Ch 19 _perinatal_stem_cells_isolated_from_complete_umbilical_cord_tissue_for_family_stem_cell_banking_and_potential_ther

Chapter 19
Perinatal Stem Cells Isolated From
Complete Umbilical Cord Tissue for
Family Stem Cell Banking and Potential
Therapeutic Use
Robert Briddell1
, Frank Litkenhaus1
, James E. Carroll2
, Mohammed Ali2
, Kate F. Girard1
, William Fodor3
and
Morey Kraus1
1
ViaCord LLC, a PerkinElmer Company, Cambridge, MA, United States; 2
Augusta University, Augusta, GA, United States; 3
Cell Therapy Group,
Madison, CT, United States
Chapter Outline
Goody BagdAfter Birth 257
Going NativedWithout Further Manipulation 258
Know-HowdThe Process Is the Product 259
Safe Deposits. What to Bank on 261
The GamishdConsistent Heterogeneity 262
Proof of Principle. A Preclinical Model With Functional
Output 263
Animal Recognition and Removal Behavior Tested with the
Forepaw Adhesive Square Method 265
Closing the LoopdBack to Birth and Banking 266
List of Abbreviations 268
References 268
GOODY BAGdAFTER BIRTH
Every baby comes into this world with a “goody bag” at his or her moment of birth. In human mothers, unlike most
peripartum mammalian females who consume the afterbirth [1], this goody bag has been summarily discarded after birth.
Scientific advancements have now made more families, and physicians aware that this goody bag, which just moments ago
was the lifeline of the fetus, contains within its mass and structure relatively large numbers of pristine perinatal stem cells
(PSCs). These cells are now candidates for preservation and are currently being explored for a wide variety of clinical
applications.
For descriptive purposes, there are four anatomically unique areas of this afterbirth from which compositionally distinct
stem cell populations can be harvested. These are the residual liquid in the umbilical vessels commonly known as cord
blood (CB); the flexible cable that connected the baby to the placenta or the cord tissue (CT); the pancake-shaped organ
implanted into the wall of the uterus or the placenta (P); and the aqueous fetal environment including its barrier or the
amniotic fluid (AF) and membrane (AM), respectively. Shortly after delivery, more than 95% of these biological goody
bags are today routinely discarded. This chapter will focus exclusively on one of the four populations; the phenotypic and
functional characterizations of PSCs from complete umbilical CT can be considered for robust isolation and cryopres-
ervation for potential future therapeutic utility.
Unlike a typical birthday party with goody bags containing sweets and toys, this original birth moment yields a goody
bag with labile biological ingredients containing, in particular, significant numbers and types of potent PSCs. These goody
bags cannot readily be harvested, carried home, and preserved by the parents. In this regard, parents are unlikely to have
Perinatal Stem Cells. https://doi.org/10.1016/B978-0-12-812015-6.00019-4
Copyright © 2018 Elsevier Inc. All rights reserved.
257

the technical means to collect, transport, isolate, preserve, or store their newborn’s PSCs in long-term cryobanking
conditions. Therefore, numerous commercial banking services also known as “private” cord blood banks or stem cell banks
or sometimes family banks, provide such services for a preagreed fee. These banks provide collection kits, transportation
logistics, processing technology, cryopreservation, and long-time storage capabilities. CB, a liquid substance composed of
the hematopoietic or blood immuneeforming lineage at the perinatal stage (i.e., also PSCs), has been for almost 20 years
the only option available to parents for banking in this manner. This is largely because of the ease of CB collection and the
early recognition that neonatal blood [2] and later CB did indeed contain significant numbers of highly potent hemato-
poietic stem cells (HSCs) that could be effectively cryopreserved for later use [3].
In the last decade, efforts have been made to commercialize additional PSC banking options. These include PSCs from
the AM [4], AF [5], placental perfusate (PP) [6], complete CT [7], complete digested CT [8], CT epithelial membrane
(EPM) [9], or the placenta (P) [10] itself. The use of these various sources is under intense investigation, for the
manufacturing of homogeneous products, as distinct cell populations have been isolated and cultured or expanded ex vivo
from each. For family banking, CT stands out due to the practicality and simplicity of collecting, processing, and storing
CT-PSCs for future use. Therefore it has become a relatively prominent offering, second only to the banking of CB in the
private banking industry. For this reason, and to inform and guide the industry to validate clinical targets and ultimately to
the realization of value for the family’s investment in commercial banking services, information regarding the phenotypic
and functional characterization of the CT-PSCs should be further elucidated. Moreover, this is critical in identifying
appropriate clinical targets in advance of any efforts to evaluate potential therapeutic interventions.
With regard to therapeutic use, particularly with respect to CT-PSCs, no assumptions should be made. As will be
discussed later in detail, CT-PSCs are composed of a heterogeneous population of cells. The particular candidate cell, the
degree of its potency, the number required, and the route of administration for therapeutic applications are presently the
subjects of much scientific investigation. Whether the larger portion of native mesenchymal stem cells (MSCs) or various
subpopulations of native endothelial, epithelial, hematopoietic, and neural stem cells or indeed a combination of all of these
constituents represents an active therapeutic dose for various proposed indications is largely untested in human clinical
studies. This chapter will review suggestions that CT-PSCs might be used in patients with brain injuries, specifically
hypoxic ischemic injuries (HIIs), based on results from experiments in a representative rat HII model. It might be a prudent
for historical context to remember that CB, once thought of exclusively as a source of reconstitution following
radiochemotherapy due to the presence of potent HSCs, is now under active clinical investigation in applications of
regenerative medicine, including acquired brain injuries, such as cerebral palsy [11] and stroke [12], or brain dysfunction,
such as autism spectrum disorder [13].
