Final Paper pdf.

Title: The Use of Menstrual Blood Stem Cells in Therapy

Date: November 2, 2017
Panama College of Cell Science Azuero Business Center, Suite 758 Avenida Perez Chitre 00395,
Panama Republica de Panama
Menstrual Blood.

Artwork by Jen Lewis, from art project, "Beauty in Blood", excerpted from the website
Gothic Life [http://gothic.life/beauty-in-blood-art-with-menstrual-blood/image/11/]
Author: Thain Yang Wey
Contact: 56 Park Ave, Lyndhurst New Jersey
Email: twey@omegabiochem.com

1
Final Paper pdf.

Table of Contents

1. Kind of Stem Cells found in Menstrual Blood …………………………… 3

2. How are Stem cells in menstrual blood isolated and collected …………. 6

3. What numbers of stem cells can be found, and in what quantity of blood…… 6

4. Are menstrual blood stem cells immuno-privileged ……………………. 8

5 Discussion: MenSCs V/s UC MSCs: ………………………………………… 11

6. MenSCs stem cell therapies being used now internationally…………. 12

7. Conclusion: ………………………………………………………………. 16

2
Final Paper pdf.

Introduction: The plasticity and immune privilege of MenSCs for potential research in
regenerative medicine.
Menstrual-derived stem cells are currently being used as a source of mesenchymal stem cells.
There is a surge of interest in clinical applications and the looming potential owing to the fact that,
MenSCs are highly proliferative, multipotent and easy to obtain from non-invasive procedures.
The human MenSCs have been recently discovered having vast potential in the therapeutic
possibilities of treating immune-mediated and neoplastic conditions. There are no ethical
dilemmas involved in using MenSCs. Human MenSCs belong to the larger family of mesenchymal
stem cells, with the exception being MenSCs are collected from menstrual fluid. MenSCs collected
from young healthy women grew at a rate of 19.4h, this is twice the rate of bone-marrow derived
cells. They have the potential to differentiate into various cell types, including bone, adipose,
cartilage, neural, cardiac, pancreatic and hepatic cel1s. In clinical applications, a good proliferation
rate is preferred due to single cell based therapies being dosed dependent with the preference of
cells from lower passages. The menstrual blood derived stem cells are experimentally observed
having characteristics such as high proliferation rate along with self -renewal capabilities and
pluripotent differentiation abilities.
The human endometrium is noted to have a population of stem cells which are responsible for
regenerative abilities. The menstrual fluid involves a population of similar cells, which upon
investigation through culturing revealed the expression of phenotypes that are of multiple lineages.
The menstrual blood-derived stem cells (MenSCs) possess stem-like characteristics such as high
proliferative potential, self-renewal as well as pluripotent differentiation abilities. Studies on
MenSCs have demonstrated their abilities of differentiating to cardiocytes. There are easy and
simple ways used in harvesting MenSCs, without necessarily conducting invasive surgical
interventions or hospitalization. [1]. The molecular profile assay for instance, allows the
expression of MenSCs as pluripotent markers. This includes the specific pluripotent markers such
as SSEA-4, Oct-4, c-kit and nanog. (See Figure: 1)
A group of researchers [2] collected menstrual blood from volunteer women aged 20-30 years.
These volunteers first signed an ethical informed consent sheet, thereafter, about 5-10 mls of
menstrual blood was collected from each of them using sterile Diva cups. The cups were inserted
to the vagina during menstruation. The menstrual blood obtained was thereafter transferred into
Falcon tube which contained phosphate buffered saline with no Ca or Mg ions which had been
supplemented with 2.5 micrograms of fungizone and 100 micrograms of streptomycin, 100-unit
millimeters of penicillin and lastly 0.5mM of EDTA. Mononuclear cells were thereafter isolated
by using Ficoll-Hypaque density gradient centrifugation and was thereafter washed in PBS. Non-
adherent cells were removed after the second day of incubation and culture adherent cells were
continued until a confluence of 70% was attained. In order to induce differentiation, the researchers
isolated MenSCs into epidermal lineage by co-culturing the isolated cells with keratinocytes that
were obtained from foreskin of healthy newborn males who were below 10 months.

3
Final Paper pdf.

Figure 1 Patel, Amit & Park, Eulsoon&Kuzman, Michael & Benetti, Federico & Silva, Francisco
&Allickson, Julie. (2008). Multipotent Menstrual Blood Stromal Stem Cells: Isolation, Characterization,
and Differentiation. Cell transplantation. 17. 303-11. 10.3727/096368908784153922.

