Method for reducing the inflammatory activity of a stem cell transplant and use thereof

Abstract

The disclosure is in the field of cell therapy, more in particular, stem cell transplantation therapy. The disclosure provides methods and compositions for improving the efficacy of stem cell transplantation therapy by reducing the inflammatory activity of a stem cell transplant. More in particular, the disclosure provides a method for preparing a stem cell transplant with reduced inflammatory activity comprising a step of suspending a composition comprising stem cells in a fibrinogen-depleted plasma and/or in a fibrinogen and C-reactive protein-depleted plasma.

Claims

1. An in vitro method for producing a hematopoietic stem cell composition with reduced inflammatory activity, the method comprising: suspending a composition comprising nucleated cells comprising at least one stem cell in plasma or serum depleted of fibrinogen and C-reactive protein, thereby obtaining a stem cell composition with reduced inflammatory activity.

2. The method according to claim 1, wherein the composition comprising nucleated cells is obtained from a tissue sample taken from a subject.

3. The method according to claim 2, wherein the subject is a human subject.

4. The method according to claim 2, wherein the plasma or serum depleted of fibrinogen and C-reactive protein is obtained from the same subject as the subject from which the tissue sample is obtained.

5. The method according to claim 1, additionally comprising a step of depleting pro-inflammatory cells from the composition comprising nucleated cells.

6. The method according to claim 1, wherein the fibrinogen and C-reactive protein depleted plasma or serum is obtained by filtration and/or chromatography.

7. The method according to claim 2, wherein the tissue is selected from the group consisting of hone marrow, peripheral blood and umbilical cord blood.

Description

DETAILED DESCRIPTION

(1) Using animal models of spinal cord injury, it was surprisingly found that certain proteins present in the plasma of bone marrow have a negative effect on the efficacy of the stem cells obtained from the bone marrow, in particular, when they are used as a stem cell transplant and transplanted intrathecally to the autologous animal.

(2) This negative effect appeared to be caused by the presence of the acute phase proteins fibrinogen and C-reactive protein in the bone marrow plasma. Removal of the two proteins by filtration optionally followed by removal of the cells with a pro-inflammatory capability, resulted in stem cell transplants with a significant improvement of the transplantation efficacy, compared with the effect obtained when a stem cell transplant was used wherein only the cells with a pro-inflammatory capability were removed.

(3) It was concluded from this surprising result that the use of stem cell transplants in which the pro-inflammatory cells (such as macrophages, dendritic cells, lymphocytes and granulocytes) and acute phase proteins (such as fibrinogen and C-reactive protein) were removed, are superior in terms of transplantation efficacy compared to stem cell transplants of the prior art.

(4) In a preferred embodiment, the disclosure, therefore, relates to a method for improving the efficacy of a stem cell transplant, wherein the stem cell transplant is depleted of fibrinogen and C-reactive protein. Optionally, the transplant is also depleted of pro-inflammatory cells.

(5) The term “improved efficacy of a transplant” refers to the capability of a transplant, in particular, a stem cell transplant, to repair damaged tissue, in particular, neuronal tissue.

(6) As used herein, the term “stem cell transplant” refers to a composition comprising stem cells, wherein the composition is suitable for administration by transplantation into a subject.

(7) The stem cell transplant of the disclosure may advantageously be obtained from a tissue biopsy, such as peripheral blood, umbilical cord blood or bone marrow. Collection of bone marrow or peripheral blood for use in autologous or allogeneic stem cell transplantation therapies is common practice, and methods to collect bone marrow or peripheral blood biopsies are well known in the art.

(8) The term “intrathecal” as used herein is an adjective that refers to something introduced into or occurring in the space under the arachnoid membrane of the brain or spinal cord.

(9) The term “pro-inflammatory capability” or the like is the capacity of cells or other components to initiate the process of inflammation in vivo, characterized by the accumulation of pro-inflammatory cytokines. The skilled person is well aware of options to determine the pro-inflammatory capacity of a compound.