Importantly, of those parents who today chose to bank their baby’s umbilical CB [14], nearly half have chosen to also
bank PSCs from the umbilical CT. Parents banking CT-PSCs are certainly early adopters who speculate that banking PSCs
from CT might provide incremental value. Nevertheless, the expectation that the cells isolated from complete umbilical CT
might be used alone or in combination with CB as a therapy for one or more indications has not been previously validated
in an animal model. The PSC banking industry is actively working on multiple approaches to enable several applications
for derivatives of the tissues available at birth. This chapter will discuss data regarding one such approach and the use of
heterogeneous populations of CT-PSCs alone and in combination with CB in a rat HII model of hypoxic brain injury.
Preliminary data suggest that enzyme-digested complete CT-PSC preparations display, through an unknown mechanism of
action, regenerative properties, resulting in the restoration of brain function and tissue in injured rats. Before the animal
model and the resulting functional data are discussed, one must first understand what is being collected, processed, and
stored. In other words, what is the product that contains a solution of heterogeneous CT-PSCs? In the cell therapy space, as
with other medicinal production methodologies, the process is the product. It is, therefore, prudent to first consider both the
final product and the process that generated the product and then move on to discuss the use of these cells in preclinical
models.
GOING NATIVEdWITHOUT FURTHER MANIPULATION
As a category, cell-based therapeutics have been focused on developing highly homogeneous products derived from
regimented clinical manufacturing processes This is in contrast to how CB and CT-PSCs are processed. In many instances,
manufacturing is a key commercialization requirement, particularly with respect to cost-of-goods, which is more often than
not a gatekeeper for commercial feasibility. Simply put, the cell therapy industry is keenly aware that making one product
for many is much more attractive, commercially speaking, than single unit processing for individualized or personalized
medicine. The former paradigm fits neatly into the pharmaceutical model that seeks to provide homogeneously processed,
packaged, and distributed medicines destined to reside over or under the pharmacist’s counter ready for purchase and use in
258 SECTION j III Umbilical CordeDerived Cells

indications of need. Awareness of this paradigm, along with empirical findings that have led to the development of many
elegant ex vivo expansion systems, has bolstered various efforts to expand cell populations and/or their constituent
components into homogeneous populations determined to become homogeneous cell therapy products or “cells in a bottle”
so to speak. MSCs specifically, whose growth is surface dependent and which literally grow like weeds, now have many
examples of products that have, unfortunately, fallen short of the mark of expected clinical efficacy [15]. This is not to say
that the effort to make such a product is without merit or that it will not reap significant value when configured
appropriately or articulated toward more precise medical indications where efficacy may ultimately be achievable.
Chimeric antigen receptoreactivated T cells or CAR-Ts are a hybrid example of a cell therapy product that has been
“manufactured” in a more traditional pharmaceutical configuration in the immunotherapy space, still at the single-unit,
single-use level [16]. Again, the product is the process.
Nevertheless, there is also scientific rationale to make a simple contextual case for the utilization of a native hetero-
geneous cellular composition. Native PSCs of various types, derived from whole CT tissue, can be investigated for the
treatment of individualized indications in regenerative medicine by virtue of their ability to repair and regenerate tissue
through engraftment through homing, expansion, and differentiation en vivo. For additional context, it is helpful to think of
CB, which is used primarily as a “treatment-ready” native composition. Of note, efforts to yield CB derivatives into larger
more homogeneous populations through subfractionation or ex vivo expansion has been challenging. In the case of native
CB and CT-PSC utilization, the expectation is that the appropriate cell types will home to the area of damage or injury, a
hallmark property of stem cells [17], and either engraft, including en vivo expansion, or provide a paracrine effect, or both.
The yield and phenotype of cells from each of the anatomical sections of the CT, namely the Wharton’s Jelly,
perivascular region, vessels, and lumen endothelium cellular regions, have been quantified in detail by Schugar et al. [18].
Going forward, CT-PSCs will be defined as a native heterogeneous composition of viable perinatal cells isolated en masse
from the complete umbilical CT after routine exsanguination of the newborn CB from the umbilical vein. Various ap-
proaches have been used by different groups to accomplish viable cell isolation, including enzyme digestion [8,19],
mechanical disruption [20], and explanted cultures [21]. Each of these methods may or may not be followed by ex vivo
expansion after isolation or after cryopreservation. However, the result of ex vivo expansion is invariably the generation of
a more homogeneous population of MSC-like cells with trilineage differentiation potential (bone, cartilage, and fat), the
hallmark of classic MSC cultures [22]. This may very well prove useful in any number of indications, but the resulting cells
are significantly different in both phenotype and function from the neat or native heterogeneous population of CT-PSCs
described herein. Markers of phenotype similarly coalesce around a homogeneous definition depending on the subsource
of perinatal cells or the ex vivo expansion method. Some are CD105 and/or CD90-positive to differing degrees, and some
are plus or minus major histocompatibility complex markers 1 and 2 (MHC 1 and MHC 2) [19], to name just a couple of
important phenotypic variables. Native CT-PSC isolates from enzyme or mechanical methods differ substantially in their
number, composition, and therapeutic potential from either explanted or seeded ex vivo expansion cultures, whether
accessed before or after cryopreservation [7]. Even differences in the enzyme used in the digestion, such as collagenase or
dispase [18], and time that the enzyme is exposed to the tissue to facilitate digestion can influence the composition of
isolates before and after ex vivo expansion [23].