The researchers further isolated MenSCs by using adherent culture plates which after 24 hours
were highly proliferated [2]. After attaining confluency of 75%, the cells depicted spindle-
morphology like fibroblasts. The researchers used cytometry analysis which allowed culturing
MenSCs. The culture results revealed expression of mesenchymal markers like CD29, CD10,
CD105 and lastly CD73. Analysis of these markers further revealed that there was lack of
hematopoietic stem cells markers. The researchers used the differentiation of culture system and
the initial morphology of MenSC which was spindle-shaped was later changed into nearly irregular
round to polygonal shape. After a fortnight, the researchers used immunostaining assay by
inducing the cells through indirect-co-culture system [2] This method revealed the expression of
K14 as well as involucrin.
Figure 2 & 3 below depicts isolated MenSCs which were adherent to culture plates after 24 hours.
The MenSCs were easily explanted and were highly proliferated. By reaching a confluence of
75%, the cells exhibited spindle-shaped morphology like fibroblasts (Figure 2).

4
Final Paper pdf.

Fig. 2: Cultured menstrual blood-derived stem cells. The adherent cells showed spindle-shaped like
morphology. (World J Plast Surg. 2016 Jan; 5(1): 26–31.0)

Fig: 3 Immuno-staining of epidermal Markers of involucrin (a) and K 14 (b) (World J Plast Surg. 2016 Jan;
5(1): 26–31.0)

Fig.: 4 Flowcytometric analysis of isolated menstrual blood-derived stem cells. The isolated cells expressed
specific markers of mesenchymal stem cells. (World J Plast Surg. 2016 Jan; 5(1): 26–31.)

5
Final Paper pdf.

Figure 5 Flowcytometric analysis of isolated menstrual blood-derived stem cells. The isolated cells did not
express specific markers for hematopoietic stem cells. (World J Plast Surg. 2016 Jan; 5(1): 26–31.)

In the pictures above, researchers used cytometry analysis which allowed culturing of MenSCs
and thereafter revealed the expression of mesenchymal markers like CD29, CD10, CD105 and
lastly CD73. (Figure 4 and 5) An analysis of these markers further revealed the lack of
hematopoietic stem cells markers when differentiation of culture system is applied, the initial
morphology of MenSC was spindle-shaped, it later changed to nearly irregular round to polygonal
shape.
Stem Cells in Menstrual Blood Isolated and Collected:
In another study, researchers used intensive methods in the procurement as well as processing of
the menstrual stem cells from females [3]. The menstrual stem cells were isolated, collected and
preserved from menstrual blood. The initial step involved collecting of menstrual blood using
procurement kit prepared by the processing facility. The kit was approved by the IRB study to be
used in the collection, processing, cryopreservation and lastly in post-thaw viability of MenSCs.
The collection of menstrual blood took less or more than 4 hours. The blood was mainly collected
in day one, two or three of menstruation Menstrual flow was heaviest during these days of the
menstruation cycle. The average menstrual blood collected was 8-10 mls in every sample. The
average collection for this was around 30 million with collection variations ranging between 0.5
to 40% of the adherent cells. In the first culture, around a million cells were only required so as to
select the adherent cells, of which around 10% of the cells depicted adherence of around 100, 000
cells being sub cultured. The cells thereafter doubled after approximately 24 hours. The samples
were collected and stored at 4 degrees after collection. They were thereafter shipped to the
laboratory by using frozen bricks to ensure the cells were not interfered with in cool temperatures.
Another menstrual blood stem cell research tried to process and cryopreserve Menstrual blood
cells by using buffered saline media (DPBS) throughout the entire procedure of cell isolation
process by using heparin [3]. The collected Menstrual blood cells were placed in a buffered saline
conical collection tube and thereafter subjected to centrifugation at 2000 rpm for a period of 7
minutes at 4 degrees Celsius. Supernatant was then used in microbiological testing. Pelleted cells
were later suspended to determine cell viability and count. The cells were thereafter prepared by
using cryopreservation and bacteriological analysis of the supernatant by performing
BacT/ALERT system. The researchers thereafter grew the cells by using either passage 6 or 9. The

6
Final Paper pdf.

menstrual blood stem cells were then cultured to add an addition of 3 passages before testing to
observe for contamination signs.
In another study, researchers collected menstrual blood-derived stem cells from healthy donors by
using silicone cups [4]. This experiment used menstrual Stem cells blood and bone marrow
mesenchymal stem cells from three donors. The menstrual blood was then transferred to 50ml tube
that had 10 ml of phosphate buffered saline which contained 0.25 mg/ml amphotericin B,
streptomycin 100 mg/m, penicillin 100U and 2 Mm of ethylenediaminetetraacetic acid (EDTA).
The menstrual blood mononuclear cells were then separated by Ficoll-Paque plus density gradient
and thereafter washed in PBS. The cells were thereafter cultured by using a T25 flask which
contained Dulbecco’s and was then modified with Eagle’s medium, high glucose and
supplemented with 1% streptomycin/ penicillin, 1% amphotericin B and lastly 1% glutamine as
well as with 15% fetal bovine serum. This culture was placed in 37 degrees Celsius and 5% carbon
dioxide so as to obtain adherent cells. The media of this culture was then changed the next day
through washing off the non-adherent cells. The cells were seeded once more.
The findings of the study by [4] showed Aliquots derived from the 2 samples of cells, passages
either 6 or 9, and grown for additional 3 passages in culture, were found to behave similarly. Cell
yields, number of adherent cells and those expressing neuronal phenotypes, graft survival and
functional effects showed near complete resemblance of the 2 cell populations. Thus, the data from
these 2 cell populations were collapsed into a single treatment condition.
A flow chart of experimental procedures is provided (Fig. 6). Cultured menstrual blood cells
display embryonic stem cell-like features Immunocytochemical assays of cultured menstrual blood
reveal that they express embryonic-like stem cell phenotypic markers (Oct4, SSEA, Nanog)
(Fig.2). Greater than 90% of the cells were positive for these pluripotent markers. They maintained
these stem-ness properties up to at least 9 passages plus the additional 3 culture passages (i.e.,
longest time point the cells were cultured in this study). In addition, their growth rate or
proliferative capacity did not change over time. Of note, human ES cells showed the typical
specific nuclear staining, but detected a few cells positive.

