WO2010049783A1

WO2010049783A1 - Stem cells in obstetrics and gynaecology

Info

Publication number: WO2010049783A1
Application number: PCT/IB2009/007227
Authority: WO
Inventors: Satish Manohar Patki; Ramesh Ramchandra Bhonde
Original assignee: Patki Research Foundation & Hospital
Priority date: 2008-10-28
Filing date: 2009-10-27
Publication date: 2010-05-06

Abstract

The invention relates to the use of an "improved culture medium" containing cord blood serum for isolating and expanding stem cells isolated from various sources. The invention also deals with the method of treating various obstetric complications such as hypertensive disorders of pregnancy, repeated abortions, placental insufficiency and intrauterine growth restrictions-using stem cells specially those isolated from bone marrow. In another embodiment, the invention describes processes of improving pregnancy rates in assisted Reproductive Technology using the improved medium.

Description

STEM CELLS IN OBSTETRICS AND GYNAECOLOGY

FIELD OF INVENTION: The invention relates to method of isolating stem cells, media enriched with cord blood serum for better isolation of stem cells and the use of stem cells specifically in the treatment of various complications in the field of Obstetrics and Gynaecology. These conditions include - hypertensive disorders of pregnancy, repeated abortions, placental insufficiency and intra-uterine growth restrictions. The invention also deals with the isolation of mesenchymal stem cells from endometrium using media enriched with cord blood serum and the use of cord blood serum enriched medium for improving implantation rate s of 'ln-Vitro fertililization'(IVF) procedures and "Intrauterine inseminations, (I.U.I)

BACKGROUND OF THE INVENTION: Stem cells:

Stem cells have the ability to divide without limit and possess great plasticity .It can be said that the term "stem cells 'represents a generic group of undifferentiated cells that possess the capacity for self renewal while retaining varying potentials to form differentiated cells and tissues.

The potential utility of stem cells in clinical medicine is well documented. Currently there is enormous interest in stem cells as a new treatment modality for regenerative medicine. Stem cell research is now in an exciting phase of development with increasing acceptance of the potential of 'Adult stem cells' in therapy in almost all branches of medicine. Adult stem cells have been identified in diverse tissues including human bone marrow, breast, prostate, brain, liver and adipose tissue etc. Mesenchymal stem cells (MSCs), the non-haematopoietic progenitor cells found in various adult tissues, are characterized by their ease of isolation and their rapid growth in vitro while maintaining their differential potential, allowing for extensive culture expansion to obtain large quantities for therapeutic use. (Chen FH;Tuan RS; Arthritis Res ther2008; 10(5)223) Stem cells from endometrium:

Research has demonstrated that human endometrium also contains a small population of cells that exhibit stem/progenitor cell behaviour in vitro;clonigenicity The progenies in these colonies have been characterized and growth factors supporting clonogenicity identified (Gargett CE Aust NZ Obstet Gynaecol.2004,Oct;44(5): 380-386).

The human endometrium is a dynamic tissue which undergoes cycles of growth and regression with each menstrual cycle. Endometrial regeneration also follows parturition and extensive resection and occurs in post menopausal women taking estrogen replacement therapy. It is likely that adult stem/progenitor cells are responsible for this remarkable regenerative capacity. Functional approaches have been used to identify endometrial epithelial and stromal stem/progenitor cells, due to lack of known specific endometrial stem cell markers.( Gargett CE et al Curr Opin Obstet Gynaecol 2007,Aug, 19 (4) :377-83 ).A recent study has identified CD146 as a marker of colony forming human endometrial stromal cells supporting the concept that human endometrium contains a population of candidate stromal stem/progenitor cells (Schwab KE et al Hum Reprod 2008.Apr;23(4);934-43 )

A potential advantage of using stem cells from an adult is that the patients own cells could be expanded in culture and then reintroduced to the patient. The use of patients own adult stem cells would mean that the cells would not be rejected by the immune system. This represents a significant advantage as immune rejection is a difficult problem that can only be circumvented with immunosuppressive drugs.