Native heterogeneous CT-PSC populations specifically discussed in the proceeding sections are the direct coisolates of
a collagenase digestion method used before cryopreservation and represent all associated umbilical CTs, including the
epithelial cord membrane itself, the Wharton’s Jelly, the endothelium of the two umbilical arteries and the umbilical vein,
pericytes surrounding the vessels, and any residual hematopoietic cells remaining in the vasculature at the time of isolation.
Therefore, this description will focus specifically on the enzymatic digestion method of isolation and cryopreservation of
the heterogeneous population of native CT-PSCs from complete tissue, the phenotypic composition of those isolates, and
their functional characterization through en vivo expansion and engraftment in at least one model of neurologic injury.
KNOW-HOWdTHE PROCESS IS THE PRODUCT
The complete human umbilical CT is composed of an EPM, a gelatinous collagenebased connective tissue known as
Wharton’s Jelly, and vascular tissue that together contain different subpopulations of PSCs of different varieties, including
mesenchymal (MSCs), epithelial (EPCs), endothelial (a.k.a. human vascular endothelial stem cells or HUVECs), and
hematopoietic (HSCs) stem cells, again collectively referred to herein as cord tissue perinatal stem cells or CT-PSCs. The
isolation and cryopreservation of CT-PSCs for potential therapeutic use is an active area of research and has taken on
several configurations in the stem cell banking industry. These methods of isolation include, but are not limited to,
prefreeze enzymatic digestion, prefreeze mechanical disruption, cryopreservation of the tissue itself followed by either
explant culture or postthaw enzymatic digestion or mechanical disruption.
Perinatal Stem Cells Isolated From Complete Umbilical Cord Tissue Chapter | 19 259

The two most prominent current practices in the banking of CT and CT-PSCs for future use will be compared.
Specifically, it is common practice today for some stem cell banks providing CT stem cell banking services deployed to
cryopreserve a fraction of the CT itself without first isolating the cells within the tissue. Harris et al. [21] described this
method in detail. Other commercial stem cell banks enzymatically digest the CT before cryopreservation as described in
detail by Briddell et al. [8] (Figs. 19.1A and B).
These two methods have been compared experimentally to determine the optimal method for processing CT-PSCs for
banking purposes. Briefly, the experimental design consisted of taking CT that was aseptically obtained and processing
parallel CT segments of equal mass from the same newborn’s CT by each of the two methods. In method 1 (the Briddell
method), CT-PSCs were isolated from partially dissected fresh CT segments by overnight collagenase digestion (2.5 mg/
mL-g), followed by filtration and centrifugation. Isolated CT-PSCs from this process were cryopreserved in a cryopres-
ervation solution (11% DMSO, 1% Dextran 40, 4% HSA in 0.9% NaCl) and then frozen and stored at À80
C
for 30 days. Cells were later rapidly thawed at 37
C, washed, and resuspended in D-PBS for enumeration and
phenotyping using flow cytometric analysis. In method 2 (the Harris method), a segment of whole CT was cryopreserved
in the same cryopreservation solution as in method 1, then frozen and stored at À80
C for 30 days. Frozen CT tissue
was later rapidly thawed at 37
C and processed by enzymatic digestion, filtration, and centrifugation as described above to
isolate the CT-PSCs. CT-PSCs recovered from method 2 were washed and resuspended in Dulbecco’s phosphate-buffered
saline solution for enumeration and limited phenotyping using flow cytometry. Comprehensive phenotyping will be
discussed shortly.
For now, a simple measure for tracking in this processing efficiency, which captures a reproducible representative
snapshot of the predominant population isolated during the process method(s), is advisable. In this case, the flow
cytometric gating for 7AAD negativity, a viability characteristic, together with an assessment of pan leukocyte marker
CD45 negativity, allows quantification of the overall viable CD45 cell population as distinct from hematopoietic, blood-
forming, cells. To further identify the composition of isolates as primitive by nature, the addition of CD90 (Thy-1)
FIGURE 19.1A Briddell method of enzymatic digestion results in a cellular suspension comprising cord tissue perinatal stem cells.
FIGURE 19.1B Harris method of cryopreserving a whole or segmented tissue comprising cord tissue perinatal stem cells.

positivity, a marker of stemness [24] and representative of the generalized population of CT-PSCs, is useful. CD105
(Endoglin) positivity, a marker for mesenchymal lineage, may also be used but with much greater variability in expression
than that before ex vivo expansion.
In this regard, we were able to quantify and compare the number of viable CD45À/CD90þ CT-PSCs after cryo-
preservation and thawing from each of the two methods described above. When CT-PSCs were isolated by enzyme
digestion before cryopreservation (method 1), an average yield of 5.04 Â 105
viable CD45À/CD90þ cells/gram of tissue
(n ¼ 15) was achieved, whereas when CT-PSCs were isolated after cryopreservation and thawing and then collagenase
digested whole CT (method 2), an average yield of 0.82 Â 105
viable CD45À/CD90þ cells/gram of tissue (n ¼ 15)
CT-PSCs was achieved. By these measures, significantly higher viable cell recoveries (8.4-fold average, 6.8-fold median,
range 2.1e18.1, P .001) were obtained when CT-PSCs were harvested by enzyme digestion (method 1) before
cryopreservation.
