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WO2024200276A1 - Cellules souches mésenchymateuses inactivées par la chaleur et leurs utilisations - Google Patents

Cellules souches mésenchymateuses inactivées par la chaleur et leurs utilisations Download PDF

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WO2024200276A1
WO2024200276A1 PCT/EP2024/057786 EP2024057786W WO2024200276A1 WO 2024200276 A1 WO2024200276 A1 WO 2024200276A1 EP 2024057786 W EP2024057786 W EP 2024057786W WO 2024200276 A1 WO2024200276 A1 WO 2024200276A1
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msc
hsc
cells
hscs
culture
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Hermanus Johannes Marco Eijken
Anne Louise Schacht REVENFELD
Bjarne Kuno MØLLER
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Aarhus Universitet
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Aarhus Universitet
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1352—Mesenchymal stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources

Definitions

  • the present invention relates to a process for expanding stem cells.
  • the present invention relates to heat-inactivated mesenchymal stem cells (HI- MSC) for use as feeder cells in a process of expanding hematopoietic stem cells (HSCs).
  • HI- MSC heat-inactivated mesenchymal stem cells
  • HSCs hematopoietic stem cells
  • HSCs Hematopoietic stem cells
  • HSCs are a type of stem cell that can differentiate into all the different types of blood cells in the body. They are responsible for maintaining the constant supply of new blood cells that the body needs to function properly. HSCs are continuously dividing in the body in order to produce new blood cells, which makes them a good candidate for gene editing. However, HSCs can also enter a state of dormancy in which they are not actively dividing. In order to ensure that gene editing is successful, it is important to expand HSCs in the laboratory in a way that promotes their proliferation, or active division. Sternness refers to the ability of a stem cell to maintain its undifferentiated state and to give rise to different types of cells. It is an important property of stem cells because it allows them to continuously regenerate and repair tissues throughout an individual's lifetime.
  • HSCs During HSC expansion, it is possible for HSCs to lose their sternness, or their ability to maintain their undifferentiated state and to give rise to different types of cells. This can occur for a variety of reasons, including the culture conditions used to expand the HSCs, the presence of certain signaling pathways or growth factors, or the accumulation of genetic mutations.
  • Loss of sternness can be a problem during HSC expansion because it can lead to the differentiation of HSCs into more specialized cell types, which reduces their ability to regenerate and repair tissues. It can also limit the potential clinical applications of the expanded HSCs, such as in bone marrow transplantation or gene editing.
  • HSCs are infused into a patient to replace their own damaged or diseased bone marrow. Expanding HSCs in the laboratory allows doctors to obtain a larger number of HSCs for transplantation, which can be especially useful in cases where the patient's own bone marrow is not able to produce enough healthy HSCs.
  • CRISPR-Cas9 Another potential use of expanded HSCs is in gene editing.
  • Gene editing is a technique that allows scientists to make precise changes to an individual's genetic material in order to correct genetic defects or to introduce new traits.
  • One of the most commonly used gene editing techniques is called CRISPR-Cas9, which uses a special enzyme called Cas9 to cut the DNA at a specific location, allowing researchers to delete, insert, or replace a specific sequence of DNA.
  • the gene edited cells can then be used to study the effects of gene editing in cell culture or animal models, or potentially to treat genetic diseases in humans.
  • the cells being edited In order for gene editing to be successful, the cells being edited must be actively dividing. This is because the changes made by the CRISPR-Cas9 system are incorporated into the genome of the cell during DNA replication, which occurs during cell division. If the cells are not dividing, the gene editing will not be successful.
  • HSCs mesenchymal stromal/stem cells
  • MSCs mesenchymal stromal/stem cells
  • A) Growth medium is often optimized for one specific cell type to keep it in the right phenotype and to support proper cell growth. Finding a growth medium meeting the requirement of all involved cell types involves substantial optimization and is often impossible resulting in comprised conditions for one of the cell types.
  • MSCs are adherent cells and must be pre-cultured to allow for cell attachment in the culture dish prior to the start of the co-culture. During the co-culture, MSCs will continue to proliferate which makes it very challenging to standardize the amount of MSCs in the co-culture. Moreover, as MSCs keep on proliferating during the entire culture period they can reach an amount that will be too high to support a proper cell culture.
  • the supporting cells need to be isolated and in vitro expanded. All regulatory requirements need to be fulfilled for the supporting cells on top of the cells of interest.
  • MSC feeder layers have been used to support HSC expansion (3, 4).
  • a feeder layer basically consists of adherent cells that are unable to divide but still maintain the ability to secrete factors that stimulate proliferation. By irradiation or mitomycin C treatment, the proliferation capacity of MSC can be limited while secretion of essential factors is kept intact.
  • the use of feeder cells eliminates the disadvantages of cell proliferation in co-cultures, the setup is still very challenging regarding the other points addressed above.
  • HI-MSCs prepared for other applications are normally used directly after heat inactivation.
  • the inventors have previously found that HI-MSC can be stored, for at least two days, in a refrigerator and used directly therefrom (stored HI-MSC).
  • Such cells are specifically identified in PCT Application No. PCT/EP2022/076148, published as WO 2023/046709.
  • an improved expansion process of HSCs would be advantageous, and in particular a process that provides an expanded HSC population in the correct growth medium with less contamination, at a lower cost, and in a shorter time would be advantageous.
  • an object of the present invention relates to the optimization of HSC expansion protocols.
  • An improved expansion process of HSCs would be advantageous, and in particular a process that provides an expanded HSC population in the correct growth medium with less contamination, at a lower cost, and in a shorter time would be advantageous.
  • the present invention has surprisingly solved these problems by the use of heat- inactivated MSCs, which in contrast to the known inactivated cells, are metabolically inactive, and were found to be able to support expansion of HSCs in diverse setups, such as from freely-diffusible co-cultures to pre-conditioned media with no transfer of intact HI-MSC cells.
  • the present disclosure relates to a process of expanding hematopoietic stem cells (HSCs), the process comprising the steps: a) providing heat-inactivated mesenchymal stem cells (HI-MSC cells), or a HI-MSC pre-conditioned HSC growth medium, wherein the pre-conditioned medium has been contacted with at least one HI- MSC cell for a time sufficient to condition the media, such as for at least 24 hours, prior to the removal of the HI-MSC cells; b) providing an HSC culture comprising HSCs and HSC growth medium; and c) contacting the HSC growth medium from step b) with the HI-MSC cells or pre-conditioned growth medium from step a) for a time sufficient to expand the HSCs.
  • HSCs hematopoietic stem cells
  • the present disclosure relates to a process of providing a pre-conditioned media as described in the first aspect, the process comprising the steps: a) providing at least one HI-MSC cell; b) contacting a media with the at least one HI-MSC cell for a time sufficient to condition the media, such as for at least 24 hours; c) removing the HI-MSC from the media, thereby providing a preconditioned media; and d) optionally diluting or concentrating the provided pre-conditioned media.
  • the present disclosure relates to a process of providing a pre-conditioned media, the process comprising the steps: a) providing at least one HI-MSC cell; b) contacting a media with the at least one HI-MSC cell for a time sufficient to condition the media, such as for at least 24 hours; c) removing the HI-MSC from the media, thereby providing a preconditioned media; and d) optionally diluting or concentrating the provided pre-conditioned media.
  • the present disclosure relates to a pre-conditioned media obtained by or obtainable by the process as described above.
  • the present disclosure relates to a container comprising the pre-conditioned media.
  • the present disclosure relates to a use of the pre-conditioned media, to expand a population of HSCs.
  • the present disclosure relates to a use of HI-MSCs, such as stored HI-MSCs, as cells for pre-conditioning HSC growth media.
  • the present disclosure relates to a kit comprising the preconditioned media as described herein, and optionally comprising instructions for expanding HSCs.
  • the present disclosure relates to a use of HI-MSCs, such as stored HI-MSCs, as feeder cells for expanding HSCs.
  • the present disclosure relates to a HSC population of cells obtained by or obtainable by the process as described herein.
  • the present disclosure relates to a container comprising the HSC population of cells as described herein.
  • the present disclosure relates to a kit comprising HI-MSC, and a HSC media, and optionally comprising instructions for expanding HSCs.
  • the present disclosure relates to the HSC population of cells as described herein, for use as a medicament.
  • the present disclosure relates to the HSC population of cells as described herein, for use in the treatment or alleviation of, a blood disorder, a cancer, such as a liquid cancer, such as leukemia, or an immune system disease.
  • the present disclosure relates to a method of treating or alleviating a blood disorder, a cancer, such as a liquid cancer, such as leukemia, or an immune system disease in a patient in need thereof, the method comprising expanding HSCs as described above, and administering the expanded HSCs to the patient in need thereof.
  • Figure 1 shows staining pattern of 7AAD changes after heat-inactivation.