Unmet needs of Obstetrics;

Though adult stem cells have been extensively used as a therapeutic tool with great success ,its potential in the field of obstetrics and gynaecology is yet to be exploited - particularly in the pregnancy related disorders such as Hypertensive disorders of pregnancy, Repeated abortions, Placental insufficiency, and Intrauterine growth restrictions. Though pregnancy is a physiological process it can become pathological or high risk in the above mentioned conditions and add to the maternal and fetal morbidity and mortality. Intrauterine growth restriction (IUGR) is a common diagnosis in obstetrics and carries an increased risk of perinatal mortality .The incidence of IUGR is estimated to be approximately 5 percent in general obstetric population. Timely diagnosis and proper management of IUGR is still a challenge. According to the National Vital Statistics Reports (2002) 37.7% of pregnancies are affected by the pregnancy related hypertensive complications-contributing to maternal and fetal mortality rates. Placental insufficiency is yet another complication of pregnancy in which placenta can not bring enough oxygen and nutrient to a baby growing in the womb. We feel there is a definite need to make use of this unique technology of stem cells in reducing the pregnancy related common complications of pregnancy.

Stem cell culture medium: The challenges One of the challenges facing the stem cell research is the need for an ideal medium for isolation and expansion of stem cells .A variety of factors are required to be incorporated into the culture medium to support specific types of stem cells, maintain their growth and specific characteristics.- as they are obtained from different tissues. Culture medium compositions typically include essential amino acids, salts, minerals, trace elements sugars, lipids. Many cell culture medium formulations are documented in the literature and a number of media are commercially available. Though conventional culture media are frequently used, it is felt that there is a definite need for a better and more efficient medium. A lot of research is being done in this area. Avoiding the use of any animal component in the stem cell culture medium seems to be another research priority.

US Patent no.6833271 describes a culture medium containing a sterol such as cholesterol stabilized by surfactant rather than serum products or phospholipid micelles.

US Patent no.4,560,655 deals with a defined serum free medium containing serum substitutes composed of fetuin, transferrin, phosphatidylcholine etc.- that is capable of growing a wide range of suspension and monolayer cells

Proceedings of the National Academy of Sciences has reported that Scientists from 'The Scripps Research institute, Novartis Research Foundation and Max Planck Institute have developed a new synthetic compound "Pluripotin" that can support growth and self renewal of embryonic cells.

( WWW. scripps. edu/newsandviews/e 20061106/ding. html) Patent no.CA2663976 describes a method of culturing stem cells in a special medium containing a ROCK (Rho-Kinase) inhibitor.

It is clear from the above data that continuous attempts are being made to improve the quality of the stem cell culture medium in order to obtain consistent results. There is definitely scope for improvement.-of the media and the method.

OBJECTS AND SUMMARY OF THE INVENTION: An object of the invention is providing an 'improved culture medium' for stem cells.

Another object of the invention is to isolate bone marrow stem cells using the 'improved culture medium' and an improved method.

Other object of the invention is to make use of stem cells specially derived from bone marrow -for the treatment of various complications of the pregnancy which include- hypertensive disorders of pregnancy , Repeated abortions, Placental insufficiency, and intra-uterine growth restrictions.

Yet another object of the present invention is to isolate menchymal stem cells from endometrium using the 'improved culture medium' and using the same for 'in vitro fertilization' procedures

A further object of the invention is to develop an improved process based on the use improved medium containing cord blood serum for treating semen samples and oocytes for better results in 'In vitro Fertilization'(IVF) and Intrauterine inseminations(IUI).

The improved culture medium contained Cord blood serum added to usual DMEM medium (Dulbecco's minimum Eagles medium) at appropriate concentrations -ranging from 10-50%. Administration of Stem cells isolated from Bone marrow to obstetric patients helped enormously in reducing pregnancy related complications. Use of Cord blood containing improved medium and an improved process for activating semen samples and oocytes helped in increasing pregnancy rates. BRIEF DESCRIPTION OF THE FIGURES:

Figure 1 : Photograph showing female genital tract. Figure 2: Microphotograph showing mesenchymal stem cells isolated from endometrium.

5 Figure 3: Microphotograph showing mesenchymal stem cells isolated from fallopian tube.

Figure 4 : Microphotograph showing mesenchymal stem cells isolated from cumulus cells.

