34 ⁺ and CD133 ⁺ HSPC among CD45 ⁺ cells

Figure 7 - MSC-associated HSPC when analyzed on a flow cytometer - distribution of size and granularity.

Figure 8 - Distribution of MSC-associated HSPCs when analyzed on a flow cytometer. Gating populations of MSCs and HSPCs by CD45 antigen.

Figure 9 - Distribution of MSC-associated HSPCs by analysis on a flow cytometer - CD34 ⁺ and CD133 ⁺ HSPC among CD45 ⁺ cells.

DETAILED DESCRIPTION OF THE INVENTION

Isolation of the mononuclear fraction of cord blood (MNC PC).

The procurement of PCs was carried out with written informed consent of the examined healthy women in labor at the departments of the Scientific Center for Obstetrics, Gynecology and Perinatology named after Acad. IN AND. Kulakova. Blood was collected in bags of the donor system with the anticoagulant TSFDA-1 and processed for 24 hours. The preparation of the “nucleated cell concentrate” was carried out by the double centrifugation method in accordance with the registered medical technology (FS2009 / 387 of 11/23/2009). After erythrocyte sedimentation and removal of excess plasma, the fraction of nucleated PC cells was resuspended in autologous plasma with the addition of 10% dimethyl sulfoxide (Sigma, USA) and 1% Dextran-40, packaged in cryovials, and subjected to program freezing to a final temperature of -90 ° C in accordance with Standard stem cell bank operating procedures. On the day of the experiment, the cells were thawed in a water bath at + 37 ° C and washed from the cryoprotectant in excess of the culture medium (see below). After assessing viability in the trypan blue test, the cell concentration was adjusted to 1.5-2.5 × 10 ⁶ cells / ml and used for 30 minutes.

Isolation and cultivation of MSCs

To obtain MSCs, the stromal-vascular fraction of human adipose tissue (zhMSC) was used. Cells were isolated according to the standard method and cultured with zMTSCs continuously at 5% O ₂ in a multigas incubator (Sanyo, Japan) in α-MEM medium (Gibco, USA) with the addition of 10% inactivated fetal bovine serum (Hyclone, USA), 100 units / ml of penicillin and 100 μg / ml of streptomycin (Biolot, Russia). By

reaching 70-80% confluency, the cells were reseeded. For experiments used MSC 2-4 passages.

Obtaining zhtMSK-associated GSPK PC

A suspension of PK MNC (10x10 ⁶ ) in 5 ml of medium was added to the MSC premonolayer in Petri dishes 60 mm in diameter. After 72 hours, non-adherent cells were selected and the culture medium was replaced with fresh. Co-cultivation led to adhesion of a portion of the MNCs of PK. These cells, upon further cultivation for 96 hours, gave rise to a new population of suspension HSPCs, substantially enriched with CD34 ⁺ undifferentiated hematopoietic precursors. At the same time, part of the GSPC remained associated with MSCs, forming two compartments — on the surface and under the monolayer of MSCs. At the end of the co-cultivation, the newly formed suspension of HSPC was selected, and the remaining MSC / HSPC complex was trypsinized. Then, samples were prepared from suspension and MSC-associated HSPCs to characterize the immunophenotype and viability using flow cytometry. The scheme for obtaining HSPC after co-cultivation of MNC PK with MSCs is shown in figure 1.

GSPK immunophenotyping was performed among the initial nucleated cells of PCs adhered to HSPC PCs and a population of cells newly formed upon further cultivation of attached cells by flow cytometry (Accuri C6, BD Biosciences, USA). We used FITC, phycoerythrin, and PerCP conjugated antibodies to CD45, CD34, CD133, as well as FITC-labeled antibodies to MSC antigen - CD90 (BD Pharmingen, USA) at the concentration recommended by the manufacturer.

Statistics . Statistical analysis was performed using the Microsoft Excel 2000 and Statistica 7.0 software package and the Mann-Whitney test. Differences were considered significant at p <0.05.

Example 1

To the pre-monolayer of zhMSC (70-80% confluency) cultivation of which was carried out at 5% O _{2, a} suspension of MNC PK was added in an amount of 10 × 10 ⁶ .

The co-cultivation of cord blood mononuclear cells and the stromal-vascular fraction of human adipose tissue was carried out at a concentration of O ₂ in the medium of 20%. PC MNCs that were not adhered for 72 hours were removed and the remaining cells were further cultured for 96 hours at an O ₂ concentration of 20% with the formation of a stroma-associated population of HSPCs.

In the course of the work, it was shown that after 7 days of ex vivo expansion in the co-culture of zhMSC / HSPC, 2 populations of hematopoietic cells can be detected. The figure 2 shows a representative image of a suspension of HSPC obtained by microscopy by phase contrast, the scale segment corresponds to 100 μm. In figure 3, red arrows show MSC-associated HSPCs on the surface of MSCs, yellow arrows show MSC-associated HSCCs under the stromal sublayer — “cells forming cobblestone pavement regions - (COOB)” in co-culture with MSCs from adipose tissue after 7 days of ex vivo expansion ( phase contrast, scale segment - 100 microns),

suspension HSPCs and stroma-associated HSPCs, which included both cells located on the surface of MSCs and cells forming cobblestone pavement areas under the stromal layer (Figure 3).