  • Figure 2 shows the concentration of stored HI-MSC remains stable.
  • the concentration of HI-MSC was determined using quantitative flow cytometry.
  • the intact cells were identified based on forward scatter (FSC) and side scatter (SSC) properties, excluding only cellular debris.
  • FSC forward scatter
  • SSC side scatter
  • Figure 3 shows HI-MSC induce dose-dependent increase of proliferation of CD34+ HSC in culture.
  • the total amount of viable CD34+ cells after 6 days of culture in HSC monoculture was compared to the number of viable CD34+ HSC in co-cultures with HI-MSC at different concentrations.
  • N 4.
  • Figure 5 shows HI-MSC support dose-dependent generation of CD34+ HSC with a relevant stem cell phenotype.
  • the total number of CD34+ cells with associated stem cell marker (CD90, CD38, CD133, CD201) was determined by flow cytometric analyses at baseline (DO) and after 6 days of culture. Mean ⁇ SD.
  • N 4.
  • Figure 6 shows more pronounced HSC over HI-MSC donor effect on the expansion of CD34+ HSC in co-cultures.
  • CD34+ HSC from four different donors were each co-cultured with four HI-MSC batches for 6 days.
  • Figure 7 A and B shows HSC donor variation has larger impact on amount of relevant CD34+ HSC than HI-MSC donor variation.
  • the stem cell phenotype of cultured CD34+ HSC was determined by flow cytometry.
  • Figure 8 shows HI-MSC prevents drop in cellular viability of cultured CD34+ HSC.
  • the cellular viability was evaluated by 7AAD staining at baseline (DO), on D3 and D6 of culture.
  • the 7AAD- are shown for each of the time points for culturing of CD34+ HSC from four different stem cell donors, using the same four HI-MSC batches for each HSC donor.
  • N 4.
  • Figure 10 shows HI-MSC boosts viability of cultured CD34+ HSC.
  • the cellular viability was evaluated with 7AAD staining of HSC at baseline (DO) and after 6 days of culture. The percentage of 7AAD- cells are reported as the viability.
  • N l.
  • Figure 11 shows HI-MSC increases production of CD34+ HSC with relevant stem cell phenotype.
  • the absolute number of viable CD34+ HSC with an associated stem marker is provided for baseline and after 6 days of culture in the HSC monoculture and in the co-cultures.
  • N l.
  • Figure 12 shows HI-MSC facilitated expansion support of CD34+ HSC is contact - independent and can also be obtained with HI-MSC primed HSC medium.
  • Different CD34+ HSC monocultures were prepared and compared to co-cultures with HI- MSC.
  • HI-MSC primed HSC medium was incubated for 24h prior to culture setup, using the same HI-MSC concentration as the co-cultures.
  • Figure 13 shows HI-MSC and their derivatives support optimal CD34+ HSC viability.
  • Figure 14 shows HI-MSC and HI-MSC derivatives induce similar stem cell phenotype in cultured CD34+ HSC.
  • Figure 15 shows priming of HSC medium with different concentrations of HI-MSC yields similar effect, when used as HSC expansion support in identical final concentration.
  • HSC was primed with two concentrations of HI-MSC (60.000/mL and 2xlO' K 6/mL) for 24h. Both were used as expansion support for CD34+ HSC in a final concentration corresponding to 20.000 HI-MSC/mL.
  • Figure 16 shows expansion of CD34+ HSC alters both number and type of colonies in CFU assay. After 6 days of culture, the differentiation potential of expanded CD34+ HSC was evaluated in an in vitro CFU assay. After 14 days of differentiation, the cells were evaluated by flow cytometric analysis.
  • Figure 17 shows HI-MSC remain stable in culture, both as a monoculture and coculture with CD34+ HSC.
  • HI-MSC was diluted to a final concentration of 20.000 HI-MSC/mL in HSC medium and incubated for 6 days after which the concentration was determined by quantitative flow cytometry. Three independent experiments.
  • Figure 18 shows that a clinically relevant HSC medium supports CD34+ expansion in both mono- and co-culture setting.
  • the RUO medium, StemSpan SFEMII was replaced by the GMP compliant StemSpan AOF.
  • Figure 19 shows that the type of inactivation method affects plastic adherence and morphology of MSC.
  • OH inactivation
  • HI- MSC heat
  • irr. MSC irradiation
  • Figure 20 shows different viability stain patterns and apoptosis levels observed for MSC inactivated with heat and irradiation.
  • N l.
  • N l.
  • Figure 21 shows that metabolic activity is maintained after irradiation of MSC, but not after heat inactivation.
  • the metabolic activity was evaluated before inactivation of MSC and at different time points after inactivation with either irradiation or heat.
  • N l.
  • Figure 22 shows that freshly prepared and pre-frozen (-80 °C) HI-MSC primed medium provides similar CD34+ HSC expansion support.
  • Monocultures of CD34+ HSC with or without addition of primed medium from HI-MSC were prepared.
  • One version on the HI-MSC-primed medium was freshly prepared, while another had been stored for -80 °C for 9 days prior to usage.
  • a co-culture of CD34+ HSC and HI-MSC was also created. All cultures had duration of 6 days.
  • N l.
  • Figure 24 shows that concentration and 7AAD staining of long-term stored HI-MSC remain stable.
  • B) The level of 7AAD staining was determined at immediately after inactivation and following storage at 4 C° for 17 and 21 months. N l.
  • Figure 25 shows HI-MSC stored for 21 months preserve supportive capabilities on HSC expansion.
  • N l.
  • NSC Mesenchymal Stem Cells
  • MSC Mesenchymal Stem Cell
  • MSCs can be isolated from numerous tissues such as bone marrow, adipose tissue, the umbilical cord, liver, muscle, and lung. MSCs adhere to plastic when maintained under standard culture conditions. MSCs express CD73, CD90, and CD105, but under standard culture conditions lack expression of CD31, CD45, CDllb, and CD19 surface molecules.
  • Heat-inactivated cells Heat-inactivated- Mesenchymal Stem Cell
  • HI-MSC cells HI-MSC cells
  • HI-MSC HI-MSC
  • the heat treatment can for example be conducted as provided in example 1.
  • cellular integrity or “maintained their cellular integrity” is to be understood as i) the metabolically inactivated cells have a clear cellular structure when viewed under a light microscope (data not shown), ii) the metabolically inactivated cells stain positive for a nuclear staining such as 7-Aminoactinomycin D (7-AAD).
  • viability is a measure of the proportion of live, healthy cells within a population of cells. Viability can be measured in different ways.
  • 7AAD staining is applied, which is a standard measure of viability, in which viable cells are not stained, and thus 7AAD negative (7AAD-), due to an intact cell membrane.
  • metabolic inactivated refers to cells which are without mitotic and metabolic activity.
  • the metabolically inactivated MSCs are metabolically inactivated and cannot reduce MTT to formazan.
  • the metabolically inactivated MSCs are shown to be metabolically inactive, such as through the use of an MTT- or XTT assay, such as CyQUANT XTT Cell Viability assay (Thermo Fischer Scientific).
  • the metabolically inactivated MSCs cannot adhere to plastic. In a further embodiment, the metabolically inactivation of the MSCs is irreversible.
  • Hematopoietic stem cell “HSC”, “Hematopoietic stem cells” or”HSCs” is understood as the rare population of multipotent cells residing in the bone marrow and other hematopoietic tissues that have a unique ability to self-renew and differentiate into all blood cell lineages, including red blood cells, leukocytes, and platelets, throughout the lifespan of an organism. HSCs are essential for maintaining the homeostasis of the hematopoietic system and for replenishing the blood cell pool after injury or infection.
  • HSCs express a variety of differentiation markers that distinguish them from other hematopoietic cell types.
  • CD45, CD34, CD90, and CD133 are commonly used to identify and isolate HSCs, while HSCs with low or no CD38 expression may have enhanced self-renewal capacity and the ability to differentiate into multiple lineages. These cells are also thought to be more quiescent and resistant to chemotherapy.
  • CD201 also known as endothelial protein C receptor (EPCR) is a transmembrane glycoprotein that is expressed on the surface of various cell types, including endothelial cells, hematopoietic cells, and some stem cells. As provided by the examples, CD201 may be induced by the inclusion of UM171 in the HSC growth medium. The expression of CD201 is currently investigated in HSCs.
  • EPCR endothelial protein C receptor
  • Pre-conditioned medium or “primed medium” are used interchangeably herein.
  • balanced salt solution is a solution made to a physiological pH and isotonic salt concentration. Solutions most commonly include sodium, potassium, calcium, magnesium, and chloride. Examples of balanced salt solutions which may find use with the present invention are:
  • EBSS Earle's balanced salt solution
  • HBSS Hanks' balanced salt solution
  • PBS Phosphate buffered saline
  • RBSS Ringer's balanced salt solution
  • SBSS Simm's balanced salt solution
  • TRIS-buffered saline TBS
  • Ringer Preferably: Ringer, Isotonic saline, Albumin supplemented sodium chloride composition.