Figure 5: Microphotograph showing differentiation of endometrial stem cells into islets0 of pancreas.

Figure 6: Microphotograph showing bone marrow stem cells. Figure 7: Photograph showing culture medium.

DETAILED DESCRIPTION OF THE INVENTION: S There are different embodiments of the present invention.

The invention deals with:

1) Isolation of stem cells from born marrow using enriched medium ii) The use of stem cells in the treatment of obstetric complications iii) Isolation of mesenchymal cells from endometrium, fallopian tube & ovary and0 use of the same for enhancing the pregnancy rates in Assisted Reproductive

Technology, iv) In another embodiment ,the present invention decribes unique processes of treating semen samples and oocytes for better results in -In vitro

Fertilization(IVF) and Intrauterine insemination (IUI). 5 i) ISOLATION OF STEM CELLS FROM BONE MARROW:

We have developed a sequential culture media for isolation of stem cells from bone marrow.

The composition of our culture media is as follows -0 1) Medium A - It contains Ficoll in Ringer Lactate base.

2) Medium B - It contains a balanced salt solution having following components - .(DMEM medium)+Cord blood serum)

Sodium Chloride Potassium Chloride Magnesium Sulfate

- Potassium Phosphate

- Calcium Lactate - Sodium Bicarbonate

L - Aspartic Acid

- Glucose Sodium Pyruvate L- Serine - EDTA

- L - Glutamine Ampicillin & Cloxacillin

- Sodium Citrate

- Phenol Red. 3) Medium B 10% - Contains 10 % tested human cord blood serum as a protein source.

4) Medium B 50% - Contains 50 % tested human cord blood serum as a protein source.

Storage - At 2 - 8°c & to be warmed to incubator temperature prior to use.

The procedure of isolation of stem cells from bone marrow -

1) 100 cc blood sample is aspirated from iliac crest under short general anesthesia.

2) The blood sample is collected in heparin to prevent clotting & is then aliquoted into aliquots of 10 ml each.

3) 5 ml of medium A is taken which is overlaid by 10 ml of the blood.

4) It is centrifuged at 1500 RPM for 30 min.

5) The buffy coat at the junction is isolated.

6) This buffy coat is taken into another tube to which medium B 10% (2ml) is added. This is again centrifuged at 1500 RPM for 15 min. to get rid of traces of medium A.

7) The pellet is finally suspended in 2 ml of medium B 50% which is ready for therapy. ii) USE OF BONE MARROW DERIVED STEM CELLS FOR OBSTETRIC COMPLICATIONS:

Procedure of stem cell therapy

This stem cell population obtained above is injected either intravenously or at the placental bed under sonography guidance. Being autologus, there is no ethical issue or question of rejection.

Our observation -

We have studied 80 cases of hypertensive disorders of pregnancy & found excellent results in the terms of reduction of maternal hypertension & improvement of fetal parameters like amniotic fluid index, Doppler findings of umbilical & uterine vessels etc.

iii) ISOLATION OF ENDOMETRIAL MESENCHYMAL CELLS & USE B) Autologus "Mesenchymal" stem cell therapy for improving implantation rates of In Vitro Fertilization procedures.

In this invention, we do endometrial biopsy of the patient, & culture & isolate the mesenchymal stem cells. We do repeated passages to get pure population of these cells. During the procedure of Embryo transfer, these cells are transferred along with the embryos. These cells improve the implantation & pregnancy rates. Being 'Autologus', there is no question of rejection or ethical issue.

Processing of Endometrium For Mesenchymal Stem Cells • Take endometrium sample in IX PBS

• Wash it 2 to 3 times to remove RBCs

• Take endometrium tissue in 0.25% TPVG (3 to 5 ml) in 15 ml Falcon tube.

• Shake it till the tissue dissolves.

• For hard tissue add collaginase (1 ml) (0.15%) • Centrifuge the tube for 10 min at 2000 RPM

• Collect the cell pellete and discard supernatant

• Resuspend the pellete in 1 ml growth medium i.e. DMEM + 20% serum.

• Centrifuge the tube for 10 min at 2000RPM. Remove the supernatant.

• Resuspend the pellete in 1 ml growth medium i.e. DMEM + 20 % serum. • Make the volume to 5 ml and seed in to tissue culture flask.