7
Final Paper pdf.

FIG.6. Cultured menstrual blood cells display embryonic stem cell-like features. Panels A and B are positive control
images taken from human embryonic stem cells expressing the phenotypic markers Oct4 and CXCR4.
Immunocytochemical assays of cultured menstrual blood reveal that these cells (75%) were CXCR4-positive, a stem cell
chemotaxis marker (Panel C). Furthermore, they express embryonic-like stem cell phenotypic markers Oct4, SSEA, and
Nanog as shown in panels D–F, respectively. Greater than 90% of the cells were positive for these pluripotent markers.
They maintained these stemness properties at least up to passage 9 plus the additional 3 passages in culture (ie, longest
time point the cells were cultured in this study). In addition, their growth rate or proliferative capacity did not change
over time. The cells were plated on a coated 10-cm dish in DMEM/F12 supplemented with ITS and medium was changed
twice a week throughout the study. STEM CELLS AND DEVELOPMENT Volume 19, Number 4, 2010 © Mary Ann
Liebert, Inc. DOI: 10.1089/scd.2009.0340

Are Menstrual Blood Stem Cells Immuno-privileged?

In view to the discussions above relating the manner in which MenSCs are collected, menstrual
blood cells have been observed to have immune-privilege capabilities. MenSCs have the
potential of being used in regenerative transplantation therapies for diverse tissues and organs.
As s a result of MenSCs overcoming various ethical challenges that are associated with human
stem cells, MenSCs have been associated with a diminished possibility of having immune
rejection. To this end, there are various possibilities of using MenSCs as sources of cell therapy
if solutions are created that will diminish the current source limitations of using human MenSCs.
Several studies have indicated the range of stem cells whenever they are transplanted in specific
microenvironments and thereafter stimulated by the niche. After the release of cell growth
factors, the cells stimulate surrounding tissues into having regeneration and differentiation
capabilities into specific organ or tissue-like cells. Using stem cells in treating medical
conditions should be effective and safe. In another study, it indicated Human derived menstrual
blood stem cells having high levels of mesenchymal stem cell biomarkers [5]. This suggests
MenSCs have the potential of being cultivated and isolated to produce various body cells. As a
result of MenSCs depicting differentiation capabilities, they are able to be stimulated and
induced in any body organ under optimal conditions and regenerate. This has been evidenced
from study [5] whereby the authors used MenSCs after stimulation to differentiate them into
cervical-like fibromuscular tissue in the microenvironment of the cervix.
8
Final Paper pdf.