Similar results were obtained and later reported by Harris et al. [21], where fresh and long-term cryopreserved CT-MSC
content were compared after 5 years of frozen storage. In that study, upon thawing previously enzymatically digested CT
and whole CT, which was then enzymatically digested under the same conditions and concentration of collagenase, there
was a sixfold reduction in cell yield defined by viable CD45À/CD90þ/CD105þ per gram of CT in the latter method.
Importantly in either case, once isolated from the extracellular matrix, CT-PSCs can be readily expanded ex vivo, either
before cryopreservation or after being properly cryopreserved. If the isolated cells have been cryopreserved, the storage bag
containing the CT-PSC cell suspension is removed from vapor phase LN2 and thawed using a 37
C water bath. The cells
are then removed from the storage bag and washed using serum-containing media. Alternatively, if the umbilical tissue is
cryopreserved without having first been processed using enzymatic or mechanical methods, the umbilical tissue would then
need to be thawed and undergo one of the aforementioned processing methods. The cell suspension can then be
phenotypically characterized using a flow cytometer. At this point the CT-PSCs are in a format analogous to the freshly
processed CT-PSCs before cryopreservation, and the next steps of tissue culture would be the same for any of the three
scenarios described above. Using the phenotype obtained from the flow cytometer analysis, the CT-PSCs are placed into
liquid culture using the same serum-containing media mentioned earlier at approximately 20,000 viable CT-PSCs per
square centimeter. The CT-PSCs are then placed into a 5% CO2 incubator at 37
C. The cells adhere to the plastic culture
dish and begin to expand. Typically, the CT-PSCs are harvested or passaged once they have expanded to cover about 90%
of the culture vessel’s surface area.
A frequently used axiom in the regulated environment of drug development is that the “process is the product.” In the
processing methods described above, either the umbilical CT is digested, cryopreserved, and thawed or alternatively it is
cryopreserved, thawed, and digested. The only real distinction resulting from these methods is that far greater numbers of
CT-PSCs per gram of CT are isolated from the Briddell method versus the Harris method. Importantly, in terms of their
cellular identity, when using the simple processing snapshot measurements as above (CD45À/CD90þ or CD45À/CD90þ/
CD105þ), the products do not appear to differ in any substantial way. Harris et al. [8] have in fact shown for the MSC
population obtained from the CT by either method that the plating efficiency, population doubling, and trilineage
differentiation functionality are not substantially different. Although this is not a proof that what has not been measured is
missing, it is a reasonable assumption that a variety of cell types are present in the digested complete CT, given the known
anatomic and cellular complexity. If native CT-PSCs are used as a regenerative bolus carrying a heterogeneous mix of stem
cells that are capable of homing and en vivo expansion, engraftment, or differentiation, the only practical limitation of CT-
MSCs from the Harris method versus the Briddell method will be the available cell dose from each banked unit. If, on the
other hand, both products are substantially the same in composition after ex vivo expansion into a homogeneous population
of MSCs, the therapeutic utility of those expanded MSCs will be relative to other allogeneic and/or nonhaploidentical MSC
populations derived from any number of MSC sources and indications for use for expanded MSCs themselves.
SAFE DEPOSITS. WHAT TO BANK ON
PSCs extracted from complete CT are processed under validated methods. Validation requires establishing documentary
evidence that procedures performed in testing a production method maintain compliance with standard operating
procedures and output measurements at all stages. Such documentation is beyond the scope of this chapter, but the output
from many processes can demonstrate either the indicative robustness or the instability of any given process over many
procedures. This is particularly true if, as in this case, the final product in each case is produced from unique starting
material. Despite the unique nature of the starting material, which is collected from uniquely individual newborns by a
subset of unique obstetricians and birthing providers, Fig. 19.2 shows that on the basis of biweekly rolling averages over a
15-month period, the average mass of 9269 CT units received for processing is remarkably stable at about 27 g.

Perhaps as important as the output from the process of enzyme digestion using a validated source of collagenase also
remains remarkably consistent over time. Fig. 19.3 shows that on the basis of 1-week rolling averages of 6429 individual
CT units over a 10 month period, the average number of cells obtained from the process is approximately 500,000 CD45
per gram of starting tissue.
THE GAMISHdCONSISTENT HETEROGENEITY
Shorthand nomenclatures for the product identity or viable cellularity, such as CD45À/CD90þ or CD45À/CD90þ/
CD105þ, are critical for the demonstration, monitoring and overall reliability of a validated process. A consistent process
results in a consistent product. It is understood that the product may contain signiﬁcant intrinsic heterogeneity. For
example, CB is typically red blood cell depleted and volume reduced. The processed CB product is by most standards then
UCL=36.32
LCL=17.501
CEN=26.91
11
16
21
26
31
36
41
1/1/2016
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2/23/2017
2/28/2017
3/5/2017
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3/17/2017
3/22/2017
3/29/2017
MSC Initial Weight
FIGURE 19.2 Control chart representing the weekly rolling averages with the upper control level (UCL ¼ 36.32 gm), lower control level
(LCL ¼ 17.50 gm), and mean values of mass in grams (26.91 gm) of cord tissue received over 15-month period of time in the processing laboratory
(N ¼ 9269).
UCL=897597
LCL=53840
CEN=475718
0
200000
400000
600000
800000
1000000
1200000
9/3/2015
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6/22/2016
CD45-/gram of Umbilcal Tissue
FIGURE 19.3 Control chart representing the weekly rolling averages with the upper control level (UCL ¼ 897,597 cells/gm), lower control level
(LCL ¼ 53,840 cells/gm), and mean values of cells per gram of cord tissue (475,718 cells/gm) processed over 10-month period of time in the processing
laboratory (N ¼ 6429).