• Incubate at 37⁰C for overnight

iv) PROCESS OF TREATING SEMEN SAMPLES AND OOCYTES

1) A NOVEL SEQUENTIAL CULTURE MEDIUM FOR ASSISTED

REPRODUCTIVE TECHNOLOGY (A.R.T.)

A.R.T. is a treatment modality for infertile couples, in which the gametes i.e. sperms & eggs & embryos are handled in the laboratory. Basically, there are two types of A.R.T. -

1) Intrauterine Insemination (I.U.I.)

Here, the sperms are activated 'In Vitro' by using culture medium & are injected in the uterus at the time of ovulation.

2) In Vitro Fertilization & Embryo Transfer (I.V.F. & E.T.) - Here, the oocytes are retrieved from the wife's ovary & fertilized with the activated sperms of the husband. The resultant embryos are then transferred to the uterus for further growth.

Culture Medium -

Culture medium is basically a balanced salt solution supplemented with protein. The commonly used balanced salt solution is Earle's Balanced salt solution

(EBSS) & commonly used protein sources are Human Serum Albumin (HAS) or Bovine Serum Albumin (BSA) or synthetic Serum Replacement (SSR). We have developed a Novel Culture medium, which contains - DMEM (Dulbecco's minimum Eagle's medium), as balanced salt solution & protein source is tested human cord serum.

(Nobody has used such medium for A.R.T. in the world). The cord serum is rich not only in proteins but also in growth factors required for sperm capacitation as wells as embryonic development & cleavage. We have 2 types of media (Hence called sequential)

1) 10 % - in which cord serum is 10 %

2) 50 % - in which cord serum is 50 % Protocols for sperm preparation in I.U.I. -

• Semen sample is taken in a test tube.

• Our 10 % medium is added to it (twice the volume of semen) & mixed well.

• Centrifugation is done at 1200 RPM for 15 min. • The supernatant is removed. This supernatant contains seminal plasma (which is fertilization inhibitor)

• The pellet contains sperms & other cells like WBCc etc.

• This pellet is again mixed with 2 ml of 10 % medium & again centrifuged for 15 min at 1200 RPM. • Again supernatant is removed to get rid of seminal plasma completely.

• The pellet is overlaid with 0.5 ml of 10 % medium (without disturbing pellet)

• The tube is kept in Carbon Dioxide incubator (at 37°C) for 30 min.

• During this time the motile sperms will "Swim Up" & come in the supernatant. » 0.5 ml of this supernatant, which contains motile fractions (capacitated) of sperms.

• This is used for I.U.I, as well as for I.V.F.

I.U.I. Procedure - Wife's ovulation is monitored by sonography & exact day of ovulation is pinpointed.

The sperms are washed by the protocol as mentioned above.

0.5 ml of this is injected in the uterus by using cannula.

Procedure is done on O.P.D. basis. Success rates by our medium -

- Traditional culture media give success rates of 20 % per cycle in I.U.I.

- We have done comparative study in 100 cycles of each by using Traditional & 10 % medium of ours.

- The pregnancy rates of 20 % are achieved when traditional medium is used. - The pregnancy rates of 40 % are achieved (DOUBLE) when our 10 % medium is used. Protocols for I.V.F. - E.T. (Test Tube Baby)

Here, the oocytes are retrieved from ovary by vaginal sonography technique & are washed by using our 10 % culture medium. The washed sperms are added to eggs (50,000 sperms per oocytes). The fertilization is confirmed after 24 hrs. The embryos of 8 - 16 cells are formed on Day - 3, which are transferred to uterus using catheters. While transferring our medium containing 50 % cord serum is used. However percentage of cord serum is used for - 1) Early embryonic development. 2) Implantation process. (Acts as embryo glue)

2) - Traditional culture media give success rates of 20 %.

- Our sequential culture media in a series of 100 cycles have given success rates of 40 % 2) Use of endometrial mesenchymal stem cells for I.V.F. E.T.

This is a novel concept for 1st time in world.

Hence, the endometrial biopsy is taken fore the same patient in previous cycle.

The mesenchymal cells are cultures by using our 10 % medium.