The viability of HSPC in both populations according to the trypan blue test was more than 90%.

In both populations, the ratio of early, middle and late undifferentiated hematopoietic precursors was determined.

The figure 4 shows a representative histogram of the distribution of suspension HSPC in size and granularity when analyzed on a flow cytometer. In the scatter plot characterizing HSPC in size and structure, one could see the presence of two subpopulations of different sizes. Smaller cells formed a dense cloud characteristic of a lymphocytic-monocytic gate. In the second subpopulation, the cells were larger with more granular cytoplasm. The number of large cells was on average 1.5 times more than small (figure 4). Figure 6 shows the distribution of suspension HSPCs when analyzed on a flow cytometer — gating of the HSPC population by the presence of CD45 antigen. The figure 7 shows a representative distribution of cells bearing CD34, CD133 and both antigens immediately among CD45 ⁺ HSPC (large cells). In samples of MSC-associated HSPCs used for cytofluorimetric analysis, both HSPC and MSC were present.

A representative distribution chart of MSC-associated HSPCs when analyzed on a flow cytometer in size and granularity is shown in Figure 7. Size / structure analysis did not reveal clearly distinguishable subpopulations. Also, as for the HSPC suspension, a population of MSCs and HSPCs was gated by the CD45 antigen, as shown in Figure 8, after which CD34 ⁺ and CD133 ⁺ HSPCs were detected among CD45 ⁺ cells, a representative distribution of which is shown in Figure 9. Almost all CD45 ⁺ cells were positively stained with antibodies against CD34 and CD133 antigens of uncommitted hematopoietic precursors.

In this work, we first analyzed the ratio of undifferentiated HSPCs of varying degrees of maturity after ex vivo expansion using MSCs from adipose tissue, cultivation of which was carried out under conditions of “physiological” hypoxia (5% O ₂ ).

Table 1 presents quantitative data on the content of HSPCs related to early, middle and late undifferentiated precursors.

As can be seen from table 1, less than half of the suspension CD45 ⁺ cells carried antigens of primitive HSPCs. Among them were about 20% of early (CD34 ^- / CD133 ⁺ ) and 30% of medium (CD34 ⁺ / CD133 ⁺ ) predecessors. Almost all CD45 ⁺ HSPCs associated with MSCs were represented by primitive hematopoietic precursors with the phenotype of medium precursors CD34 ⁺ / CD133 ⁺ (more than 90%). In both populations, there was practically no late CD34 ⁺ / CD133 ^- HSPK.

The effectiveness of the proposed method, allowing the cultivation of HSPC, promotes the proliferation of hematopoietic precursors with the phenotype CD34 ⁺ / CD133 ⁺ without their further differentiation in a short time (7 days).

In our work, among the CD34 ⁺ / CD133 ⁺ HSPCs associated with MSCs, the majority were represented by COOB, which was confirmed by microscopic analysis (Figure 3), which revealed COOB under MSCs, as well as flow cytofluorimetry data on the presence of large CD34 ⁺ / CD133 ⁺ cells in the general population of MSC-associated HSPCs. We found that HSPCs in co-culture with zhMSCs occupy the same compartments with respect to the stromal layer as in the case of bone marrow-derived MSCs.

Since the time in the co-culture for which we obtained these cells was 168 hours (7 days), it can be assumed that in the cohort hierarchy the described cells belong to cohob-7, capable of committing to multi- and bipotent CFUs: CFU-HEMM, CFU GM, PFU-E in vitro, as well as CFU-C in vivo [de Haan and van Zant, 1997].

Thus, we have proposed an effective method for obtaining and increasing the number of undifferentiated HSPCs with the complex phenotype CD34 ⁺ / CD133 ⁺ associated with the stromal sublayer of

multipotent stromal cells (MSCs) of adipose tissue, while the population of the resulting cells is more than 90% represented by hematopoietic progenitors with the CD34 ⁺ / CD133 ⁺ phenotype and with the formation of cells forming cobblestone regions (COOB) with the phenotype of primitive predecessors.

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Claims (1)

A method for producing a population of undifferentiated hematopoietic umbilical cord blood precursors (HSPCs) comprising HSPCs with the CD34 ⁺ / CD133 ⁺ phenotype associated with the stromal sublayer from multipotent stromal adipose tissue cells (MSCs), comprising preparing a stromal sublayer from MSCs from stromal-vascular tissue fat constantly cultivating at a concentration of O ₂ in a medium of 5%, the addition of a mononuclear fraction of umbilical cord blood (MNC PK) to the MSC monolayer, their joint cultivation for 72 hours with further selection from MNC P To undifferentiated hematopoietic precursors due to adhesion to MSCs at 20% O ₂ , continued cultivation of MSCs with PC MNCs attached to them for 96 hours at a concentration of O ₂ in a 20% medium to form a HSPC population including HSPCs with the CD34 ⁺ / CD133 ⁺ phenotype associated with the stromal sublayer from MSCs of adipose tissue.

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