  • Additives may include antibiotics, vasoconstrictors, growth factor, salt, sugars, or other stimulants.
  • the present disclosure relates to a process of expanding hematopoietic stem cells (HSCs), a process of providing a pre-conditioned media, and the pre-conditioned media obtainable by thereby, uses of HI-MSCs and the pre-conditioned media and the expanded HSC population of cells, for use as a medicament.
  • HSCs hematopoietic stem cells
  • HSCs hematopoietic stem cells
  • the inventors have surprisingly realized that the use of HI-MSCs, were found to be able to support expansion of HSCs in diverse setups.
  • the HSCs can either be expanded by
  • HI-MSCs • by adding HI-MSCs into a container having separate chambers, a first chamber comprising HSC growth medium and the HSCs to be expanded and a second chamber comprising HSC growth medium and the HI-MSCs, the chambers being separated from each other by a membrane having pores allowing for a non-cellular diffusion between the two chambers,
  • the present disclosure relates to a process of expanding hematopoietic stem cells (HSCs), the process comprising the steps: a) providing heat-inactivated mesenchymal stem cells (HI-MSC cells), or a HI-MSC pre-conditioned HSC growth medium, wherein the preconditioned medium has been contacted with at least one HI-MSC cell for a time sufficient to condition the media, such as for at least 24 hours, prior to the removal of the HI-MSC cells; b) providing an HSC culture comprising HSCs and HSC growth medium; and c) contacting the HSC growth medium from step b) with the HI-MSC cells or pre-conditioned growth medium from step a) for a time sufficient to expand the HSCs.
  • HSCs hematopoietic stem cells
  • the time sufficient to expand the HSCs is at least 1 day, such as at least 2 days, such as at least 3 days, such as at least 4 days, such as at least 5 days, such as at least 6 days.
  • the present invention can be performed in several different variations.
  • a variant is to conduct a "classical" co-culture system where the cells are only in fluid connection, and thus cannot directly contact each other.
  • the HI-MSC cells and the HSCs are placed in separate chambers in fluid connection, such as a transwell co-culture, such as a non-contacting co-culture.
  • the fluid connect is often established by the use of specific filters, that allows for a free dissociation of particles under a specific size, as shown by the examples the present inventors have conducted their experiments using 0,4 pm separation, however the skilled person will be able to select other filters, with the same expected result.
  • the chambers are separated with a filter, such as a filter having a pore size in the range 0,2 pm to 2 pm, such as in the range 0,4 pm to 1 pm.
  • Another variation to the setup can be to place the cells in direct contact.
  • the HI-MSC cells are placed in spatial contact with the HSCs.
  • the examples in the present application are conducted with 12-well plates, however, especially when the process is introduced into a GMP compliant environment, where the output is for use in a clinical setting, the working volumes will be significantly larger.
  • the skilled person can use different measures to arrive at the number of cells to be used, and will in general scale- up the process in accordance to the volume or the surface used. Since the cells are in suspension, a volumetric measure may be preferred, and thus in a preferred embodiment, 20.000 HI-MSC/mL are provided.
  • HI-MSC/mL between 10.000-500.000 HI-MSC/mL are provided such as providing 30.000 HI-MSC/mL, such as 50.000 HI-MSC/mL, such as 80.000 HI- MSC/mL, such as 100.000 HI-MSC/mL, such as 200.000, such as even providing 400.000 HI-MSC/mL.
  • a ratio is selected corresponding to a ratio of 0.4: 1 - 10: 1 (HI-MSC:HSC).
  • At least 1000 HI-MSC cells are provided, such as at least 5000, such as at least 10,000, such as at least 20,000 HI-MSC cells are provided, such as even up to 6*10 6 HI-MSCs, such as even 12*10 6 HI-MSC cells are provided. In one embodiment of the present disclosure, between 40.000/cm 2 and 70.000/cm 2 HI-MSC cells are provided, such as 50.000/cm 2 , such as even 60.000/cm 2 HI-MSC cells are provided, preferably 60.000/cm 2 HI-MSC cells are provided. In one embodiment of the present disclosure, at least 1000 HSCs are provided, such as at least 5000, such as at least 10,000, such as at least 20,000, such as at least 30,000, such as at least 40,000, such as at least 50,000 HSCs are provided.
  • a good measure on the amount of HI-MSCs that should be provided is the relative amount of HI-MSCs in relation to HSCs.
  • the process functions over a wide range of ratios, since as low as 4000 HI-MSC in relation to 50000 HSCs provide a sufficient expansion, and even up to 100000 HI-MSC in relation to 50000 HSCs also provide expansion.
  • at least 5% HI-MSC cells are provided in relation to the amount of HSCs provided in step b, such as at least 8%, such as at least 40%, such as at least 100%, such as up to 200% HI-MSC cells provided in relation to the amount of HSCs provided in step b.
  • HI-MSC may be prepared by various methods known to the skilled person. Freshly harvested MSC were centrifuged at 440 x g for 5 min. and resuspended in MEM-a to reach a concentration of 3-5x10 ⁇ 6 cells/mL. The cell suspension was added to sterile 1,5 mL tubes (max 1 mL in each) and heat-inactivated at 50 °C for 35 min. Heat inactivation was stopped by cooling the tubes in ice bath for 5 min. Cell concentration and viability were evaluated, using flow cytometry.
  • Each HI-MSC batch was stored at 4 °C and used at day 3-4 post-inactivation, in StemSpan SFEM II (STEMCELL Technologies) or saline with 2-5% human serum albumin (HSA) at a final concentration of approximately 5x10 ⁇ 6 HI-MSC/ mL. Prior to use in co-cultures, the concentration and degree of 7AAD staining was determined for each HI-MSC batch, using flow cytometry.
  • MSCs can be inactivated by different means of heat treatment.
  • the cells may be inactivated by heating to a temperature in the range 40-75°C, such as 40-60°C, preferably at 45-55°C, more preferably at about 50°C, for a period from 5 minutes to 2 hours, 10 minutes to 1 hour, preferably such as 15-45 minutes, more preferably for a period of about 30 minutes or such as at least 10 minutes at 45-75°C.
  • the heat inactivation may be stopped by cooling the tubes in ice bath, such as for around 2-10 minutes, preferably around 5 minutes.
  • the MSCs can be provided from different tissues.
  • the MSCs are selected from the group consisting of adipose derived MSCs, human umbilical cord MSCs, bone marrow derived MSCs, dental pulp MSC and induced pluripotent mesenchymal stem cells, preferably the MSCs are adipose derived MSCs.
  • the MSCs are adipose derived or bone marrow derived.
  • the invention is applicable to both these types of MSCs, showing that the method is generally applicable to MSCs.
  • the adipose derived MSCs are derived from a stromal vascular fraction (SVF).
  • SVF stromal vascular fraction
  • the MSCs may be modified in different ways before being inactivated.
  • the MSCs are primed and/or pretreated MSCs and/or genetically modified MSCs.
  • priming or pre-treatment are i) incubation with cytokines, interleukins, growth factors such as VEGF or other secreted factors such as damage-associated molecular patterns (DAMPs), ii) pharmacological or chemical agents, iii) exposure to hypoxic conditions, iv) exposure to other cells types such as injured endothelial cells v) expansion of MSCs in 3D conditions.
  • the priming/pretreatment is selected from the group consisting of incubation with factors such as TNFalpha, INFgamma, ILlbeta, and damage-associated molecular patterns (DAMPs), incubation with pharmacological or chemical agents, exposure to hypoxic conditions, exposure to other cells types such as injured endothelial cells, and expansion of MSCs in 3D conditions.
  • factors such as TNFalpha, INFgamma, ILlbeta, and damage-associated molecular patterns (DAMPs)
  • DAMPs damage-associated molecular patterns
  • the MSCs are derived from a mammal, and preferably a human being.
  • the MSCs are expanded (such as 1-8 passages) or nonexpanded MSCs.
  • the MSCs are thawed cryopreserved mesenchymal stem cells or freshly harvested mesenchymal stem cells. Both options work equally well.
  • the MSCs are inactivated in a liquid, such as saline or a balanced salt solution (BSS), such as PBS.
  • BSS are considered pharmaceutical acceptable mediums.
  • the media comprises carrier protein such as human serum albumin (HSA), such as 0.5%-20% HSA, such as 0.5%-10% HSA, such as 0.5% to 5% HSA, such as 1-3% HSA by weight.
  • HSA human serum albumin
  • HSCs can be derived from multiple different sources, and the process as described herein will be able to expand the HSCs, no matter the source of origin.
  • the source providing the highest amount of HSCs for expansion is where HSCs are chemically mobilized from bone marrow and released into the peripheral blood.