(10) As used herein, the term “reduced inflammatory activity” is to be interpreted as the inflammatory activity of a composition in comparison to a reference composition. A suitable reference composition in respect of a preferred embodiment of the disclosure (i.e., a composition comprising a stem cell in a plasma depleted of fibrinogen and/or C-reactive protein) would be a composition comprising stem cells resuspended in a normal plasma or a plasma not depleted of fibrinogen and/or C-reactive protein.

(11) The term “plasma” is used herein to refer to the pale-yellow liquid component of blood that normally holds the blood cells in whole blood in suspension. It makes up about 55% of the body's total blood volume. It is the intravascular fluid part of extracellular fluid (all body fluid outside of cells). It consists of mostly water (up to 95% by volume), and contains dissolved proteins (6-8%) (i.e., serum albumins, globulins, and fibrinogen), glucose, clotting factors, electrolytes (Na.sup.+, Ca.sup.2+, Mg.sup.2+, HCO.sub.3.sup.−, Cl.sup.−, etc.), hormones, and carbon dioxide (plasma being the main medium for excretory product transportation). Serum is blood plasma without clotting factors. The terms “serum” and “plasma” are used interchangeably herein, i.e., where it reads “serum,” it also refers to “plasma,” and vice versa.

(12) The inflammatory activity of a composition may be determined by routine assays available to the skilled person, such as immunological and histochemical methods. Inflammatory activity may also be measured by immunochemical methods in samples taken from the transplantation site, wherein the presence of inflammatory markers such as the cytokines interleukin 1 and interferon gamma is assessed.

(13) In a preferred embodiment, the composition according to the disclosure has an inflammatory activity that is less than the inflammatory activity of a composition comprising the same cells in a plasma or serum that is not depleted of fibrinogen and/or C-reactive protein. “Less” in this respect, means at least 10% less, such as 20%, 30% or 50% less. Preferably, the inflammatory activity of a composition according to the disclosure is less than 40% of the reference composition, such as 30%, 20% or 10% or less. Most preferred is an inflammatory activity close to zero or not detectable.

(14) This disclosure, therefore, also relates to a method for reducing the inflammatory activity of a stem cell transplant comprising a step of removing fibrinogen and C-reactive protein from the transplant. A composition comprising stem cells for use as a transplant may also be obtained by resuspending stem cells in serum or plasma depleted of fibrinogen and/or C-reactive protein.

(15) Such may effectively be accomplished by using a step of filtration. Hence, the disclosure relates to a method as described above wherein the fibrinogen and C-reactive protein are removed by filtration. In another embodiment, the fibrinogen and C-reactive protein may be removed by density-gradient centrifugation. In yet another embodiment, the fibrinogen and C-reactive protein may be removed by chromatography.

(16) The method according to the disclosure is particularly useful when the stem cell transplant is also depleted of pro-inflammatory cells. The disclosure, therefore, also relates to a method as described above, additionally comprising a step of removing cells with a pro-inflammatory capability, such as the macrophages, dendritic cells, lymphocytes and granulocytes.

(17) The cells may be removed by negative cell selection or any other method known in the art per se. The different steps of the method may be conducted in a random order.

(18) Negative cell selection may involve a step of reacting a stem cell transplant containing multiple cell lineages with an antibody composition containing antibodies specifically binding to the antigens CD235a (glycophorin A), CD2 and/or CD3, CD14 and/or CD16 and CD19 and/or CD 20 for selective depletion of the target cell erythrocytes, lymphocytes, macrophages, dendritic cells and granulocytes. The antibody composition added includes the suspension medium of the antibodies, mainly consisting of a buffered saline solution for each antibody.

(19) The antibodies in the antibody composition may be magnetically labeled, so that the target cells present in the stem cell transplant can be selectively labeled and removed by retention upon the application of magnetic force to the sample.

(20) A method is herein provided for the selective depletion of cell lineages defined by antibody-mediated recognition of specific epitopes, preferably, but not limited to, erythrocytes, lymphocytes, macrophages, dendritic cells and granulocytes from stem cell transplants obtained from the human body, preferably, but not limited to, human bone marrow.