Stimulation of human derived MenSCs in the cervix of pregnant women with cervical
complications such as cervical incompetence [5]. demonstrated that by introducing MenSCs, so as
to transfer collagen fibers as well as muscle fibers or stimulate them in the cervix, the results from
this study demonstrated there were no statistical findings in which the patients showed signs of
infection. Rather, MenSCs is a main source of proteinase-nexin-1, a product which has strong
antimicrobial properties. The same study studied on aquaporin by having cervical smear, a first of
its own in literature. The results obtained vindicated MenSCs caused improved rates of skin graft
survival. MenSCs was also observed as having a rich source of early pregnancy factor that has
immunosuppressive as well as growth factor properties.
Another research demonstrated menstrual blood cell injections as having the capacity to offer
restorative therapy in patients with stroke, hence decreased disability among the affected patients
[6]. The therapeutic agents relied on the migration from the site of injury secreted neutrophilic
factors and immunomodulators as being the major footholds of therapeutic agents. A comparison
of bone-marrow derived cells and menstrual blood cells demonstrated that menstrual blood cells
as presenting more immature phenotype as well as behaviors and at the same time maintained the
characteristic abilities of adult stem cells safety. Many experimental studies have also
demonstrated the advantages of menstrual blood cell administration towards functional
improvement and tissue repair not only in the central nervous system but also in the ischemic limbs
and the heart.
The proliferative characteristics of human menstrual blood-derived cells were shown in [7] as
having high proliferative rates as well as immature phenotypes by expressing themselves through
embryonic cell markers which had remained unaltered after passing through 20 passages. In trying
to study the immune-suppressive characteristics of MenSCs in comparison with the more advanced
bone marrow derived MSCs by using in vitro proliferation assays [8], demonstrated that MenSCs
had lower suppressive effects on the peripheral blood mononuclear cells specifically on the pro-
inflammatory CD4 IFN-y as well as CD8 IFN-y cells than the bone marrow derived-MSCs. The
authors experimented on the immunosuppressive characteristics of the MenSCs by assessing their
capacity towards modulating the proliferative response of the CFSE-labeled PMMCs on the
stimulation of PHA. The MenSCs however failed in exerting therapeutic effects on the CIA model
that was used [9].
Regarding neurovascular disorders [3], demonstrated menstrual blood transplantation in an
experiment of stroke showed that once the cells had been isolated, expanded and thereafter selected
for CD117, markers that are associated with migration and high proliferation as well as survival
were present. The expression of embryonic like stem cell phenotypic markers like SSEA-4, Nanog
and Oct4 were cultured in 9 passages and thereafter induced to express neutral markers. After an
evaluation of this experiment, some human cells were found to have migrated hence depicting cell
differentiation of menstrual blood stromal cells.
Current Stem Cell Therapies:
The undifferentiated cells that can replicate themselves without differentiating are known as stem
cells. Stem cells have the potency to differentiate into various cell types such as adipocytes,

9
Final Paper pdf.

osteoblasts, hepatocytes and neurons. Furthermore, under specific conditions stem cells can be
induced to undergo mitosis to produce more functional specialized cells [10]. As a result of these
properties, stem cell therapy by transplantation has become a potential therapeutic option in the
near distant future.
Current research is therefore focusing on the hematopoietic cell transplantation (HCT), which has
the potential to cure a variety of hematologic malignancies. The use of allogeneic mesenchymal
stem cells (MSC) has evolved substantially due to the advances in selection of sources of stem
cells from donors. Along with this, new approaches have been also introduced to prevent
complications, toxicity or rejection during transplantation. This has in turn resulted to patient count
for HCT being significantly expanded.
Many research studies carried on both animals and human beings support the immune privileged
state of MSCs, due to their unique immune-modulatory properties and the abilities of being
harvested from young and healthy donors. In another study, the authors mentioned a new source
of highly proliferative stem cells within menstrual fluid which display novel properties [11]. The
authors identified an alternative to MSCs that has the abilities to differentiate under specific
conditions into specific-tissue cells of three germ layers known as menstrual-derived stem cells
(MenSCs). MenSCs have many advantages over the conventional MSCs as they can be easily
accessed without any surgical procedures which in turn make them free from ethical dilemmas [1]
Immuno-phenotype of MenSCs:
It is observed that MenSCs have positive expression for certain MSC markers including CD9,
CD29, CD105, CD73 and for embryonic markers such as SSEA-4, Nanog. They have negative
expression for CD34, CD45 and CD133 hematopoietic markers. It was observed that MenSCs
mildly express some receptors such as chemokine receptor and receptor for SDF-1 that are
involved in MSC migration. [10]
The menstrual blood derived stem cells have undergone nuclear karyotype analysis to observe
visible abnormality at the chromosomal level. It has been found that the telomerase activity is 50%
greater in MenSCs than embryonic stem cells and MenSCs can be expanded without any mutation
[10].
Advantages of MenSCs:
The highly proliferative nature of MenSCs has been investigated for immune suppressive
properties in comparison with bone marrow MSC. The researchers estimated at 40-45 h in early
passage and observed twice faster growth rate of MenSCs in menstrual fluid of young healthy
women than BM-MSCs [1].
Recently, in vitro proliferation assay, found out that MenSCs have lower suppressive effect on
peripheral blood mononuclear cells and pro inflammatory CD41IFN-c1 and CD8IFN c1cells as
compared to BM-MSCs. MenSCs activated with IFN-c and IL-1B produced lower amounts of
immunosuppressive factors including IDO, PDL-1, PGE2 and Activin A and lower expression of
IFN-c also observed. A study carried out in collagen induced arthritis model where MenSCs