  • HSCs are present in the peripheral blood, the cells are subsequently obtained therefrom.
  • Such a mobilization can for instance be done by the use of Plerixafor.
  • the HSCs are derived from umbilical cord and/or derived from bone marrow, preferably from bone marrow, even more preferably HSCs mobilized from bone marrow and subsequently obtained from peripheral blood.
  • the HI-MSCs can be derived from multiple different sources.
  • the HI-MSCs are adipose derived, bone marrow derived, and/or umbilical-cord derived.
  • the adipose derived MSCs are derived from a stromal vascular fraction (SVF).
  • HSCs As presented in the background, expansion of HSCs are needed for multiple different purposes, and thus in some instances, other up-stream and/or downstream processes (such as expansion/gene-editing/enrichment) are performed prior to and/or after contact with the HI-MSCs. In other embodiments, the HSCs are manipulated further after they have been expanded. In one embodiment of the present disclosure, the HSCs have been genetically modified prior to, during, and/or after expansion, such as by the use of CRISPR-Cas9.
  • the expanded HSCs are to be used in bone marrow transplantation.
  • the expanded HSCs are to be used in gene editing, such as CRISPR-Cas9.
  • the gene edited cells can then be used to study the effects of gene editing in cell culture or animal models, or to treat genetic diseases in humans.
  • the MSCs are derived from a mammal, and preferably a human being.
  • the mammals may also be commercially relevant mammals, such as cattle, pigs, horses, sheep, goats, mink, ferrets, hamsters, cats and dogs, as well as birds.
  • the MSCs themselves may also have been manipulated in different ways, before or after their inactivation.
  • the MSCs are expanded MSCs.
  • the examples, in particular example 2 and example 7, furthermore show the surprising stability of the HI-MSCs, and thus show the cells can be stored at different temperatures.
  • the HI-MSC cells are provided directly after heat inactivation, or the HI-MSC cells have been stored at e.g. below 10°C such as below 5°C, such as in the range 10°C-0.1°C, between -24°C and 5°C, or have been stored at -80°C, such as having been cryogenically stored.
  • the HI-MSC cells have been stored at 4 °C and used 2 days post in-activation such as 3-4 days post-inactivation, such as even weeks or months post inactivation, such as even after 1 month post inactivation, 2 months post inactivation, 3 months post inactivation, such as even 4 months post inactivation, such as the cells have been stored for at least 1 month post inactivation, at least 2 months, at least 3 months, such as the cells have even been stored for at least 4 months post inactivation.
  • 2 days post in-activation such as 3-4 days post-inactivation, such as even weeks or months post inactivation, such as even after 1 month post inactivation, 2 months post inactivation, 3 months post inactivation, such as even 4 months post inactivation, such as the cells have been stored for at least 1 month post inactivation, at least 2 months, at least 3 months, such as the cells have even been stored for at least 4 months post inactivation.
  • HI-MSCs A particular advantage of using HI-MSCs is that the risk of injecting contaminating compounds is reduced.
  • the HI-MSC and/or the HSC medium is free from chemical cell inactivation agents, such as mitomycin C.
  • HI-MSCs Another advantage of using HI-MSCs is that a precise feeder: responder cell ratio can be employed. Since HI-MSC remain intact in culture, this ratio can be easily adjusted over time as well, when HSC expand. This provides the ability to have a more standardized cell-to-cell ratio than with other types of feeder cells.
  • the HI-MSCs are not able to actively secret compounds to their surrounding medium, and the HI-MSCS are for instance metabolically inactive. In one embodiment of the present disclosure, the HI-MSC cells are metabolically inactivated.
  • the HI-MSC can maintain their cellular integrity for sustained periods of time.
  • the provided HI-MSCs in step a) has maintained their cellular integrity, such as maintained their cellular integrity
  • At least 2 days such as at least 15 days, such as at least 30 days or such as at least 60 days, such as at least 4 months, when stored in a temperature range between 0.1 and 10°C, such as 1-8°C, such as 2-6°C, such as 3-5°C, or such as around 4°C; and/or for at least 2 days, such as at least 7 days, such as at least 15 days, such as at least 30 days, such as at least 60 days, when stored in a temperature range 11-28°C, such as 15-28°C, or such as 18-25°C.
  • an upper limit to the storage has currently not been identified.
  • cells can be stored for at least 17 or even 21 months, and maintain their cellular integrity as well as their function as feeder cells when stored in a temperature range between 0.1 and 10°C, such as 1-8°C, such as 2-6°C, such as 3-5°C, or such as around 4°C.
  • cells can even be stored for years, such as 1 years post inactivation, such as even 2 years post inactivation.
  • the cells are not stored for more than 3 years, such as not more than 4 years, such as not more than 5 years.
  • HSC growth media preferably one or more of the components rhSCF, rhTPO, rhFLT3L, rhIL-6, UM171, are used in the HSC growth media, to expand the HSCs.
  • UM171 can be left out of the HSC growth medium when HI-MSC are applied to expand HSCs.
  • HI-MSC thus supports similar expansion fold and viability of cultured CD34+ HSC both in the presence and absence of UM171 in the HSC growth medium.
  • the expression of CD201 was completely absent, when UM171 was removed from the HSC medium.
  • a slightly different stem cell phenotype arises, when UM171 is not applied in coculture, yet substantial amounts of CD34+ HSC with a relevant phenotype is still produced.
  • all the components rhSCF, rhTPO, rhFLT3L, rhIL-6, and UM171 are added to the HSC growth medium, whilst in another embodiment UM171 is not used, thus only rhSCF, rhTPO, rhFLT3L, and rhIL-6 are added to the HSC growth medium.
  • Antibiotics are most often also applied in the process, and preferred options may be streptomycin and/or penicillin, however the skilled person will know that the process is not influenced by the choice of antibiotics.
  • the HSC growth media may be StemSpan SFEM II or StemSpan AOF.
  • the HSC growth media comprises or consist of a HSC growth media supplemented with one or more of the following rhSCF, rhTPO, rhFLT3L, rhIL-6, UM171, streptomycin or penicillin, such as a HSC growth media selected from StemSpan SFEM II and StemSpan AOF.
  • the HSCs can be expanded by supplementing their growth medium with a "pre-conditioned" medium.
  • a pre-conditioned medium is also provided, as well as its different uses.
  • the process may either provide the pre-conditioned media to be used in the first aspect of the disclosure, or the process may provide the preconditioned medium for any further use.
  • the present disclosure relates to a process of providing a pre-conditioned media as described in the first aspect, the process comprising the steps: a) providing at least one HI-MSC cell; b) contacting a media with the at least one HI-MSC cell for a time sufficient to condition the media, such as for at least 24 hours; c) removing the HI-MSC from the media, thereby providing a preconditioned media; and d) optionally diluting or concentrating the provided pre-conditioned media.
  • the present disclosure relates to a process of providing a pre-conditioned media, the process comprising the steps: a) providing at least one HI-MSC cell; b) contacting a media with the at least one HI-MSC cell for a time sufficient to condition the media, such as for at least 24 hours; c) removing the HI-MSC from the media, thereby providing a preconditioned media; and d) optionally diluting or concentrating the provided pre-conditioned media.
  • the media is selected from
  • a growth media such as a HSC growth media
  • a liquid cell medium such as a balanced salt solution (BSS), such as PBS, preferably isotonic saline and/or a pharmaceutical acceptable composition.
  • BSS balanced salt solution
  • the HSC growth media is described in more detail above, such as StemSpan SFEM II or StemSpan AOF, as well as the different components the skilled person may add.
  • the HSC growth media is supplemented with one or more of the following rhSCF, rhTPO, rhFLT3L, rhIL-6, UM171, streptomycin or penicillin, such as a HSC growth media selected from StemSpan SFEM II and StemSpan AOF.
  • the preconditioned media is prepared in advance, such as for bulk storage or when offered for sale it may be advantageous to leave out the additional components, and only add one or more of the following rhSCF, rhTPO, rhFLT3L, rhIL-6, UM171, streptomycin or penicillin, when the pre-conditioned add the additional components.
  • the skilled person may find it advantageous to leave out these components entirely, since the pre-conditioned media might be added in minute amounts, i.e.
  • the HSC growth medium provided in step b will comprise the correct amounts of rhSCF, rhTPO, rhFLT3L, rhIL-6, UM171, streptomycin and/or penicillin.
  • the pre-conditioned medium can be prepared using various amounts of HI-MSC.
  • at least 60.000 HI-MSC/mL medium is added, such as up to 2x10 ⁇ 6 HI-MSC/mL medium, preferably 2x10 ⁇ 6 HI- MSC/mL medium.
  • the present disclosure relates to a pre-conditioned media obtained by or obtainable by the process as described above.
  • the present disclosure relates to a container comprising the pre-conditioned media as described above.