(21) In a more preferred embodiment, a stem cell transplant is reacted with an antibody composition containing antibodies capable of binding to the antigens CD3, CD14, CD19 and CD235a. The antibodies may be magnetically labeled, so that target cells can be selectively labeled and removed by retention upon the application of magnetic force to the sample.

(22) In a preferred embodiment of a method according to the disclosure, unwanted cell populations are removed from a full volume of a stem cell transplant by the application of specific magnetically labeled antibodies. In an even more preferred embodiment, those antibodies comprise, but are not limited to, antibodies specific for CD2 and/or CD3 to remove T-lymphocytes, antibodies specific for CD19 and/or CD20 to remove B-lymphocytes, CD14 and/or CD 16 for removal of granulocytes, monocytes, dendritic cells and macrophages. In another embodiment, antibodies are specific for CD235a and/or Glycophorin A to remove erythrocytes.

(23) The disclosure also relates to a stem cell transplant obtained in a method as described above. These methods yield a stem cell transplant in which fibrinogen and C-reactive protein are removed. The disclosure, in particular, relates to a stem cell transplant as described above, in which cells with a pro-inflammatory capability are removed.

(24) The terms “removal,” “depletion,” “removed,” “depleted,” or the like, refer to a reduction in the protein amount or cell number in the stem cell transplant of the disclosure of at least 90 percent or 99 percent, compared to the protein amount or cell number in the tissue biopsy such as the bone marrow. The fibrinogen concentration in the blood plasma ranges between 1.5 to 4 grams per liter. In a preferred embodiment, the transplants of the disclosure contain less than 0.4 grams of fibrinogen per liter. In a more preferred embodiment, a plasma preparation suitable for use in a method according to the disclosure, contains less than 40 milligrams of fibrinogen per liter, such as less than 4 milligrams per liter. In a further preferred embodiment, a composition comprising stem cells for use according to the disclosure comprises less than 0.4 gram of fibrinogen per liter, such as less than 40 milligrams or less than 4 milligrams per liter.

(25) The C-reactive protein concentration in blood plasma varies considerably among individuals, but generally ranges between 10 to 100 milligrams per liter. In a preferred embodiment, the transplants of the disclosure contain less than 10 milligrams of C-reactive protein per liter. In a more preferred embodiment, the transplants of the disclosure contain less than 1 milligram of C-reactive protein per liter.

(26) The lymphocyte number in the blood plasma ranges between 1 to 4 thousand per microliter. In a preferred embodiment, the transplants of the disclosure contain less than 400 lymphocytes per microliter. In a more preferred embodiment, the transplants of the disclosure contain less than 40 lymphocytes per microliter. The granulocyte number in the blood plasma ranges between 2.5 to 7.5 thousand per microliter. In a preferred embodiment, the transplants of the disclosure contain less than 750 granulocytes per microliter. In a more preferred embodiment, the transplants of the disclosure contain less than 75 granulocytes per microliter. The dendritic cell and macrophage number in the blood plasma ranges between 10 to 800 per microliter. In a preferred embodiment, the final transplants of the disclosure contain less than 80 dendritic cells or macrophages per microliter. In a more preferred embodiment, the transplants of the disclosure contain less than 8 dendritic cells or macrophages per microliter.

(27) In a preferred embodiment, the disclosure relates to a method as described above, wherein the stem cell transplant is suitable for hematopoietic tissue regeneration. Such a stem cell transplant is particularly suited for restoring hematogenesis. Hence, the disclosure also provides a method for restoring hematogenesis in a subject with a stem cell transplant as described herein.

(28) In another preferred embodiment, the disclosure relates to a method as described above, wherein the stem cell transplant is suitable for vascular tissue regeneration. Such a stem cell transplant is particularly suited for restoring vascularization. Hence, the disclosure also provides a method for restoring vascularization in a subject with a stem cell transplant as described herein.

(29) In a particularly preferred embodiment, the disclosure relates to a method as described above, wherein the stem cell transplant is suitable for neural tissue regeneration. Such a stem cell transplant is particularly suited for neurogenesis. Hence, the disclosure also provides a method for restoring or improving neurogenesis in a subject by treating the subject with a stem cell transplant as described herein.