10
Final Paper pdf.

injection did not induce potent therapeutic effect but BM-MSC administration provided
therapeutic effect by decreasing pro-inflammatory T cell frequency in lymph nodes [8]
Differentiation Potential and Regenerative Properties:
MenSCs have great potential of differentiating to different cell types. The multi-potent nature of
MenSCs allows them to differentiate into respiratory epithelial cell, endothelial cells and many
other cell types.
A group of researchers [8], revealed immortalization by the help of human telomerase reverse
transcriptase on MenSCs when co-cultured with fetal mouse cardiomyocytes. MenSCs also
showed higher expression of TNNT2 cardiac marker after cardiogenic differentiation in a scaffold
culture when compared with induced BM-MSCs. In chronic liver disease the production of hepatic
cells from BM-MSCS by differentiation is a well-known cell based therapy, but a recent research
suggested that MenSCs can express many hepatic markers including albumin, cytokeratin 18 and
tyrosine amino transferase and hence using MenSCs based therapy is a safe alternative to BM-
MSCs. MenSCs not only express hepatic cell marker or cardiac marker but also up regulate glial
fibrillary acidic protein, Olig-2 and down regulate Nestin protein [8].
Discussion: MenSCs V/s UC MSCs:
In allogeneic therapy, it was observed that both MenSCs and UC MSC’s are good sources, but still
there is a debate between researchers to understand which is a better source of MSC’s to be used
in human therapy without any adverse effect. During menstrual period from the uterine lining
(endometrium) MenSCs are secreted. MenSCs can be harvested easily in a painless and
noninvasive manner.
Researchers found out that cord blood stem cells and menstrual stem cells have the abilities to
differentiate into various kinds of cell lineages. Furthermore, the researchers discovered both stem
cells have abilities to contribute in cell survival due to their immunologically immature nature.
In support with the fact that MenSCs can differentiate into various cell types, a research
successfully carried out to investigate the improvement in behavioral function when MenSCs were
infused into an animal model of stroke [3]. It was observed that MenSCs differentiated into nerve
cells. The chief scientists at Cryo-Cell International, Julie Allickson found stem cells from
menstrual blood. She further explained that the collection of both umbilical cord blood and
menstrual blood is easy but in case of menstrual blood it is very necessary to collect it in a hygienic
way. Collection of menstrual blood requires a medical grade silicon cup being inserted up to 3
hours in the vagina during the period of heaviest flow of a woman which thereafter collects about
10 to 20 milliliters of blood. The collected blood is poured into container in a collection kit Cryo-
Cell for further processing and stored in menstrual blood bank [3].
Current Stem Cell Therapies Being Used Now Internationally:
Menstrual blood has been indicated as a source of mesenchymal stem cells. This finding represents
the fact that menstrual blood carries unlimited potential as being a source of stem cells which will
be used in regenerative medicine. Menstrual blood stromal cells have been observed in various
studies as being clinically relevant in the expression of multipotent markers at the cellular and

11
Final Paper pdf.

molecular level. This suggests the potential of MenSCs in playing a critical role in transplantation
therapies for various diseases. MenSCs were recently used internationally in human therapy and
it indicates that MenSCs takes part in playing various roles which is further discussed in this part.
1. Clinical usage of MenSCs was first reported, when four patients were administered allogenic
injections with aggregate dosage of sixteen to thirty million cells. The four patients who were
diagnosed with multiple sclerosis did not display any physical or serological abnormalities during
follow up diagnostics [12]. Recent reports reveal that a stem cell company, Medistem, used
MenSCs in phase II clinical trials that it launched, embarking on ambitious plans to get 60 patients
on board. The plan was to administer the 60 patients who were diagnosed with congestive heart
failure with dosages that increased up to a staggering two hundred million cells; and all from a
single donor. A report that was published in 2013 revealed statistics that seventeen patients who
received the injections did not experience or report treatment related adverse effects [13]. FDA
clearance has been granted to Medistem to commence Phase I trials. The trials to be conducted in
the U.S, are intended to treat critical limb ischemia, which is a manifestation of periphery artery
disease.
2. Autologous menstrual blood-derived stromal cells transplantation for severe Asher man’s
syndrome [14], investigated on the autologous transplantation of menstrual blood-derived stromal
cells (MenSCs) of stromal fibroblasts and mesenchymal stem cells. The authors investigated
whether they could regenerate women’s endometrium with severe Asherman Syndrome and
thereafter facilitated healthy parturition. The prevalence of Asherman ranges from 2-22% and is
regarded as the primary cause of secondary infertility among women. The authors recruited
infertile women diagnosed with severe Asherman Syndrome.The traditional curative methods of
Asherman Syndrome mainly aim at restoring uterine fertilityas well as parturition. This is achieved
by doing surgical resections such as hysteroscopy and hormonal therapies so as to regenerate the
endometrium. In severe cases, the traditional therapeutic interventions are limited in regenerating
new cells potentially leading to regeneration failure and formation of adhesions. In an attempt to
come up with novel medical interventions, stem-like supplementation is required in women
suffering from severe Asherman Syndrome. Faced with this challenge, the authors recruited
women with severe Asherman Syndrome and investigated if autologous transplantation of
MenSCs would regenerate in the endometrium hence facilitate healthy parturition. They conducted
autologous MenSCs transplantation and followed it with HRT. The endometrial thickness was
monitored using the frozen embryo transfer technique [14].
The researchers [14] isolated and cultured MenSCs through collecting samples of
menstrual blood by using catheters that had been rinsed by streptomycin and penicillin from the
patients after day two of their menses. The samples collected were thereafter transferred to
phosphate buffered saline which contained heparin, streptomycin and penicillin. Mononuclear
cells were fractioned by using the Ficoll’s solution and thereafter cultured by using Dulbecco’s
modified eagle medium. A supplement of HyClone Logan was added with 10% autologous serum.
This culture medium was changed after three to five days with the cells being passage through
trypsin digestion so as to reach a confluence of 80-90%. After this process, the culture supernatant
of MenSCs was sampled after the medium being changed at least twice and thereafter sent for