characterized by total number of viable total nucleated cells and the percent and number of CD45þ/CD34þ. Notably,
T cells, B cells, megakaryocytes, eosinophils, macrophages, neutrophils, nucleated red blood cells, and other
subpopulations are not enumerated as such within the process to product designation but are expected to be present
nevertheless. Furthermore, although CD34þ may be the “active ingredient” with respect to transplant biology, it is not
clear that it is such in regenerative medicine indications such as brain injury, for instance. Similarly, CT-PSC isolated from
the complete enzymatic digestion of the CT before cryopreservation demonstrates a wide range of cellular constituents, not
just garden variety MSCs.
In fact, because of its unique anatomical composition, CT possesses additional stem/progenitor cell types of hetero-
geneous lineages, including hematopoietic, endothelial, epithelial, and neuronal. A comprehensive phenotypic analysis for
cells enzymatically processed from CT consisting of 31 univariate cell surface markers to determine the degree of
heterogeneity within the general population of the isolated cells was undertaken. Before cryopreservation, complete
umbilical cord tissues, including the Wharton’s Jelly, perivascular regions, vasculature, and EPM, were processed by
overnight collagenase digestion, a process designed to release all of the cellular components. Following centrifugation, the
cells were concentrated into a single cell preparation and then both analyzed before cryopreservation and cryopreserved for
later analysis. With the use of antibody reagents and flow cytometry, processed CT cell preparations were shown to contain
a heterogeneous cellular composition with multiple tissue-type hierarchies. In addition, it was demonstrated that the cell
surface proteins remained consistent pre- and postcryopreservation. Table 19.1 illustrates that complete enzymatically
digested CT cell preparations contain a heterogenic composition of lineage-specific stem/progenitor cells that includes
hematopoietic (CDs 14,19, 34, 38, 45, 117, and 133), endothelial (CDs 31, 62e, 140b, 146, 184, and 309), neuronal (CDs
271 and GFAP), and mesenchymal (CDs 73, 90, 105, 106, 29, and STRO-1) stem cell lineages.
Given the heterogeneous nature of native PSCs isolated directly from complete CT as described above and in
Table 19.1, it is worthwhile to distinguish these compositions from any number of homogeneous derivatives of said
isolates. Obviously, any specific subtype of CT-PSCs might be routinely isolated from flow-activated sorting or immune-
magnetic separation, but more commonly homogeneous derivatives refer to the output of tissue cultures, more specifically
ex vivo expansion cultures, that provide selective conditions for the propagation of specific clonal or oligoclonal cell types
such as either mesenchymal (MSCs) or endothelial stem/progenitor cells (MSCs and EPCs). In the case of MSCs, there is a
clear timewise outgrowth of phenotypically homogeneous CD45À/CD105þ/CD90þ/CD73þ cells with functionally
homogeneous characteristics of trilineage differentiation into endodermal tissues of bone, fat, and cartilage. In fact, the
working definition of MSCs is, for better or worse, based on a homogeneous population of expanded cells with these very
attributes. In the case of CT-PSCs that are not expanded in vitro, the expectation is that they will home to sites of
degeneration or damage, and expansion and differentiation will occur en vivo. This is the dynamic that enables HSC
transplantation from CB where unexpanded HSCs from CB are infused and lead to stable long-term engraftment. Ex vivo
expansion protocols may lead to the elimination of certain populations of cells and adulteration of this potential in certain
indications as will be discussed in the next section.
PROOF OF PRINCIPLE. A PRECLINICAL MODEL WITH FUNCTIONAL OUTPUT
Human umbilical CT has been identified as a potential clinical source of MSCs for therapeutic applications because of its
practical accessibility, expansion potential, immune-modulating properties, and plasticity. This thinking has largely been
driven by the association of CT with homogeneously derived MSCs and their constituent properties. On the other hand, it
has yet to be determined how best to prepare and administer a heterogeneous population of CT-PSCs or for what in-
dications they might be best suited. Studies have been undertaken and described herein to empirically determine whether or
not the preparation of a native heterogeneous population of extracted and cryopreserved CT-PSCs, or a homogeneous
population of expanded CT-MSCs from the same extracted and cryopreserved CT-PSCs, can alone or in combination with
CB be administered intravenously in a rat model of HII to achieve a functional benefit.
Briefly, pregnant SpragueeDawley rats were obtained approximately 1 week before parturition. Neonate rats were kept
in a regular light/dark cycle (lights on 8 a.m.e8 p.m.) with free access to food and water for 7 days after birth. Seveneday-
old rats averaging 12e14 g weight underwent unilateral ligation of left carotid artery using 6-0 silk suture via a midline
neck incision after giving anesthesia with 0.2 mL of inhalation of isoflurane. After surgery, the rats were kept in an
incubator for 10e15 min for observation at 36
C. After regaining normal movement, they were returned to their mother
for nursing for 2 h. These rat pups were placed in a hypoxic chamber of 8% oxygen/92% nitrogen maintained at 36
C for
10 h. Seven days after surgery, at the age of 14 days, rat pups were given IV infusion of CT-PSCs at 50,000 and 100,000
viable cells per pup versus ex vivoeexpanded CT-MSCs from CT-PSCs at 100,000, 300,000 and 500,000 viable cells per

pup. At 1 month and 2 months, various measurements were performed to evaluate the efﬁcacy of native heterogeneous CT-
PSCs alone, ex vivoeexpanded homogeneous CT-MSCs alone, and the combination of CT-PSCs and CB.