These cells are transferred along with embryo during embryo transfer. These cells help for implantation by following mechanisms-

1) These cells are immunosuppresant.

2) They help in implantation by reducing rejection.

3) These cells produce growth factors required for embryonic development & implantation. In our series of 100 cycles, the pregnancy rates are 40 % (double than observed by using traditional medium)

We found that the mesenchymal stem cells obtained from Endometrium, Fallopian tube or Ovary are useful for the treatment of various medical disorders as an allogenic stem cell therapy. These medical disorders include -

Gynaecological disorders like infertility, pregnancy induced hypertension, orthopedic disorders like osteoarthritis and Neurological disorders like stroke and spinal cord injuries. Our enriched medium and the process described above further helps in obtaining adequate quantity of quality mesenchymal cells from tissues like endometrium.

Claims

WE CLAIM:

1. An enriched medium for supporting growth, maintenance and the therapeutic efficacy of cells of the human body 'in -vitro' comprising of the conventional medium used for tissue culture and stem cell culture such as Dulbecco's minimum Eagle's medium(DMEM ) with added Cord blood serum.

2. The enriched medium as per claim 1 wherein the concentration of the Cord blood serum used is in the range of 10-50%

3 The use of the enriched medium as per claim lor claim 2 for processing human semen samples for improved Intrauterine Insemination (IUI)and In vitro Fertilization (IVF)

4. The use of enriched medium as per claim 3 wherein the concentration of the Cord blood serum used in the medium is in the range of 10-50% and preferably 10%

5. An improved method of preparing semen samples using enriched medium containing Cord blood serum for Intrauterine Insemination (IUI) and 'In Vitro

Fertilization' (IVF) comprising the steps of : a) taking Semen sample in a test tube and adding 10 % medium ie.DMEM medium with 10% Cord blood serum to it (twice the volume of semen) and mixing the same; b) centrifuging the above solution at 1200 RPM for 15 min; c) removing the supernatant; d) mixing the pellet containing sperms & other cells like WBCc in 2 ml of 10 % medium (of step a) and again centrifuging for 15 min at 1200 RPM; \ (e) removing the supernatant to get rid of seminal plasma completely;

(f) overlaying the pellet with 0.5 ml of 10 % medium (without disturbing pellet);

(g) keeping the tube in Carbon Dioxide incubator (at 37⁰C) for 30 min; (h) using the 0.5 ml of this supernatant, which contains motile fractions (capacitated) of sperms for I.U.I, as well as for I.V.F.

6. The use of the enriched medium as per claim lor claim 2 for washing the oocytes and transferring the embryo to uterus during the 'In vitro fertilization' process.

7. The use of enriched medium as per claim 6 wherein the concentration of Cord blood serum used in the medium for washing the oocytes is 10% and for transferring the embryo to the uterus is 50%

8. An improved method of treating oocytes for enhancing pregnancy rates during

IVF, comprising steps of :

(a) washing the oocytes retrieved from the ovary with ' 10% culture medium' i.e. DMEM containing 10% Cord blood serum;

(b) using "50% culture medium' ie. DMEM containing 50% Cord blood serum for transferring the fertilized oocytes (embryo) to uterus.

9. An improved culture medium comprising of DMEM medium with added cord blood serum in a range of 10-50% for activating semen samples during the In vitro fertilization (FVF ) and Intrauterine insemination (IUI) procedure

10. The improved culture medium as per claim 9 wherein the concentration of added

Cord blood serum is 10%

1 1. An improved culture medium comprising of DMEM medium with added cord blood serum in a range of 10-50% for washing and activating oocytes during the in vitro fertilization procedure(IVF)

12. The improved culture medium as per claim 1 1 wherein the concentration of Cord blood serum used is 10%.

13. An improved culture medium comprising of DMEM with added Cord blood serum in the range of 10-50% for transferring the embryo to the uterus during the in vitro fertilization (IVF) procedures.

14. The improved culture medium as per claim 13 wherein the concentration of cord blood serum is 50%

15. The use of the enriched medium as per claim lor claim 2 for isolation and expansion of stem cells from bone marrow.

16 A method of treating Obstetric complications by administering bone marrow derived stem cells intravenously or at the placental bed preferably under sonography guidance.