12
Final Paper pdf.

testing of fungi, bacteria. Hepatitis virus and lipopolysaccharides in a clinical laboratory. Detection
of any microbial contamination lead to abandoning the cells. The cells were thereafter cultured for
up to 14 days and thereafter MenSCs were transplanted before reaching the third passage. (figure:
7) Figure 7: Study design of this research. ET; endometrial thickness.

The researchers conducted flow cytometry to identify the phenotypes of the MenSCs before
doing transplantation. On the sixteenth day of the current menstrual cycle the patient was
transplanted with MenSCs. The MenSCs were trypsinised thereafter washed twice, counted and
then re-suspended by using PBS. A scratch on the endometrium was done thereafter
transplantation of MenSCs was delivered to the fundus of the uterus. The hormonal replacement
therapy was thereafter adopted so as to stimulate endometrial growth [14]. The subjects were
given oral oestradiol tablets after day five of menstruation up to day 14. The thickness of the
endometrium was monitored and increases of more than 7mm indicated a triple-line appearance.
The results of the seven patients investigated with severe Asherman Syndrome showed all of
them having positive results for MenSC culture. The autologous cells transplanted back to the
uterus after hormonal stimulation suggested significant increases in ET in all the participants.
The results of ultrasound indicated recovery of the endometrial morphology into the normal
status [14]. 5 women had thickness of the endometrium reaching 7-8mm and indicated triple-line
appearance.

13
Final Paper pdf.

Endometrial thickness before and after MenSCs transplantation. Data were shown as mean values ±SEM,

***P <0.00.

At least 43% of the participants were able to conceive successfully. These results suggested that
the autologous MenSCs transplantation could be among the best treatment modalities for
endometrial regeneration as long as enough MenSCs would be cultured for treatment. The cells
that had been cultured appeared as being adherent as well as indicated clonal cell phenotype. The
analysis of the flow cytometry indicated that the cells were positive for the CD 105, CD90, CD73
and CD 44. The findings obtained in this research supported the speculation that MenSCs confer
unique advantages as well may be the best alternative in the future than bone marrow stem cells.
The transplantation in this research of MenSCs was successful and it increased the ET of the
patients ailing from Asherman Syndrome.
This article demonstrates the usage of autologous MenSCs transplantation as being a potential
alternative of treating women affected with severe Asherman Syndrome in the future. The data
further obtained from this study indicated that cell therapy is a promising intervention that will be
applied in the treatment of severe Asherman Syndrome. There is need for further research to
investigate the proliferation and differentiation of MenSC under hormonal stimulation in in- vitro
and in vivo studies. The limitations of this study can however be summarized a; the study is not
yet adequate in evaluating the application of MenSCs therapy due to investigating only 7 patients.
There is need of having long-term randomized control studies with several patients. The outcome

14
Final Paper pdf.

from pregnancy in this study was not very successful probably due a low number of implanted
MenSCs at the endometrium.
3.Treatment of Refractory (Resistant) Cerclage by Human Menstrual Blood Stem Cells-
Modern Trend. [5] discuss resistant refractory cartilage, a concept which entails repeated failure
of vaginal cerclage which has been done more than 3 times by expert gynecologists without
contradictions for the procedure. Human MenSCs are noted to exhibit progenitor and stem cell
properties and may be used in the repair of various types of cells in vivo. Human MenSCs have
high levels of expression of the mesenchymal stem cells such as CD49f, CD 117, CD 105, CD 90
and CD44 as well as the embryonic stem cell markers such as SSEA3/4 and Oct4. Since MenSCs
are easily available than bone marrow stem cells, their usage as a potential donor source for stem
cell therapy was attempted in resistant cerclage.
The study conducted by the authors entailed preparation of human menstrual blood stem cells by
using the methods of culturing cells, western blotting, flow symmetry analysis and
immunofluorescence staining. A total of 10 patients with refractory cerclage who participated in
the research and had been picked with varying gestations between 8-10 weeks were subjected to
autologous intracervical injection of human menstrual blood cells. An ultrasound was done before
the procedure and thereafter every fortnight. A cervical smear for aquaporin as well as cervical
mucous for the detection of collagenase and IL8 were conducted before the procedure and during
delivery. The detection of IL8 at cervical mucous before the procedure was done and thereafter
monthly, detection of aquaporins AQP5, AQP4 and AQP3 were also tested in the cervical smear
by using immunohistochemistry.