  • a pre-conditioned medium prepared as described above can be stored at -80 °C prior to usage. As such, batches may easily be prepared and stored for long periods of time. The skilled person would not expect any further degradation to occur by prolonged periods of -80 °C storage.
  • the processes for expanding HSCs and obtaining a HI-MSC pre-conditioned medium have now been introduced.
  • the present disclosure relates to several further aspects, including but not limited to different uses of HI-MSCs, expanded HSCs, and pre-conditioned media.
  • the present disclosure relates to a use of HI-MSCs, such as stored HI-MSCs, as feeder cells for expanding HSCs.
  • the present disclosure relates to a use of HI-MSCs, such as stored HI-MSCs, as cells for pre-conditioning HSC growth media.
  • the present disclosure relates to a use of the pre-conditioned media as described above, to expand a population of HSCs.
  • the present disclosure relates to a HSC population of cells obtained by or obtainable by the process as described herein.
  • the HSC cells provided herein are positive for the CD45, and CD34 markers. Further additional markers will also often be present. As presented by the examples, in particular example 5, the inclusion of UM171 in the HSC growth medium results in a cell that is positive for the CD201 marker, and resultantly the exclusion resulting in a lack of CD201 expression. Thus, in one embodiment of the present disclosure, the cells are positive for the markers CD45, CD34 and
  • the cells are positive for the markers CD45, CD34, and positive for at least one of the markers selected from the group CD90, and CD133, and/or negative for CD38 and CD201.
  • the viability of CD34+ HSC is markedly higher in HI-MSC co-cultures compared to HSC monocultures after 6 days of culturing.
  • the viability on DO, D3, and D6 of the culture it was possible to identify that HI-MSC seemingly reduces a drop in CD34+ HSC viability, as measured in D3 ( Figure 8).
  • the HSC population of cells has an increased viability, when compared to a HSC monoculture.
  • the HSC population of cells may be comprised in a contained, thus in another aspect, the present disclosure relates to a container comprising the HSC population of cells as described herein.
  • Kits may also be provided comprising the components to conduct the processes as described herein, thus in another aspect, the present disclosure relates to a kit comprising HI-MSC, and a HSC media, and optionally comprising instructions for expanding HSCs.
  • the pre-conditioned media may also be provided in a kit, thus in another aspect, the present disclosure relates to a kit comprising the preconditioned media as described above, and optionally comprising instructions for expanding HSCs.
  • the present disclosure also provides the uses of these cells as medicaments and/or treatments of various diseases.
  • the present disclosure relates to the HSC population of cells as described herein, for use as a medicament.
  • a related aspect is thus, the HSC population of cells as described herein, for use in the treatment or alleviation of, a blood disorder, a cancer, such as a liquid cancer, such as leukaemia, or an immune system disease.
  • a likewise related aspect is the method of treatment of such disease.
  • the present disclosure also relates to a method of treating or alleviating a blood disorder, a cancer, such as a liquid cancer, such as leukaemia, or an immune system disease in a patient in need thereof, the method comprising expanding HSCs as described above, and administering the expanded HSCs to the patient in need thereof.
  • the leukemia is a leukemia selected from the group consisting of Multiple myeloma (MM), Myelodysplastic syndrome (MDS), Acute lymphoblastic leukemia (ALL), Acute myeloid leukemia (AML), Chronic lymphocytic leukemia (CLL), Chronic myeloid leukemia (CML), Hodgkin lymphoma, and Non-Hodgkin lymphoma (NHL), including: Diffuse large B-cell lymphoma (DLBCL), Follicular lymphoma, Mantle cell lymphoma, Burkitt lymphoma, and T-cell lymphoma.
  • MM Multiple myeloma
  • MDS Myelodysplastic syndrome
  • ALL Acute lymphoblastic leukemia
  • AML Acute myeloid leukemia
  • CLL Chronic lymphocytic leukemia
  • CML Chronic myeloid leukemia
  • NHL Hodgkin lymphoma
  • NHL Non-Hodgkin lympho
  • the blood disorder is a blood disorder selected from the group consisting of sickle cell disease, thalassemia, aplastic anemia, and Fanconi anemia.
  • the immune system disease is an immune system disease selected from the group consisting of severe combined immunodeficiencies (SCID), chronic granulomatous disease (CGD), Wiskott-Aldrich syndrome (WAS), hemophagocytic lymphohistiocytosis (HLH), severe autoimmune diseases and selected inherited metabolic disorders.
  • SCID severe combined immunodeficiencies
  • CCD chronic granulomatous disease
  • WAS Wiskott-Aldrich syndrome
  • HSH hemophagocytic lymphohistiocytosis
  • HSC heat-inactivated mesenchymal stromal cells
  • the cell culture medium consisted of Minimum Essential Medium (aMEM) (Gibco-Thermo Fisher Scientific), 5% PLTGold Human Platelet Lysate (Mill Creek Life Sciences), 2 mM L-Glutamin (Gibco-Thermo Fisher Scientific) and 50 U/mL Penicillin-Streptomycin (Gibco- Thermo Fisher Scientific).
  • aMEM Minimum Essential Medium
  • PLTGold Human Platelet Lysate Mill Creek Life Sciences
  • 2 mM L-Glutamin Gibco-Thermo Fisher Scientific
  • 50 U/mL Penicillin-Streptomycin Gibco- Thermo Fisher Scientific
  • AD-MSCs at first, second, or third passage were harvested using TrypLETM Select (Gibco-Thermo Fischer Scientific) at 90% confluency and cryopreserved using CryoStor CS10 (Stemcell Technologies) at - 80°C.
  • MSC medium consisting of MEM- a, supplemented with L- Glutamine, 50 U/mL Penicillin-Streptomycin, and 5% PLTGold human platelet lysate. After centrifugation at 440xg for 5 min, cells were resuspended in MSC medium and seeded in T175 flasks, using approximately 1x10 ⁇ 6 cells/flask. The cells were cultured in a CO2-incubator at 37 °C and 5% CO2 until 80-90% confluence was reached.
  • Freshly harvested MSC were centrifuged at 440 x g for 5 min. and resuspended in MEM-a to reach a concentration of 3-5x10 ⁇ 6 cells/mL.
  • the cell suspension was added to sterile 1,5 mL tubes (max 1 mL in each) and heat-inactivated at 50 °C for 35 min. Heat inactivation was stopped by cooling the tubes in ice bath for 5 min. Cell concentration and viability were evaluated, using flow cytometry.
  • Each HI-MSC batch was stored at 4 °C and used at day 3-4 post-inactivation, in StemSpan SFEM II (STEMCELL Technologies) or saline with 25 human serum albumin (HSA) at a final concentration of approximately 5x10 ⁇ 6 HI-MSC/ mL. Prior to use in co-cultures, the concentration and degree of 7AAD staining was determined for each HI-MSC batch, using flow cytometry.
  • Live MSC or HI-MSC were stained with pre-diluted 7AAD (1:5 in PBS) for 5 min.
  • the acquisition of stained cells was performed on a NovoCyte 3000 (Agilent Technologies), using the NovoExpress Software (version 1.5.0, Agilent Technologies). See Materials and methods section in Example 2 for more details. Technical triplicates were measured and the mean value for the absolute number and the fraction of 7AAD+ and 7AAD- cells were used.
  • the level of 7AAD staining is normally used as a measure of cell viability and as such viable cells are not stained, and thus 7AAD negative (7AAD-), due to an intact cell membrane.
  • 7AAD- cells rapidly drops immediately after heat-inactivation and is further reduced 3-4 days post-heat inactivation ( Figure 1). This indicates that the HI-MSC are dead and have a permeable cell membrane.
  • Example 2 Dose-response, co-culture of HI-MSC and HSC
  • HSC hematopoietic stem cells
  • a standard leukapheresis procedure was applied at the Blood Bank of Aarhus University Hospital, Aarhus, Denmark. HSCs were mobilized through selfadministration of 960 pg G-CSF subcutaneously once daily for four days leading up to harvest day. Donor Medical Evaluation and leukapheresis harvest procedure was performed following the JACIE and WMDA standards. The final leukapheresis product was stored for up to 24h at 4 °C until use.
  • CD34+ HSCs were isolated from leukapheresis products by immunomagnetic separation.
  • the CD34+ enrichment was performed on the CliniMACS Prodigy® (Miltenyi Biotec), using the predefined "CD34 enrichment" standard procedure, according to the instructions by the manufacturer.
  • the purity, yield, and viability of the isolated cells were evaluated by flow cytometry.
  • the CD34+ HSC were resuspended in CryoStor CS10 (STEMCELL Technologies), and stored at -140 °C until further use. Dose-response co-culture of HI-MSC and CD34+ HSC
  • CD34+ HSC Cryopreserved CD34+ HSC were thawed in a 37 °C water bath and transferred to pre-warmed StemSpan SFEM II followed by centrifugation at 300xg for 5 min. The cells were resuspended in StemSpan SFEM II and the post-thaw absolute concentration of viable CD34+ HSC was determined by flow cytometry. 50.000 viable CD34+ HSC /mL were seeded in 12-well plates to a total volume of 1.5 mL in each well.