The assessment of efﬁcacy was based on both functional (behavioral) performance measurements and biomarkers. For
measures of functional performance, the adhesive tape removal (ATR) test was performed at 4e6 and 8e10 weeks of age
depending on the comparative. A small square of adhesive was placed on the ventral surface of each forepaw, and the time
for the animal to recognize the square (i.e., recognition) and the total time required for the animal to remove the square (i.e.,
removal) were measured. Both forepaws were tested individually. The procedure is repeated for 5 days. The rat must
perform one trial per day for 5 days before performing the real test to decrease anxiety related to the test.
TABLE 19.1 Intrinsic Heterogeneous Phenotype of Cord Tissue Perinatal Stem Cells
Cell Surface Protein (N [ 5, Postthaw) % Expression (Univariate) Perinatal Stem Cell Compartment
CD73 (MSC) 5.7 Æ 0.7 Mesenchymal
CD90 (Thy-1; MSC) 61.3 Æ 8.0
CD105 (endoglin; MSC) 16.6 Æ 5.7
CD106 (VCAM-1; endothelial) 1.6 Æ 0.6
CD29 (integrin b1 chain) 56.6 Æ 4.4
STRO-1 (stem/progenitor) 8.8 Æ 4.4
CD14 (hematopoietic) 5.2 Æ 3.5 Hematopoietic
CD19 (hematopoietic) 1.6 Æ 1.1
CD34 (stem/progenitor) 3.0 Æ 1.4
CD38 (cyclic ADP ribose hydrolase) 2.5 Æ 1.0
CD45 (hematopoietic) 3.6 Æ 0.1
CD117 (stem/progenitor; c-kit, SCF receptor) 11.8 Æ 4.3
CD31 (PECAM-1; endothelial) 4.3 Æ 1.8 Endothelial
CD62e (E-selectin; endothelial) 12.2 Æ 4.7
CD140 b (PDGFR-b; endothelial) 47.8 Æ 2.0
CD146 (MCAM; endothelial) 22.0 Æ 6.0
CD184 (CXCR4, fusin; endothelial) 5.8 Æ 2.0
CD309 (VEGFR-2, FLK-1; endothelial) 4.3 Æ 2.3
CD271 (LA-NGFR; stem/progenitor) 3.5 Æ 1.7 Neural
GFAP (neuro) 1.1 Æ 0.8
CD44 (H-CAM; hyaluronan-binding protein) 17.6 Æ 3.3 Various other phenotypes of
interestCD49d (integrin a4 chain) 6.3 Æ 2.6
CD55 (DAF) 59.4 Æ 10.1
CD59 (MACIF) 62.2 Æ 10.5
CD200 (OX2) 44.8 Æ 13.6
CD243 (MDR; P-glycoprotein) 10.0 Æ 4.4
CD71 (transferrin receptor) 19.3 Æ 4.1
CD338 (ABCG2; stem cells) 11.1 Æ 5.1
HLA-DR (MHC class II) 3.6 Æ 1.1

The biomarker assessment consisted of immunohistochemical observations and quantitative measurements of brain
tissue, including neuronal cell counts in predesignated regions of the hippocampus. Immunohistochemistry was performed
to visualize the neurons in the rat brain by using neuronal antibody (NeuN-green) and DAPI stain (blue) for nuclei.
Selected sections were then stained with human nuclear antigen (HNA-red) to visualize the human-derived cells in the
hippocampus. The counting of neurons was performed in three areas of the rat hippocampus: dentate gyrus (DG), CA1, and
CA3. Three sections from each animal were counted, and the numbers were averaged for each result. To obtain these
measurements at the conclusion of the behavior testing but no later than 10 weeks of age, the animals were sacrificed under
isofluorane anesthesia by exposing the heart and perfusing the animal with 100 mL PBS followed by 4% paraformaldehyde
(PFA). The brains were removed and placed in 4% PFA and cut after 3 days. A Tissue-Tek processor (Sakura) was used to
process the tissue. Six-micrometer-thick paraffin sections were cut with the microtome.
Statistics for both functional and biomarker findings were performed using ANOVA for the experiments with three or
more groups. Significant differences between pairs of groups were determined with Tukey’s Honest Significant Differ-
ences. The student’s T test was used for the combination study and the control group.
Results of these experiments can be summarized as follows.
In repeated experiments using IV tail vein infusions of previously cryopreserved native heterogeneous CT-PSCs in the
rat HII model, at 2 months posttreatment, the presence of cells located within microinfarct areas of the harvested rat brains
staining triple positive for intact nuclei (DAPI), neuronal phenotype (NeuN), and HNA was apparent as shown in the
representative confocal micrograph in Fig. 19.4.
Importantly, total endogenous cell counts in the DG, CA1, and CA3 regions of the brain showed a statistically sig-
nificant increase in the numbers of cells per equivalent fields examined, suggesting a paracrine effect may also be at play.
In head-to-head comparisons of native heterogeneous CT-PSCs at 50,000 and 100,000 viable cells per pup and
ex vivoeexpanded CT-MSCs from CT-PSCs at 100,000, 300,000, and 500,000 viable cells per pup, the former population
resulted in a statistically significant improvement equating to amelioration of the HII in both the recognition and removal
functions of the ATR test, whereas the expanded homogeneous CT-MSCs did not show a statistical improvement even at
5- to 10-fold the viable cell dose. Graphs 19.1A and B represent this result.