(30) Recipients of the stem cell transplants of the disclosure can be autologous or allogeneic. In a preferred embodiment, the stem cell transplant of the disclosure is an autologous transplant. In another embodiment, the stem cell transplant of the disclosure is an allogeneic transplant.

(31) This disclosure broadly contemplates a process for enriching and recovering human stem and progenitor cells for the therapeutic treatment of different indications, preferably by tailoring a composition suitable for injection, comprising a cell composition derived from blood, umbilical cord blood or bone marrow. The skilled person is well aware of the metes and bounds of such a composition or method regarding product volume and qualitative and quantitative cell content.

EXAMPLES

Example 1: Preparation of a Human Stem Cell Transplant

(32) Bone marrow was collected from a healthy volunteer by aspiration using a syringe with a five-hole bone marrow needle with two bone punctures. The needle was repositioned after every filled syringe. In total, a biopsy of 50 ml of bone marrow was obtained. The biopsy was centrifugated in a SEPAX® II cell separator (Biosafe) using a low centrifugal force (slow speed) according to the recommendations of the manufacturer. In that way, three fractions were obtained. First, the plasma fraction, containing all soluble proteins of the biopsy. Second, a fraction containing the erythrocytes and thrombocytes was obtained. The third fraction was a composition comprising the nucleated bone marrow cells (also often referred to as the “buffy coat”), including the hematopoietic and mesenchymal stem cells. This is hereinafter referred to as “stem cell composition A.”

(33) The plasma fraction was depleted of fibrinogen and C-reactive protein by filtration using a THERASORB® Fibrinogen-specific filter (Miltenyi). The resulting plasma, from which the fibrinogen and C-reactive protein was removed, was subsequently used to resuspend the nucleated bone marrow cells from the third fraction (stem cell composition A) in a Cell Preparation Bag (Miltenyi). In that way, a stem cell composition was obtained that is herein further referred to as “stem cell composition B.”

(34) The stem cell composition B was contacted with magnetically labeled antibodies against CD3, CD14, CD19 and CD235a (Miltenyi). The bag was subsequently connected to a magnetic cell separation device (CLINIMACS® PLUS System, Miltenyi) in order to produce a stem cell composition depleted of erythrocytes, thrombocytes, lymphocytes, granulocytes, dendritic cells, macrophages, fibrinogen and C-reactive protein. This stem cell composition is hereinafter referred to as “stem cell composition C.”

(35) Stem cell composition D consisted of stem cell preparation A resuspended in the plasma fraction from the same subject, not depleted of fibrinogen and C-reactive protein. This composition acted as a reference composition.

Example 2: Allogeneic Transplantation Experiment

(36) An experimental animal model was used for spinal cord injury to compare the efficacy of the various compositions comprising stem cells. The animal model was a T-lymphocyte-deficient rat. Traumatic compression lesions of the spinal cord were induced by balloon dilation. Three days thereafter, the stem cell compositions were administered intrathecally in close proximity of the spinal cord lesion in the animal. The animals were monitored for thirty-five days. In this period, the body weight was measured and the animals underwent the catwalk and rotarod (Panlab) tests as measures of the neurological damage. After 35 days, the animals were sacrificed and the spinal cord lesions were examined histologically.

(37) In this experimental set-up, animals received stem cell transplants comprising stem cell compositions A, B, C and D, with normal plasma and untreated animals as a control. It was scored how the four compositions were able to repair the neurological damage after induction of traumatic spinal cord lesions.

(38) After 35 days, it was observed that the degree of neurological improvement of the lesioned animals treated with a stem cell transplant according to the disclosure (stem cell compositions B and C) was significantly higher than that of the untreated animals or those treated with plasma, or stem cell compositions A and D. Furthermore, the neurological improvement of the animals treated with stem cell composition C was significantly higher than that of animals treated with stem cell composition B.