  • HSC medium consisting of StemSpan SFEM II supplemented with 30 ng/mL rhSCF (STEMCELL Technologies), 10 ng/mL rhTPO (STEMCELL Technologies), 10 ng/mL rhFLT3L (STEMCELL Technologies), 10 ng/mL rhIL-6 (STEMCELL Technologies), 35 nM UM171 (STEMCELL Technologies), and 20 mg/mL streptomycin and 20 U/mL penicillin.
  • Dose-response co-cultures were prepared by addition of varying concentrations of HI-MSC to the relevant wells. Cell cultures were maintained in a humidified CO2-incubator at 37 °C and 5% CO2 for 6 days after which the viability, CD34+ expansion fold, and stem cell phenotype of the cultured HSC were determined by flow cytometry.
  • CD34-PE (clone 581), CD38-BV605 (clone HB7), CD45-FITC (clone 2D1), CD90-PE-Cy (clone: 5E10), CD133-APC (clone: W6B3C1), Brilliant Stain Buffer. From Bio Legend: CD201-APC (Clone: RCR-401). 7AAD was used to identify non-viable cells.
  • HSC HSC were harvested, washed, and resuspended in PBS with 0.1% BSA.
  • the acquisition of stained cells was performed on a NovoCyte 3000 with a 3-laser configuration (405 nm, 488 nm, 637 nm) (Agilent Technologies), using the NovoExpress Software (versions 1.5.0-1.5.6, Agilent Technologies). Calibration of the cytometer was performed daily, using the NovoCyte QC particles (Agilent Technologies). Compensation was performed monthly, using the compensation beads (anti-Mouse Ig, K, cat: 51-90-9001229 and Negative Control, cat: 51-90- 9001291, BD Biosciences) and 2 uL of each relevant antibody. All gating strategies were based on fluorescence Minus One (FMO) controls.
  • FMO fluorescence Minus One
  • the gating strategy involved securing a stable acquisition flow in a time vs. forward scatter (FSC) plot, followed by removal of doublets in a FSC-A vs. FSC-H plot.
  • FSC forward scatter
  • the parent population of CD34+ HSC is viable CD45+ cells.
  • the expansion fold of the CD34+ HSC was determined, defined as the total number of viable CD34+ HSC present after culturing relative to starting number of cells.
  • the co-cultures displayed a dose-dependent increase in the expansion (Figure 3).
  • the induction or preservation of a relevant stem cell phenotype is also dose-dependent.
  • the dose of 20.000 HI-MSC/mL was chosen as the optimal dose, as only a subtle advantage was obtained using a 5x higher dose of HI-MSC.
  • HI-MSC can be stored for sustained periods, whilst preserving their expansion capabilities.
  • Example 2 • The optimal HI-MSC dose determined in Example 2 is applied.
  • the support of HI-MSC on HSC expansion depends to a higher degree on the HSC donor than on the HI-MSC donor variation.
  • Example 3 Identical to Example 3, with an additional sampling point on D3 of the culture.
  • HI-MSC has a protective- or rescue effect on cellular viability of cultured CD34+
  • HI-MSC can serve as a substitute for a small molecule, UM171, which is widely used for CD34+ HSC expansion ex vivo.
  • UM171 and cytokines are used in HSC monoculture and in a co-culture, and compared to a co-culture without UM171, but still with cytokines.
  • HI-MSCs are here stored in saline/2% HSA.
  • the sternness of the generated CD34+ HSC was evaluated by an associated stem cell phenotype. Comparing the co-cultures, the number of CD34+ CD133+ HSC was somewhat affected by the omission of UM171, yielding similar levels as the HSC monoculture ( Figure 11). Still, there was a 7,5x increase in the absolute number of CD34+ CD133+ HSC in the co-culture without UM171, compared to baseline (monoculture vs. baseline: 6x; co-culture + UM171 vs. baseline 12x). The expression of CD201 was completely absent, when UM171 was removed from the HSC medium. From several published studies, it has been shown that CD201 expression is induced by UM171, thus confirming the observations from the current study. Finally, the number of CD34+ CD90+ and CD34+ CD38- HSC produced in the co-cultures was slightly lower, when UM171 was omitted.
  • CD34+ CD90+ HSC 2x more CD34+ CD90+ HSC was produced compared to HSC monoculture and 132x more than at baseline. A slightly higher, yet similar trend was observed for the amount of CD34+ CD38- HSC.
  • HI-MSC supports similar expansion fold and viability of cultured CD34+ HSC both in the presence and absence of UM171 in the HSC medium.
  • a slightly different stem cell phenotype arises, when UM171 is not applied in co-culture, yet substantial amounts of CD34+ HSC with a relevant phenotype is still produced. This has the potential to pave the way for new clinically relevant expansion protocols for CD34+ HSC, which eliminates the need of UM171 for clinical use.
  • Example 6 Supportive effect of HI-MSC derivatives on HSC expansion
  • HI-MSC-primed HSC medium can exert the same effect as the HI-MSC.
  • Primed HSC medium is prepared with both new HI-MSC (3-4 days post-HI) and old (3-5 weeks post-HI), using HI-MSC from the same donor that have merely been heat-inactivated at different time-points. The priming is performed for 24h and the final concentration of the applied primed HSC medium corresponds to the same dose of HI-MSC used in the co-cultures (20.000 HI-MSC/mL).
  • HSC medium was prepared. Initially, the 7AAD staining and concentration of HI-MSCs were evaluated by flow cytometry. Subsequently, HI-MSCs were diluted to a concentration of 60.000 HI-MSC/mL, using StemSpan SFEM II, and transferred to a T25 culture flask. The HI-MSC suspension was stored in the CO2-incubator at 37°C and 5% CO2 for 24 hours. Next day, the cell suspension was centrifuged at 440 x g, and the supernatant was collected to be used in the culture setup as HI-MSC-primed HSC medium.
  • priming of the HSC medium was performed with 2x10 ⁇ 6 HI- MSC/mL, but otherwise as described above.
  • HSC monocultures Three different monocultures were prepared for each culture setup, all with the same CD34+ HSC seeding density and culture medium supplements as described in Example 2. One monoculture was prepared exactly as described in Example 2. A second monoculture was set up with StemSpan SFEMII medium, which was pre-incubated without supplements for 24h in a CO2 incubator prior to culture setup, to serve as a control for the third monoculture setup. This last CD34+ HSC monoculture setup was prepared with the HI-MSC primed HSC medium. Here, 0,5 mL of primed HSC medium was added to each relevant well in a 12-well plate.
  • the final volume in the well was 1,5 mL, thus diluting the primed HSC medium corresponding to a final concentration of 20.000 HI-MSC/mL, to match the concentration of HI-MSC used in the contact-dependent setup.
  • 15 uL of the concentrated, HI-MSC-primed HSC medium was used, again diluting the primed HSC medium corresponding to a final concentration of 20.000 HI-MSC/mL.
  • 50.000 viable CD34+ HSC /mL and 20.000 HI-MSC were used, to match the condition of the contact-dependent co-culture.
  • the insert was removed from the well, allowing harvest of the cultured CD34+ HSC.
  • CFU colony-forming unit
  • StemMACSTM HSC-CFU Assay Kit (Miltenyi Biotec) was applied. The assay was performed according to the manufacturer's instructions. Briefly, the total number of CD34+ cells was determined by flow cytometry, as described above, and the cell concentration was adjusted to 250 CD34+ cells/mL with StemSpan SFEM II. Cells were seeded in a 96 round-bottom well plate with an average of 2,5 cells/well and the assay medium was added. Plates were placed within a humidity chamber in a CO2- incubator (37 °C and 5% CO2).
  • Table 1 Surface marker evaluation for determination of colony types. The five different colony types are determined based on the marker distribution pattern indicated in the table.
  • the total amount of CD34+ HSC expressing relevant stem cell markers was also markedly higher in the cell cultures containing HI-MSC or HI-MSC derivatives as compared to HSC monocultures with no HI-MSC components ( Figure 14). Similar to the CD34+ expansion fold, the HI- MSC induced CD34+ stem cell phenotype does not require physical contact or even direct presence of HI-MSC.
  • HI-MSC primed medium As a culture supplement, a priming was set up, using 2x10 ⁇ 6 HI-MSC/mL in HSC medium. This more concentrated HI-MSC primed HSC medium was subsequently diluted in the CD34+ HSC culture, to match the use of a final concentration of 20.000 HI- MSC/mL. Compared to the HI-MSC primed HSC medium, which was primed with a smaller concentration of HI-MSC, a similar CD34+ expansion fold ( Figure 15 (A)) and CD34+ stem cell phenotype ( Figure 15 (B)) was produced.
