STEM CELLS AND INFERTILITY

Dr. Gayathiri Ganesan RamMS(O.G), Fellow in Reproductive Medicine and Andrology.

Consultant Reproductive Medicine SpecialistARC Fertility Hospitals

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Edwards and Bavister fertilize first human egg

Mouse embryonic stem cells derived

Non-human primate ES cells derived

Thomson et al derive and culture first hES cells

Woo-suk Hwang fakes human stem cell cloning

Yamanaka et al derive human iPS cells

First IVF baby Louise Brown is born

First donated blastocysts for research

Dolly, the cloned sheep, is born at the Roslin

Institute

Mouse embryonic stem cells derived by SCNT

Yamanaka et al derive mouse iPS cells

A Walk Through the History…

Human embryonic stem cell: Also known as a human pluripotent stem cell, one of the "cells that are self-replicating, are derived from human embryos or human fetal tissue, and are known to develop into cells and tissues of the three primary germ layers."

(From the National Institutes of Health Guidelines for Research Using Human Pluripotent Stem Cells.)

Definition

Human Embryonic Stem Cells.

• Definition: Stem cells are clonogenic,undifferentiated, self replicating, progenitor cells, capable of self regeneration and also of multilineage differentiation and generation of several other tissues and organs in the body

Types of Stem Cells.

• 1) Toti potent stem cells.• 2) Pluri potent stem cells.• 3) multi potent stem cells.• 4) Unipotent stem cells- oxymoron?

Totipotent Stem Cells.

• Natural identical twinning is due to division of an early embryo into two embryos.

• One form of cloning would be by dividing an early embryo into two or more embryos.

Toti Potent Stem Cells

• The embryonic cells in the early stages are toti potent. Eg: two celled embryo,four celled embryo.

Pluripotent Stem Cells

• Pluri potent cells are capable of producing all the cells, tissues and organs in the body- but not an embryo.

• Cells obtained from the blastocyst -from the inner cell mass are-pluri potent.

Pluri Potent Stem Cells

• The cells from the inner cell mass are pluri potent. They are capable of producing any organ or tissue in the body-but not the placenta.

Stem Cell Cultivation

• The slide briefly describes embryonal stem cell cultivation.

• University of Wisconsin was the first one to cultivate human embryonic stem cells.

Embryonic Stem Cells

Multi Potent Stem Cells

• Adult stem cells, cord blood stem cells,and cells obtained from late stage embryos are multi potent.

• Multi potent stem cells are capable of generation of several tissues, but not all tissues.

Uni potent Stem Cells

• Oxymoron?• The stem cells found in many organs like

liver,bone marrow,testes serve as reserve cells for regeneration of appropriate tissue.

• Under proper in vitro/in vivo conditions these cells can be coaxed to become other tissues of the body.

Embryonic Stem Cells

• These cells are obtained usually from the inner cell mass of a blastocyst- day 6/7 embryo.

• These embryos are usually spare embryos, research embryos or orphaned embryos,obtained from an infertility clinic.

Sources of Stem Cells

• 1)Embryonal stem cells.• 2)Adult stem cells.• 3)Cord blood stem cells.• 4)Induced pluripotent stem cells• 5)Induced multipotent stem cells (transdifferentiation)• 6)Somatic cell nuclear transfer (Dolly)• 7)Oocyte, somatic cell fusion -

http://news.sciencemag.org/sciencenow/2011/10/an-egg-citing-recipe-for-human-s.html

Adult Stem Cells

• These cells are generally obtained from bone marrow.They may also be obtained from peripheral blood.

• Stem cells are probably present in most of the tissues and organs in the body.

• It is difficult to identify, isolate culture and grow adult stem cells.

Adult Stem Cells

• Identifying and isolating adult stem cells from many tissue would be worse than the proverbial search for ‘needle in a haystack.’

• If this becomes possible, this would avoid many of the legal, ethical and moral problems associated with the use of Embryonal stem cells.