An evaluation of the staining intensity was thereafter performed and a grading scale ranging from
0-3 whereby 3 was intense staining, 2-moderate staining, 1-faint staining and 1 with no staining
was performed. It was noted that in all the smears the number of cells that were stained was the
same. The results indicated 7 of the women who participants as having full term deliveries through
the cesarean section while two of them ended delivering at 32 and 34 weeks, the last one had an
abortion at 16 weeks. The ethical issues of this research were compiled in accordance to the
declaration of Helsinki [5].
This study demonstrated that MenSCs improved the skin graft survival as well as it was a source
of early pregnancy indicator whereby it acted immunosuppressed and the growth factor. The
method conducted by the authors illuminates a number of advantages of using MenSCs over the
15
Final Paper pdf.

current treatment modalities. The method of enrichment and isolation of the human MenSCs is
convenient [5]. The human MenSCs that are derived from the endometrium were shown to have a
high level of stemness hence their isolation and enrichment is simple. The technique reemployed
in this study was non-surgical, required no need of antibiotics or anesthesia. The method can be
further conducted at the outpatient clinic At least 70% of the patients reached full term and 20%
reached pre-term, the remaining 10% were the failed cases. Cesearean delivery was the primary
method of delivery used in all the patients. There was a positive effect on the fetus, had strong
antimicrobial properties, easy and cheap to learn it. The implications of this study implored that
the management of refractory cerclage through cervical injection of autologous human menstrual
blood stem cells as being effective, safe and cheap with no fetomaternal complications, positive
fetal effects. There is the need of further research in this area however that is required before the
elucidation and the effectiveness of the technique in women.
4.MenSCs Stem Cell Therapy in Stroke: A Review Literature [15] discuss the various methods
which are used in stem cell therapy in patients with stroke. It discusses on the various routes and
types of administration which are used in using stem cells as an alternative intervention of treating
neurologic diseases. The authors conducted an autologous experiment. Stem cell therapy as
applied in stroke indicated there were a number of germ cell lines which had the abilities of
differentiating into neurons as well as have widespread migration into other sites other than the
anatomical locations [15]. MenSCs obtained from menstrual blood were expanded in a certain
study thereafter expanded and presented with multipotential and clonogenic characteristics. The
MenSCs expressed a number of pluripotency markers like SSEA-4 and Oct-4. MenSCs had a high
affinity of autologous transplantation, lacked ethical dilemmas and had low immunogenicity
because they lacked expression of MHC class II, secretion of neutrophilic factors like angiogenic
and VEGF, immune-dilation characteristics and lastly the capacity to differentiate or express its
neural markers. These characteristics exhibited by MenSCs suggest they may serve in restorative
therapy for patients being rehabilitated from stroke.
Clinical trials conducted over MenSCs had indicated promising results towards the treatment of
stroke. The efficacy of MenSCs in transplantation in stroke patients was done in 5 patients for the
intravenous transplantation of autologous MenSCs on patients with ischemic stroke. This is due
to the evidence that had been observed early of menstrual stem cells being highly proliferative and
that the stromal cells that could be obtained from menstrual blood could be presented as clonogenic
as well as multipotent properties. The implication of this study indicates the potential of MenSCs
being used in future treatment modalities in the transplantation efficacy of stem cells in patients
with stroke. However, there is further progress required in order to ascertain as whether stem cells
will be the best option for patients with stroke.
Conclusion: Menstrual blood derived stem cells MenSCs are emerging as an alternative source
for the isolation of MSCs such as ADMSCs, BM-MSCs and UCMSCs. MenSCs have immune
modulatory properties that allow them to become immune privilege for allogeneic as well as
autologous transplantation. MenSCs have great potential for self-renewal, plasticity as well as
longer undifferentiated proliferation rate. It is reported that MenSCs and ADMSCs have increased
proliferation rate as compared to BM-MSCs and UCMSCs. In a clinical study it was observed that

16
Final Paper pdf.

MenSCs and ADMSCs have remarkable cell- cell contact inhibition, whereas in case of UCMSCs
it was insignificant. The isolation rate and the cell volume are higher in MenSCs relating to
umbilical cord blood 1.8 million stem cells are shown positive expression of stem cell marker
CD34 and the frequency of stem cell in the bone marrow increases to 1 in 1000 to 1 in 2000 cells
in humans and mice. The differentiation ability of MenSCs showed to be effective as compared
to BM-MSCs for the treatment of chronic liver disease. The MenSCs have shown
immunosuppressive properties like any other sources of MSCs. MenSCs produce indoleamine
dehydrogenase (IDO) which mediate the suppression by exhaustion of tryptophan. In addition,
MenSCs can be easily accessible and can differentiate into different cell types including neuronal
cell, pancreatic cells and osteoblasts. Therefore, MenSCs possess specific characteristics and
immune profiles that make it a potential cell based therapy in the field of regenerative medicine.
MenSCs collection procedures are non-invasive and doesn’t have surgical intervention, unlike
adipose tissue derived stem cell, bone marrow derived stem cells and umbilical cord blood derived
stem cells. Although UCBSCs can be collected without harming the donor, but the collection is
restricted only at the time of birth by contrary MenSCs can be collected from menstrual blood once
a month from a reproductive woman frequently to ensure higher therapeutic doses of low passages
MSCs in cell based therapy.
The most crucial fact that I have found in this research is that the collection of Menstrual blood
can be done at the privacy and convenience of home by using a 'Menstrual Cup' - a conical shaped
medical grade silicon cup which can collect up to 30 ml of menstrual blood. Since there is no
surgical procedure or equipment involved in the process underdeveloped countries can also obtain
menstrual blood and harvest for the isolation of MenSCs cells in an inexpensive and noninvasive
way.