  • CD34+ HSC The sternness of the CD34+ HSC was further evaluated by investigating the differentiation potential of the cultured CD34+ HSC, a functional in vitro colony forming unit (CFU) assay was performed.
  • This assay supports differentiation of the myeloid progenitor cells and not the lymphoid. Nevertheless, it estimates the presence of five different colony types; Burst-forming unit-erythroid (BFU-E), CFU-granulocyte (G), CFU-granulocyte macrophage (GM), CFU-macrophage (M), and CFU-granulocyte erythrocyte macrophage megakaryocyte (GEMM).
  • BFU-E Burst-forming unit-erythroid
  • G CFU-granulocyte
  • GM CFU-granulocyte macrophage
  • M CFU-macrophage
  • GEMM CFU-granulocyte erythrocyte macrophage megakaryocyte
  • HI-MSC and HI-MSC primed HSC medium supports higher CD34+ expansion fold and production of more CD34+ HSC with a relevant stem cell phenotype than HSC monocultures without HI-MSC components. Moreover, the HI-MSC induced effect in co-cultures is seemingly not contact-dependent.
  • the expanded CD34+ HSC preserve a differentiation potential, showing that functionally competent HSC are produced after expansion.
  • the HI-MSC primed HSC medium contains a vesicular component, which may contribute to the supportive effect of the primed HSC medium.
  • HI-MSC that have been stored for several weeks at 4 C° can be used for priming of HSC medium and support similar expansion and CD34+ phenotype as using freshly prepared HI-MSC for the medium priming.
  • HI-MSC or perhaps even primed medium/solution could be offered as a cell culture supplement.
  • Example 7 Stability of HI-MSC when used as expansion support
  • HI/mL was seeded in StemSpan SFEMII medium in a 12-well plate, using a final volume of 1,5 mL in each well.
  • the plate was placed in a humidified CO2- incubator at 37 °C and 5% CO2.
  • HI-MSC counts and 7AAD staining was evaluated after 6 days of incubation.
  • HI-MSC remain stable in cell culture medium for several days, both in HI-MSC monoculture and when used in co-culture with CD34+ HSC.
  • Example 8 Support of CD34+ expansion in mono- and co-culture in a clinically relevant growth medium
  • the basal growth medium is replaced in all steps with StemSpan AOF (STEMCELL Technologies). All cytokines were from PeproTech.
  • the expansion fold of the CD34+ HSC was determined, defined as the total number of viable CD34+ HSC present after culturing relative to starting number of cells.
  • the expansion fold of CD34+ cells in culture is highest in co-culture relative to the HSC monoculture ( Figure 18A). In the co-culture, approximately 50% more cells are obtained after 6 days of expansion, compared to the matching monoculture. The viability after 6 days was 95% and 97% in mono- and coculture, respectively (data not shown).
  • CD34+ cells with a relevant phenotype was produced in both mono-and co-culture ( Figure 18B), with the highest amount of cells produced in the co-culture.
  • Freshly harvested MSC were centrifuged at 440xg for 5 min. and resuspended in 20 mL saline + 2% HSA to reach a concentration of 3x10 ⁇ 6 cells/mL.
  • 5 mL of the cell suspension was added to a sterile 50 mL tube and irradiated (14 Gy).
  • Cell concentration, viability, apoptosis, metabolic activity, and plastic adherence were evaluated immediately after irradiation.
  • 5x10 ⁇ 5 cells were seeded in T75 culture flasks for evaluation on day 1 and day 3 post-irradiation.
  • MSC control
  • HI-MSC HI-MSC
  • irradiated MSC were seeded immediately postinactivation at a concentration of 2x10 ⁇ 4 cells/mL in MSC medium (see Example 1) in T25 flasks, in a total volume of 5 mL, to test for plastic adherence.
  • Culture flasks were placed in a humidified CO2-incubator at 37°C and 5% CO2. On day 1 and 3 after inactivation, the culture flasks were inspected by light microscopy, to evaluate adherence and cellular morphology.
  • the seeded cells were harvested to facilitate evaluation of viability, metabolic activity, and apoptosis levels.
  • the irradiated cells were harvested as described in Example 1, Cultivation of MSC.
  • HI-MSCs were stored at 4°C in saline + 2% HSA in cryotubes, containing 1 mL of 5x10 ⁇ 6 cells/mL.
  • HI- MSC were sampled directly from the tube and used in the analyses.
  • Apoptosis levels were evaluated using Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 & propidium-iodide (PI) (Thermo Fisher Scientific). The assay was performed according to the instructions by the manufacturer. In detail, 1x10 ⁇ 5 cells were transferred to an 1.5 mL tube, washed with PBS + 0.1% BSA, and centrifuged at 440xg for 5 min. After removing the supernatant, the pellet was resuspended in 100 pL lxAnnexin-binding buffer (to reach a concentration of 1x10 ⁇ 6 cells/mL).
  • PI propidium-iodide
  • the 100 pL cell suspension was transferred to a tube, suitable for flow cytometry, containing 1 pL PI (100 pg/mL) and 5 pL Alexa Fluor 488 Annexin V and incubated in darkness for 15 min. Following incubation, 400 pL lxAnnexin-binding buffer was added, and the sample was analyzed by flow cytometry. The gating strategy was performed as advised by the manufacturer, identifying live cells as Annexin V- PT, apoptotic cells as Annexin V + PT, and dead cells as Annexin V + PI + .
  • XTT reagent and electron coupling reagent were thawed according to the instructions by the manufacturer.
  • HI-MSC and irradiated MSC were added to a 96-well plate in triplicates, with each well containing 5x10 ⁇ 4 cells.
  • XTT working solution was prepared by adding 6 mL XTT reagent and 1 mL electron coupling reagent.
  • Working solution was used immediately by adding 70 pL to each of the wells containing cells, followed by 2 hours of incubation in a humidified CO2 incubator. Medium controls without cells were included in this analysis. After 2 hours of incubation, the absorbance was analyzed with an absorbance plate reader at 450 nm.
  • Inactivation of MSC with irradiation creates cells that maintain several features of non-inactivated/control MSC, namely plastic adherence, spindle-shaped morphology, and metabolic activity.
  • heat-inactivation of MSC produces cells which have lost the ability to adhere to plastic, have a different morphology, and have no metabolic activity, when compared to their noninactivated counterpart.
  • Example 10 Primed medium from HI-MSC stored at -80 °C prior to usage maintain expansion support on HSC
  • Co-cultures of CD34+ HSC and HI-MSC were performed as described in Example 2, but only using a dose of 20.000 HI-MSC/mL.
  • all cytokines were changed to GMP-compliant versions from Miltenyi Biotec and the base medium was the clinically compatible version, StemSpan AOF, described in Example 8.
  • Cells were seeded in a 24 well plate, with a total volume of 1 mL in each well.
  • HI-MSC evaluated here has been stored in StemSpan SFEMII with an addition of 8,4 pL pen/strep per mL of HI-MSC solution, giving a final pen/strep concentration of 20 U/mL.
  • HI-MSCs remain stable in terms of concentration and viability staining after longterm storage at 4°C.
  • long-term stored HI-MSCs maintain their supportive capacity of CD34+ HSC expansion.
  • a process of expanding hematopoietic stem cells comprising the steps: a) providing heat-inactivated mesenchymal stem cells (HI-MSC cells), or a HI-MSC pre-conditioned HSC growth medium, wherein the pre-conditioned medium has been contacted with at least one HI-MSC cell for a time sufficient to condition the media, such as for at least 24 hours, prior to the removal of the HI-MSC cells; b) providing an HSC culture comprising HSCs and HSC growth medium; and c) contacting the HSC growth medium from step b) with the HI-MSC cells or pre-conditioned growth medium from step a) for a time sufficient to expand the HSCs.
  • the time sufficient to expand the HSCs is at least 1 day, such as at least 2 days, such as at least 3 days, such as at least 4 days, such as at least 5 days, such as at least 6 days.
  • HI-MSC cells are provided, such as at least 5000, such as at least 10,000, such as at least 20,000 HI-MSC cells are provided, such as even up to 6*10 6 HI- MSCs, such as even 12*10 6 HI-MSC cells are provided.
  • HSCs such as at least 5000, such as at least 10,000, such as at least 20,000, such as at least 30,000, such as at least 40,000, such as at least 50,000 HSCs are provided.
  • HI- MSC cells are provided in relation to the amount of HSCs provided in step b, such as at least 8%, such as at least 40%, such as at least 100%, such as up to 200% HI-MSC cells provided in relation to the amount of HSCs provided in step b.
  • the HSCs are derived from umbilical cord and/or derived from bone marrow, preferably from bone marrow, even more preferably HSCs mobilized from bone marrow and subsequently obtained from peripheral blood.