Adult Stem Cells

http://stemcells.nih.gov/

Cord Blood Stem Cells

• Cord blood is a good source of stem cells.• It may be a good idea to store cord blood

routinely at all deliveries for autologous stem cell transplant or HLA compatible stem cell transplant- where the older sibling/ other family members are afflicted with haematological abnormalities/ malignancy.

Indications for Stem Cell Therapy.

• Stem cell therapy can be used in many degenerative diseases.

• Stem cells can be used in the regeneration of many diseased organs.

• Stem cells can be used in the correction of many metabolic and haematologic disorders.

Indications for Stem Cell Therapy.

• Current:• 1)diabetes mellitus- insulin dependent.• 2)Parkinson’s disease.• 3)heart disease- both ischaemic and

degenerative.

Indications for Stem Cell Therapy.

• 4)spinal cord injuries.• 5)Haematologic disorders- like Fanconi’s

anaemia,Thalassaemia.• 6)malignancy.• 7)autoimmune diseases.• 8)genetic diseases.

Indications for Stem Cell Therapy.

• Virtually any diseased organ or tissue can be replaced with stem cell therapy, obviating the need for transplants.

• Future research holds very great promise for correcting many diseases.

• A new specialty is emerging “regenerative medicine’ thanks to the advent of stem cell therapy.

Mode of Administration of Stem Cells.

• 1)direct injection into the organ.• 2)systemic administration and the stem cells

colonize the organ/tissue.• 3)direct surgical placement of stem cells.

Somatic Cell Nuclear Transfer

• Allows for avoidance of immune problems• Provides patient-specific disease models for

research

• Could lead to reproductive cloning• Needs the donation of eggs

SCNT

Stem cells and the reproductive tract

• Stem cells and the endometrium• In humans, regeneration of the endometrium

occurs monthly during reproductive life and beyond under appropriate ovarian steroid priming.

• Cyclic replenishment of the cellular compartments of the endometrial functionality by adult stem cells (ASC) is essential for the preparation of this organ for its main function, i.e. allowing implantation and pregnancy to proceed

• ASC present a MSC phenotype (Chan et al., 2004; Schwab and Gargett, 2007; Schwab et al., 2008; Cervello et al., 2010)

• They functionally contribute to human endometrial regeneration in vitro and in vivo (Cervello et al., 2010, 2011; Matsuda and Shi, 2010).

• Bone marrow-derived stem cells (BMDSCs) have been shown to contribute as an exogenous source to tissue repair and regeneration of different organs and tissues (Pittenger et al., 1999).

• In the human, BMDSCs are also a source of non-hematopoietic cells in the different endometrial cellular compartments (stroma, glandular epithelium, and luminal epithelium).

• They contribute mainly to the formation of endometrial stromal compartment cells and to a much lesser extent to the glandular and luminal epithelium (Du and Taylor, 2007; Mints et al.,2008; Ikoma et al., 2009; Cervello et al., 2012).

• It has been recently proposed that BMDSC infusion might improve endometrial regeneration in a murine model of AS (Alawadhi et al., 2014; Jing et al., 2014; Kilic et al.,2014; Zhao et al., 2015).

• However, peripheral blood stem cell transplant(PBSCT) did not result in engraftment of donor stemcells in the recipient uterus in a macaque model, or human (Wolff et al., 2013).

SPERMATOZOA• Obtaining PGCs (primodial germ cells) from pluripotent cells is

the first step in the differentiation toward post-meiotic spermatozoa. However, further differentiation has proven challenging in vitro.

• The strategies employed to favor it include supplementing the differentiation medium with the growth factors bone morphogenetic protein (BMP)-4, -7, -8b (Geijsen et al.,2004; Tilgner et al., 2008), N2B27, activin, Fibroblast growth factor 2 (FGF2) (Hayashi et al., 2011), retinoid acid (Nayernia et al., 2006;Eguizabal et al., 2009, 2011; Cai et al., 2013; Peng et al., 2013),R115866 (Eguizabal et al., 2011), and hormones such as Insulin andTestosterone (Easley et al., 2012).