17
Final Paper pdf.

Bibliography
1. Khoury M, Alcayaga-Miranda F, Illanes S, Figueroa F. The Promising Potential of Menstrual
Stem Cells for Antenatal Diagnosis and Cell Therapy. Frontiers in Immunology. 2014;5.
2. Faramarzi, H., Mehrabani, D., Fard, M., Akhavan, M., Zare, S., Bakhshalizadeh, S., Manaf,
A., Kazemnejad, S. and Shirazi, R. The Potential of Menstrual Blood-Derived Stem Cells in
Differentiation to Epidermal Lineage: A Preliminary Report. www.wjps.ir /Vol.5/No.1/January
2016
3. Borlongan C, Kaneko Y, Maki M, Yu S, Ali M, Allickson J Et al. Menstrual Blood Cells
Display Stem Cell–Like Phenotypic Markers and Exert Neuroprotection Following
Transplantation in Experimental Stroke. Stem Cells and Development. 2010;19(4):439-452.
4. Alcayaga-Miranda F, Cuenca J, Luz-Crawford P, Aguila-Díaz C, Fernandez A, Figueroa F et
al. Characterization of menstrual stem cells: angiogenic effect, migration and hematopoietic stem
cell support in comparison with bone marrow mesenchymal stem cells. Stem Cell Research &
Therapy. 2015;6(1).
5. Ali F, Farid, l. Ali Y. Ali M. Treatment of Refractory (Resistant) Cerclage by Human
Menstrual Blood Stem Cells-Modern Trend. 2017; ISSN 2229-5518
6. Rodrigues M, Glover L, Weinbren N, Rizzi J, Ishikawa H, Shinozuka K Et al. Toward
Personalized Cell Therapies: Autologous Menstrual Blood Cells for Stroke. Journal of
Biomedicine and Biotechnology. 2011; 2011:1-7.
7. Patel A, Park E, Kuzman M, Benetti F, Silva F, Allickson J. Multipotent Menstrual Blood
Stromal Stem Cells: Isolation, Characterization, and Differentiation. Cell Transplantation.
2008;17(3):303-311.
8. Luz-Crawford P, Torres M, Noël D, Fernandez A, Toupet K, Alcayaga-Miranda F et al. The
immunosuppressive signature of menstrual blood mesenchymal stem cells entails opposite
effects on experimental arthritis and graft versus host diseases. STEM CELLS. 2015;34(2):456-
469.
9. Ulrich D, Muralitharan R, Gargett C. Toward the use of endometrial and menstrual blood
mesenchymal stem cells for cell-based therapies. Expert Opinion on Biological Therapy.
2013;13(10):1387-1400.
10. Lin J, Xiang D, Zhang J, Allickson J, Xiang C. Plasticity of human menstrual blood stem
cells derived from the endometrium. Journal of Zhejiang University SCIENCE B.
2011;12(5):372-380.
11. Faiella W, Atoui R. Immunotolerant Properties of Mesenchymal Stem Cells: Updated
Review. Stem Cells International. 2016; 2016:1-7.
12. Zhong Z, Patel A, Ichim T, Riordan N, Wang H, Min W Et al. Feasibility investigation of
allogeneic endometrial regenerative cells. Journal of Translational Medicine. 2009;7(1):15.

18
Final Paper pdf.

13. Bockeria L, Bogin V, Bockeria O, Le T, Alekyan B, Woods E Et al. Endometrial

regenerative cells for treatment of heart failure: a new stem cell enters the clinic. Journal of
Translational Medicine. 2013;11(1):56.
14. Tan J, Li P, Wang Q, Li Y, Li X, Zhao D Et al. Autologous menstrual blood-derived stromal
cells transplantation for severe Asherman's syndrome. Human Reproduction. 2016;31(12):2723-
2729.
15. Meamar R, Dehghani L, Ghasemi M, Khorvash F, Shaygannejad V. Stem Cell Therapy in
Stroke: A Review Literature. International Journal of Preventive Medicine. 2013;4(Suppl 2):
S139-S146.

19