  • HI-MSCs are adipose derived, bone marrow derived, and/or umbilical-cord derived.
  • adipose derived MSCs are derived from a stromal vascular fraction (SVF).
  • HI-MSC cells are provided directly after heat inactivation, or the HI-MSC cells have been stored at e.g. below 10°C such as below 5°C, such as in the range 10°C-0.1°C, between -24°C and 5°C, or have been stored at -80°C, such as having been cryogenically stored.
  • At least 2 days such as at least 7 days, such as at least 15 days, such as at least 30 days, such as at least 60 days, when stored in a temperature range 11-28°C, such as 15-28°C, or such as 18-25°C.
  • the HSC growth media comprises or consist of a HSC growth media supplemented with one or more of the following rhSCF, rhTPO, rhFLT3L, rhIL-6, UM171, streptomycin or penicillin, such as a HSC growth media selected from StemSpan SFEM II and StemSpan AOF.
  • a process of providing a pre-conditioned media comprising the steps: a) providing at least one HI-MSC cell; b) contacting a media with the at least one HI-MSC cell for a time sufficient to condition the media, such as for at least 24 hours; c) removing the HI-MSC from the media, thereby providing a preconditioned media; and d) optionally diluting or concentrating the provided pre-conditioned media.
  • a process of providing a pre-conditioned media comprising the steps: a) providing at least one HI-MSC cell; b) contacting a media with the at least one HI-MSC cell for a time sufficient to condition the media, such as for at least 24 hours; c) removing the HI-MSC from the media, thereby providing a preconditioned media; and d) optionally diluting or concentrating the provided pre-conditioned media.
  • a growth media such as a HSC growth media
  • a liquid cell medium such as a balanced salt solution (BSS), such as PBS, preferably isotonic saline and/or a pharmaceutical acceptable composition.
  • BSS balanced salt solution
  • PBS isotonic saline
  • HSC growth media is supplemented with one or more of the following rhSCF, rhTPO, rhFLT3L, rhIL-6, UM171, streptomycin or penicillin, such as a HSC growth media selected from StemSpan SFEM II and StemSpan AOF.
  • a container comprising the pre-conditioned media according to item 26.
  • HI-MSCs such as stored HI-MSCs, as feeder cells for expanding HSCs.
  • HI-MSCs such as stored HI-MSCs
  • pre-conditioning HSC growth media Use of HI-MSCs, such as stored HI-MSCs, as cells for pre-conditioning HSC growth media.
  • 30. Use of the pre-conditioned media according to item 26, to expand a population of HSCs.
  • a HSC population of cells obtained by or obtainable by a process according to any of the preceding items 1-19 or 25.
  • HSC population of cells according to any of items 31-33, wherein the HSC population of cells has an increased viability, when compared to a HSC monoculture.
  • a container comprising the HSC population of cells according to any of items 31-34.
  • kits comprising HI-MSC, and a HSC media, and optionally comprising instructions for expanding HSCs.
  • a kit comprising the pre-conditioned media of item 26, and optionally comprising instructions for expanding HSCs.
  • the HSC population of cells according to any of items 31-34 for use as a medicament.
  • MM Multiple myeloma
  • MDS Myelodysplastic syndrome
  • ALL Acute lymphoblastic leukemia
  • AML Acute myeloid leukemia
  • CLL Chronic myeloid leukemia
  • CML Chronic myeloid leukemia
  • NHL Hodgkin lymphoma
  • NHL Non-Hodgkin lymphoma
  • Diffuse large B-cell lymphoma Diffuse large B-cell lymphoma (DLBCL), Follicular lymphoma, Mantle cell lymphoma, Burkitt lymphoma, and T-cell lymphoma.
  • SCID severe combined immunodeficiencies
  • CCD chronic granulomatous disease
  • WAS Wiskott-Aldrich syndrome
  • HH hemophagocytic lymphohistiocytosis
  • severe autoimmune diseases and selected inherited metabolic disorders.
  • a method of treating or alleviating a blood disorder, a cancer, such as a liquid cancer, such as leukemia, or an immune system disease in a patient in need thereof comprising expanding HSCs as described above, and administering the expanded HSCs to the patient in need thereof.
  • a process of expanding hematopoietic stem cells comprising the steps: a) providing heat-inactivated mesenchymal stem cells (HI-MSC cells), such as at least 1000 HI-MSC cells, such as at least 5000, such as at least 20,000 HI-MSC cells, or a HI-MSC pre-conditioned HSC growth medium, wherein the pre-conditioned medium has been contacted with at least one HI-MSC cell for a time sufficient to condition the media, such as for at least 24 hours, prior to the removal of the HI-MSC cells; b) providing an HSC culture comprising HSCs and HSC growth medium; and c) contacting the HSC growth medium from step b) with the HI-MSC cells or pre-conditioned growth medium from step a) for a time sufficient to expand the HSCs, such as at least 1 day, such as at least 2 days, such as at least 3 days, such as at least 4 days, such as at least 5 days, such as at least 6
  • HI-MSC cells and the HSCs are placed in separate chambers in fluid connection, such as a transwell coculture, such as a non-contacting co-culture or a contacting co-culture, optionally wherein the chambers are separated with a filter, such as a filter having a pore size in the range 0,2 pm to 2 pm, such as in the range 0,4 pm to 1 pm.
  • a filter such as a filter having a pore size in the range 0,2 pm to 2 pm, such as in the range 0,4 pm to 1 pm.
  • the HSCs are derived from umbilical cord and/or derived from bone marrow, preferably from bone marrow, even more preferably HSCs mobilized from bone marrow and subsequently obtained from peripheral blood, or wherein the HI-MSCs are adipose derived, bone marrow derived, and/or umbilical-cord derived.
  • the HSC growth media such as a HSC growth media selected from StemSpan SFEM II and StemSpan AOF, comprises or consist of a HSC growth media supplemented with one or more of the following rhSCF, rhTPO, rhFLT3L, rhIL-6, UM171, streptomycin or penicillin.
  • a process of providing a pre-conditioned media comprising the steps: a) providing at least one HI-MSC cell, such as 60.000 HI-MSC/mL medium, such as up to 2x10 ⁇ 6 HI-MSC/mL medium, preferably 2x10 ⁇ 6 HI-MSC/mL medium; b) contacting a media with the at least one HI-MSC cell for a time sufficient to condition the media, such as for at least 24 hours; c) removing the HI-MSC from the media, thereby providing a preconditioned media; and d) optionally diluting or concentrating the provided pre-conditioned media.
  • at least one HI-MSC cell such as 60.000 HI-MSC/mL medium, such as up to 2x10 ⁇ 6 HI-MSC/mL medium, preferably 2x10 ⁇ 6 HI-MSC/mL medium
  • HSC growth media such as a HSC growth media selected from StemSpan SFEM II and StemSpan AOF
  • HSC growth media such as a HSC growth media selected from StemSpan SFEM II and StemSpan AOF
  • rhSCF HSC growth media selected from StemSpan SFEM II and StemSpan AOF
  • rhSCF HSC growth media selected from StemSpan SFEM II and StemSpan AOF
  • HI-MSCs such as stored HI-MSCs stored at 4 °C and used 2 days post in-activation, as feeder cells for expanding HSCs.
  • HI-MSCs such as stored HI-MSCs stored at 4 °C and used 2 days post in-activation, as cells for pre-conditioning HSC growth media.
  • a HSC population of cells obtained by or obtainable by a process according to any of the preceding clauses 1-7, optionally wherein the HSC population of cells are positive for the markers CD45, CD34 and
  • a cancer such as a liquid cancer, such as leukemia, or an immune system disease.

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Abstract

La présente divulgation concerne un procédé de multiplication cellulaire des cellules souches hématopoïétiques (CSH), un procédé de fourniture d'un milieu préconditionné, et le milieu préconditionné ainsi obtenu, des utilisations de CSH-HI et du milieu préconditionné et la population de cellules CSH multipliée, pour une utilisation en tant que médicament.
PCT/EP2024/057786 2023-03-29 2024-03-22 Cellules souches mésenchymateuses inactivées par la chaleur et leurs utilisations Pending WO2024200276A1 (fr)

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Citations (3)

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WO2023046709A1 (fr) 2021-09-23 2023-03-30 Aarhus Universitet Agent thérapeutique à base de cellules souches prêt à l'emploi

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US20190359941A1 (en) * 2016-08-18 2019-11-28 National University Of Singapore Substituted azole derivatives for generation, proliferation and differentiation of hematopoietic stem and progenitor cells
US20230027247A1 (en) * 2019-12-16 2023-01-26 Edigene (Guangzhou) Inc. Small molecule compounds for amplifying hematopoietic stem cells, and combination thereof
WO2023046709A1 (fr) 2021-09-23 2023-03-30 Aarhus Universitet Agent thérapeutique à base de cellules souches prêt à l'emploi

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