Animal Recognition and Removal Behavior Tested with the Forepaw Adhesive Square
Method
Results from follow-up experiments testing the efficacy of CB at 1 million (1 Â 106
) viable cell and CT-PSCs at 50,000
(5 Â 104
) viable cell dosages per animal showed that although both CB and CT-PSCs were effective at reducing the impact
of HII, the combination of both sources of stem cells appears to be synergistic.
Blue = DAPI
Green = Neural
(Fox-3)
Red = Human
Nuclear Antigen
FIGURE 19.4 Confocal image of human neural cells located in the microinfarct regions of injured rat brain. Putative human cells derived from cord
tissue perinatal stem cell IV tail vein injections are present in the rat brain 2 months postinjection and staining positively with human nuclear antigen (red),
neural Fox-3 (green), and nonspecific nuclear DNA (DAPI blue).

CLOSING THE LOOPdBACK TO BIRTH AND BANKING
Insurance is, at its heart, speculative gambling, where the insured gambles or speculates that they will experience hardship,
loss, sickness, or even death, whereas the insurer gambles or speculates that they will not. Only by spreading risk is
insurance feasible. It may seem odd to some, but saving or banking PSCs at birth is not insurancedit is banking. It is
banking because one saves something that is uniquely precious and valuable at the time it is banked with the hope and/or
understanding that which has been banked will be at least as valuable, and perhaps more valuable, at some time in the
future when it is most desired. Think of a goody bag filled with precious jewels in a safety deposit box.
Upon the birth of a child, PSCs, including those found in the CB, AF, AM, PP, P, and CT-PSCs, are available for a
brief moment in time. Without proper methods of harvesting, shipping, processing, and storing these potent cells, they
inevitably perish as a consequence of their detachment from the intact being supported. In the history of humankind, there
may have been no evolutionary reason or technical means to preserve these by-products of conception. As hunters and
gatherers, brain injuries may have been frequently fatal or otherwise costly to the guardian individuals, parents, or clan.
Today, babies and even adults are more routinely rescued from near-fatal brain injury in ways and by technology
unanticipated by our biology. A newborn’s PSCs are a uniquely pristine raw biological material derived directly from the
early branches of stemness. Stemness, or essential characteristics that distinguish a stem cell from other cells, at the
developmental branch after embryogenesis and fetal development, at the very moment the newborn arrives, results in a rich
source of pristine, proliferative, pluripotent PSCs that are clonally identical to that newborn and often haploidentical to
biologically related family members. As research progresses, it seems that these by-products of conception may be more
valuable in forestalling or repairing injury and degeneration than had previously been suspected.
Unlike embryonic stem cells (ESCs), or inducible pluripotent stem cells (iPSCs), PSCs do not necessarily require sig-
nificant ex vivo manipulations or directed differentiation, which may compromise the safety and efficacy of the stem cells
themselves [17], but rather serve as a single treatment ready dose or regenerative boluses if you will, which can home,
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Minutes
Two Months Recognition Time
Significant
Improvement*
*
GRAPH 19.1A The time for animals to recognize the presence of the tape on the right forepaw was significantly less in those treated with the cord tissue
perinatal stem cells (white) versus injured but not treated (solid). ANOVA is significant at P ¼ .007. There was no statistically significant benefit in
animals who received up to five times the dose of cord tissue mesenchymal stem cells (striped).
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Minutes
Two Months Removal Time
Significant
Improvement*
*
GRAPH 19.1B The time for animals to remove the tape on the right forepaw was significantly less in those treated with the cord tissue perinatal stem
cells (CT-PSCs; white) versus those injured but not treated (solid). ANOVA is significant at P ¼ .038. There was no statistically significant benefit in
animals who received up to five times the viable cell dose of CT-MSCs (striped).

expand or differentiate en vivo according to signals from the microenvironment of damage or degeneration. For this purpose,
the banking of heterogeneous populations of PSCs, in addition to CB, with mixed stemness for future medical interventions
to repair, restore, and regenerate tissue may be a particularly timely consideration while couples are pregnant. The scope of
CB use has recently expanded beyond transplant medicine to brain injuries and atypical developmental disorders such as
cerebral palsy and autism spectrum disorder, respectively. These profound developments are taking place nearly a quarter of
a decade after the earliest adopters of CB banking ﬁrst stored their newborn’s CB-PSCs. It is very provocative to imagine CB
units in storage today might eventually be used for medical rescue, regeneration, gene editing, blood/immune boosters,
genetic and epigenetic information, drug design, or even life extension by the child or his or her closely biologically related
family. Although the mechanism for transplants is known to depend ultimately on total number of CD34þ stem cells to
rescue blood formation and establish long-term engraftment, the mechanism for these new regenerative applications of CB is
not yet clear but does not appear to correlate to the number of CD34þ stem cells in the CB units [11]. Similarly, native
heterogeneous mixed populations of PSCs from CT offer a unique biologic cocktail and are preserved the very life stage
which is preprogrammed to support tremendous growth and development in the newborn. Given these attractive stem cell
features, it would seem to be very valuable and worthwhile to consider preserving them for future use.