17. A method as per claim 16 wherein the Obstetric complications include:

Hypertensive disorders of pregnancy, repeated abortions, Placental insufficiency, Intrauterine growth restrictions.

18. A novel process of obtaining stem cells from bone marrow comprising the steps of: a) aliquoting the heparinized blood obtained from iliac crest it into aliquots of 10ml each; b) taking 5 ml of 'medium A' i.e., Ficoll in Ringers lactate base and overlaying the same by 10 ml of the blood; c) centrifuging the above at 1500 RPM for approximately 30 min; d) isolating the buffy coat at the junction; e) taking the buffy coat into another tube to which (approx, 2ml)'medium B 10%' i.e., DMEM medium containing 10% cord blood serum) is added; f) centrifuging the above at 1500RPM for 15 min to get rid of traces of medium A; g) suspending the pellet finally in 2 ml of medium B i.e., 50% DMEM medium enriched with 50% cord blood serum i.e., which is ready for therapy

19. The use of enriched medium as per claim 1 or claim 2 for isolation and processing of mesenchymal stem cells from endometrium

20. A process of isolating mesenchymal stem cells from the Endometrium comprising the steps of : a) Taking the endometrium sample in IX PBS; b) washing it 2 to 3 times to remove RBCs; c) taking endometrium tissue in 0.25% TPVG (3 to 5 ml) in 15 ml Falcon tube; d) shaking it till the tissue dissolves; e) adding collaginase for hard tissue (1 ml) (0.15%); f) centrifuging the tube for 10 min at 2000 RPM; g) resuspending the pellet in 1 ml growth medium i.e. DMEM + 20% cord blood serum; h) centrifuging the tube for 10 min at 2000RPM remove the supernatant; i) resuspending the pellet in 1 ml growth medium i.e. DMEM + 20 %Cord blood serum; j) making the volume to 5 ml and seed in to tissue culture flask; k) incubating at 37⁰C for overnight.

21. A method of improving implantation and pregnancy by transferring the embryo along with autologus mesenchymal stem cells obtained from endometrium preferably in an enriched medium containing Cord blood serum during 'In Vitro

Fertilization' procedures.

22. The method as per claim 21 wherein the concentration of cord blood serum used in the enriched medium is 50%

23 The use of mesenchymal stem cells obtained from Endometrium, Fallopian tube, or Ovary for the treatment of various medical disorders as an allogenic stem cell therapy.

24. The use of mesenchymal cells as per claim 23 wherein the medical disorders include-Gynaecological disorders like infertility, pregnancy induced hypertension, Orthopedic disorders like osteoarthritis, Neurological disorders like stroke and spinal cord injury and other degenerative disorders.

25. The use of mesenchymal cells obtained from endometrium as per the process of claim 20 for the treatment of the medical disorders which include- Gynaecological disorders like infertility, pregnancy induced hypertension, Orthopedic disorders like osteoarthritis, Neurological disorders like stroke and spinal cord injury and other degenerative disorders

26. An improved method of In vitro Fertilization (IVF) comprising the steps of: a) taking Semen sample in a test tube and adding 10 % medium ie.DMEM medium with 10% Cord blood serum to it (twice the volume of semen) and mixing the same; b) centrifuging the above solution at 1200 RPM for 15 min; c) removing the supernatant; d) mixing the pellet containing sperms & other cells like WBCc in 2 ml of 10 % medium (of step a) and again centrifuging for 15 min at 1200 RPM; e) removing the supernatant to get rid of seminal plasma completely; f) overlaying the pellet with 0.5 ml of 10 % medium (without disturbing pellet); g) keeping the tube in Carbon Dioxide incubator (at 37⁰C) for 30 min; h) using the 0.5 ml of this supernatant, which contains motile fractions

(capacitated) of sperms for I.U.I, as well as for I.V.F; i) washing the oocytes retrieved from the ovary with ' 10% culture medium' i.e., DMEM containing 10% Cord blood serum; j) the washed sperms are added to eggs (50,000 sperms per oocytes)(fertilization); k) using "50% culture medium 'i.e., DMEM containing 50% Cord blood serum for transferring the fertilized oocytes (embryo) to uterus.