(39) TABLE-US-00001 TABLE 1 Stem Cell Negative Negative Positive Experimental Composition(*) Control Control Control condition A B C D (plasma) (lesioned rat) (normal rat) Catwalk +/− + ++ +/− − − +++ Rotarod +/− + ++ +/− − − +++ Body weight +/− + ++ +/− − − +++ Histological +/− + ++ +/− − − +++ findings

(40) The performance or condition of the lesioned rats has been put on 0 percent (baseline) and the normal rats on 100 percent.

(41) −=equal or worsening of the performance or condition (−15 to 5 percent compared to baseline).

(42) +/−=about the same or slightly better performance or condition (between −15% to 10% compared to baseline)

(43) +=slight improvement of the performance or condition (5 to 10 percent compared to baseline)

(44) ++=large improvement of the performance or condition (10 to 40 percent compared to baseline)

(45) +++=Normal performance or condition of untreated rats (90 to 100 percent compared to baseline)

Example 3: Preparation of a Rat Stem Cell Transplant

(46) Stem cell compositions E, F, G and H were prepared from normal Wistar rats in an analogous way as described in Example 1 for their human equivalents. Sample E contained the nucleated bone marrow cells without plasma; sample F were the nucleated bone marrow cells in plasma depleted of fibrinogen and C-reactive protein; and sample G is composition F additionally depleted of erythrocytes, thrombocytes, lymphocytes, granulocytes, dendritic cells and macrophages. Sample H is a control sample corresponding to human sample D wherein the cells from sample E were resuspended in normal rat plasma comprising fibrinogen and C-reactive protein.

Example 4: Autologous Transplantation Experiment

(47) Essentially, the same experimental set-up was used as described in Example 2 to compare the efficacy of the various compositions comprising rat stem cells obtained in Example 3. The animal model was a normal immunocompetent Wistar rat. Traumatic compression lesions of the spinal cord were induced by balloon dilation. Three days thereafter, the stem cell compositions were administered intrathecally in close proximity to the spinal cord lesion in the animal. The animals were monitored for thirty-five days. In this period, the body weight was measured and the animals underwent the catwalk and rotarod (Panlab) tests as measures of the neurological damage. After 35 days, the animals were sacrificed and the spinal cord lesions were examined histologically.

(48) In this experimental set-up, animals received stem cell transplants comprising stem cell compositions E, F, G and H with normal plasma and untreated animals as a control. It was scored how the four compositions were able to repair the neurological damage after induction of traumatic spinal cord lesions.

(49) After 35 days, it was observed that the degree of neurological improvement of the lesioned animals treated with a stem cell transplant according to the disclosure (stem cell compositions F and G) was significantly higher than that of the untreated animals or those treated with plasma, or stem cell compositions E and H. Furthermore, the neurological improvement of the animals treated with stem cell composition G was significantly higher than that of animals treated with stem cell composition F.

(50) Three days after transplant, intrathecal fluid from the lesion site was obtained and tested for the presence of an inflammatory marker, e.g., interferon gamma using a rat IFN gamma ELISA kit (Pierce protein biology products). It was found that interferon gamma levels were high in the intrathecal fluid from animals receiving stem cell compositions E and H. The interferon gamma levels in the intrathecal fluid from animals receiving stem cell compositions F and G were considerably less, 30% and 10%, respectively, of the level in animals receiving stem cell composition H.

(51) TABLE-US-00002 TABLE 1 (CONTINUED) Stem Cell Negative Negative Positive Experimental Composition(*) Control Control Control condition E F G H (plasma) (lesioned rat) (normal rat) Catwalk +/− + ++ +/− − − +++ Rotarod +/− + ++ +/− − − +++ Body weight +/− + ++ +/− − − +++ Histological +/− + ++ +/− − − +++ findings

(52) The performance or condition of the lesioned rats has been put on 0 percent (baseline) and the normal rats on 100 percent.

(53) −=equal or worsening of the performance or condition (−15 to 5 percent compared to baseline).

(54) +/−=about the same or slightly better performance or condition (between −15% to 10% compared to baseline)

(55) +=slight improvement of the performance or condition (5 to 10 percent compared to baseline)

(56) ++=large improvement of the performance or condition (10 to 40 percent compared to baseline)

(57) +++=Normal performance or condition of untreated rats (90 to 100 percent compared to baseline)