Although there are multiple tissues available after birth, the current approach has been to offer preservation services for
CB with the additional option to collect and preserve an entirely unique heterogeneous population of native stem cells from
the CT-PSCs. Both CB and CT-PSCs are uniquely different bankable biologics with the common genetic identity rep-
resenting individualized cocktails. Each banked product might be used alone or in combination in the donor child or his or
Tape Recognition Time, after Two Months of cell Injection Right Palm
C
O
N
TR
O
L
C
B
C
B
+C
T
C
T
0.0
0.1
0.2
0.3
0.4
Tape Recognition time in Right palm after 2 months of Vehicle injection in Control and 1 Million
Cord Blood(CB),1Million Cord Blood+50,000 Cord Tissue(CB+CT) and 50,000 Cord Tissue(CT)
injected in Test Rats
TimeinMinutes
Tape Recognition Time, after Two Months of cell Injection Left Palm
C
O
N
TR
O
L
C
B
C
B
+C
T
C
T
0.0
0.1
0.2
0.3
0.4
Tape Recognition time in Left palm after 2 months of Vehicle injection in Control and 1 Million
Cord Blood(CB),1Million Cord Blood+50,000 Cord Tissue(CB+CT) and 50,000 Cord Tissue(CT)
TimeinMinutes
Improvement
Tape Removal Time, after two Months of cell Injection Right Palm
C
O
N
TR
O
L
C
B
C
B
+C
T
C
T
0.0
0.1
0.2
0.3
0.4
0.5
Tape Removal time in Right palm after 2 months of Vehicle injection in Control and 1 Million Cord
Blood(CB),1Million Cord Blood+50,000 Cord Tissue(CB+CT) and 50,000 Cord Tissue(CT)
TimeinMinutes
Tape Removal Time, after two Months of cell Injection Left Palm
C
O
N
TR
O
L
C
B
C
B
+C
T
C
T
0.0
0.1
0.2
0.3
0.4
Tape Removal time in Left palm after 2 months of Vehicle injection in Control and 1 Million cord
Blood(CB),1Million Cord Blood+50,000 Cord Tissue(CB+CT) and 50,000 Cord Tissue(CT)
TimeinMinutes
(A)
(C) (D)
(B)
GRAPH 19.2 (A) Tape recognition time, right palm. (B) Tape recognition time, left palm. (C) Tape removal time, right palm. (D) Tape removal time,
left palm.

her haploidentical siblings, parents, or grandparents with potential regenerative capacity across a wide variety of medical
indications. The preliminary data discussed above suggest that cells present in human CT-PSCs, when injected into the tail
vein of rats with hypoxic ischemic injury, were able to traverse the circulation, ultimately crossing the bloodebrain barrier,
and move on to form neural components within microinfarctions in the injured animals’ brains, and there appear to have
furthered endogenous cell recovery. When compared with injured control rats with degenerated recognition and removal
responses, injured treated rats demonstrated an amelioration of the effect in those rats receiving just 50,000 viable CT-
PSCs, as compared with 1 million viable CB cells. Furthermore, this effect was significantly improved for both CB and
CT-PSCs when used in combination.
At what time will any one individual or his or her family wish to access CT-PSCs? For what indication might these
PSCs be most useful? Although answers to these questions are still unknown, methods and means for collecting,
processing, and storing CT-PSCs in a responsible manner have been established. Additionally, preliminary data now exist
to provide some phenotypic and functional characterizations of this unique heterogeneous composition of matter that is
available to every baby at the very moment of birth. Further characterization will be required to bring these compositions
into medical use by way of controlled clinical studies to assure safety and verify any presumed regenerative function in
human subjects. One might imagine these studies to look similar to those recently undertaken using a child’s own CB to
treat brain injuries and atypical developmental disorders. In other words, those studies where the unmet need for medical
approaches to degenerative conditions within child or the child’s immediate family, those without proven effective
regenerative interventions, require visionary clinicians with access to a storehouse of cryopreserved native PSCs, in this
case CT-PSCs [7,20,21].
LIST OF ABBREVIATIONS
AF Amniotic fluid
AM Amniotic membrane
CAR-T Chimeric antigen receptoreactivated T cell
CB Cord blood
CT Cord tissue
CT-MSCs Cord tissue mesenchymal stem cells
CT-PSCs Cord tissue perinatal stem cells
DAPI 40
6-Diamidino-2-phenylindole stain
DMSO Dimethyl sulfoxide
EPM Epithelial membrane
EPSs Epithelial progenitor cells
ESCs Embryonic stem cells
FDA Food and Drug Administration
HCT/Ps Human cell tissue/product
HII Hypoxic ischemic injury
HNA Human nuclear antigen
HSCs Hematopoietic stem cells
HUVECs Human umbilical vascular endothelial cells
iPSCs Inducible pluripotent stem cells
MHC 1 Major histocompatibility complex 1
MHC 2 Major histocompatibility complex 2
MSCs Mesenchymal stem cells
NeuN Neuron-specific marker
P Placenta
PP Placental perfusate
PSCs Perinatal stem cells
REFERENCES
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[3] Koike K. Cryopresevation of pluripotent and committed hemopoietic progenitor cells from human bone marrow and cord blood. Acta Paediactrica
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[8] Briddell R, Litkenhaus F, Foertsch G, Fuhrmann A, Foster K, Falcon Girard K, Fiscus B, Boehm A, Brown M, Pettit M, Rigas Bridges A,
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[9] See harvesting newborn stem cells from cord lining with Cell optima provided by Cordlife at the Parents Guide to Cord Blood website. https://
parentsguidecordblood.org/en/news/harvesting-newborn-stem-cells-cord-lining-celloptima.
[10] See what is placenta tissue? Banking placenta tissue with Americord. https://www.americordblood.com/banking/placenta-tissue/.
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April 5, 2017:1e8. Duke ASD ICBS/ASH Dawson/Kurtzberg.
[14] ViaCord data on ﬁle.
[15] Prochymal was approved for treatment of refractory Graft versus Host Disease in Canada and New Zealand but not in the U.S. at this time. Updated
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