STEM CELLS AND VAGINAL RECONSTRUCTION

• The use of stem cells for vaginal recovery has been poorly investigated so far, but most work has been carried out with muscle-derived stem cells (MDSC).

• Recently, muscle has been identified as an alternative source of adult stem cells, different from satellite cells, possessing the capacity to differentiate into different cell lineages.

• MDSCs appear as promising candidates for the treatment of muscular, cardiac and urological disorders.

STEM CELLS AND ERECTILE DYSFUNCTION

• Penile erection is a neurovascular response involving an increase in arterial inflow, relaxation of corpora smooth muscles, and a reduction invenous outflow.

• Erectile dysfunction can be caused by conditions such as diabetes, neuropathies, cavernous nerve injuries, neurotransmitterimbalances, vascular diseases, inflammation diseases (Peyronie

• disease) or surgery.

• The results obtained indicated that the stem cell injections brought benefits in terms of restored erectile function and penile physiology (Zhang et al., 2012); interestingly, the improvement in erectile function seems to be due to the paracrine factors secreted by the injected cells (cytoprotective, anti-fibrotic and anti-apoptotic molecules),rather than direct grafting/differentiation (Albersen et al., 2010).

• No informationis available on the duration of the beneficial effects.

• The only preclinical study involving patients was carried out with intracavernosal injections of umbilical cord blood stem cells in seven patients with diabetic-erectile dysfunction: patients reported an improvement in penile erection and an increase in penile rigidity, especially when in combination with PDE5 inhibitors; the duration of the effect was variable but after a few months all patients experienced a regression

• (Bahk et al.,2010).

Current Research

• 1) current research is aimed at identifying, isolating, culturing and growing embryonal stem cells and adult stem cells.

• 2) identifying organizers/ growth factors which direct stem cells to grow into different tissue and organs.

Current Research

• 3)modifying adult multi potent stem cells to pluri potent stem cells.

• 4) engineering stem cells to avoid expression of HLA antigen which often lead to immune rejection.

• 5) optimizing the conditions for inducing pluripotyency by expressing transgenes in somatic cells

• 6) carefully characterizing the genetic and epigenetic abnormalities of current stem cells to avoid malignancy in the patient

2002 ◦ Stem Cells "Cure" Diabetes in Mouse – Stanford◦ Stem Cells Improve Motor Function in Rat Model of Parkinson's Disease -

NIH2003 ◦ Mouse Embryonic Stem Cells Develop Into Sperm – Japan◦ Human Embryonic Germ Cells Restore Movement to Paralyzed Rats –

Johns Hopkins2004 ◦ Heart Muscle Cells Produced from Human Embryonic Stem Cells Can

Replace Biological Pacemaker – Israel◦ Mouse Sperm Generated from Stem Cells Able to Fertilize Mouse Egg -

Harvard

Research Highlights of the Field

2005◦ Embryonic Stem Cells Cure Mouse Model of Hemophilia – UNC Chapel

Hill◦ Alternative Method for Deriving Stem Cells Proven in Mice – MIT

2006◦ Scientists Generate Human Embryonic Stem Cell Lines from Single Cells

– Advance Cell Technology◦ Scientists Reprogram Adult Mouse Skin Cells by Adding Defined Factors

– Japan 2007 ◦ Reprogrammed Mouse Skin Cells Cure Mouse Sickle Cell Anemia – MIT◦ Human Skin Cells Reprogrammed – Japan

Research Highlights of the Field

• 2008– Scientists Generate Stem Cell Line from Patient

with ALS Disease – Harvard– Adult Stem Cell Lines Created for 10 Additional

Human Diseases – Harvard• 2009– Geron Receives FDA Clearance to Begin World's

First Human Clinical Trial of Embryonic Stem Cell-Based Therapy

Research Highlights of the Field