University of Southern California Dissertations and Theses

Cryopreserved umbilical cord mesenchymal stem cells therapy for the treatment of knee osteoarthritis: in-vitro evaluation and phase I clinical trial protocol

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Cryopreserved umbilical cord mesenchymal stem cells therapy for the treatment of knee osteoarthritis: in-vitro evaluation and phase I clinical trial protocol

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Content Running Head: Cellistem-F® 1
Thesis Title: Cryopreserved Umbilical Cord Mesenchymal Stem Cells Therapy for the
Treatment of Knee Osteoarthritis: In-vitro Evaluation and Phase I Clinical Trial Protocol

Author: Francisca Javiera Orrego Garbin, MD
Conferring Program: Clinical, Translational and Biologic Investigations Program, Keck School
of Medicine
Degree: Master of Science
University of Southern California
Degree conferral date: May 2018

Cellistem-F® 2
Table of Contents
1. Introduction ....................................................................................................................... 3
2. Background and Significance .......................................................................................... 3
3. Preliminary Data ............................................................................................................... 5
4. Study Scheme .................................................................................................................... 8
5. Study Design and Methods ............................................................................................... 9
5.1 Stage 1: In vitro evaluation of Cellistem-F® ……………………………………..9
5.2 Stage 2: Phase I Clinical Trial …………………… ….. …………………… …….15
6. References ........................................................................................................................ 30
7. Figures .............................................................................................................................. 33
8. Appendix 1 ....................................................................................................................... 37

Cellistem-F® 3
1. Introduction
Hip and knee Osteoarthritis (OA) are among the leading causes of global disability (Kang
et al., 2013). Current treatment focuses on symptomatic relief and relies on the use of analgesics
and anti-inflammatory drugs (Zhang et al., 2008). However, the unsatisfactory effects and side
effects associated with traditional OA drugs warrant a continued search for potential new
interventions. Recent evidence points towards the safety and efficacy of Mesenchymal Stromal
Stem Cells (MSCs) for the treatment of knee OA, and its disease modifying potential (Zhang,
Ouyang, Dass, & Xu, 2016). We propose a two-stage process to evaluate an existing
standardized, cryopreserved version of umbilical cord (UC)- MSCs therapy, Cellistem-Frozen®
(Cellistem-F®) using in vitro studies to measure viability, differential potential to cartilage and
anti-inflammatory effect, (Stage 1), and a subsequent phase I clinical trial to test the safety and
estimate effect size of Cellistem-F® among patients >55 years of age with grade I-III knee
osteoarthritis (Stage 2).

2. Background and Significance
OA is a disease characterized by the loss of articular cartilage associated with the
presence of degenerative changes in the joint and in the subchondral bone (Moon & Beier,
2015). Symptomatic OA causes pain and progressive reduction of joint function, ultimately
leading to physical and occupational disability (Whittaker, Woodhouse, Nettel-Aguirre, &
Emery, 2015). According to the Global Burden of Disease (GBD) Study of 2010, OA accounts
for approximately 0.6% of all disability adjusted life years (DALY) and 10% of DALY related to
chronic conditions. It also accounts for 2.2% of global years living with disability (YLDs) and
Cellistem-F® 4
10% of all YLDs for musculoskeletal disorders (Vos et al., 2012). Furthermore, recent evidence
shows that it is an independent risk factor for premature death (Liu et al., 2015).
OA that affects the knee is one of the most prevalent forms of OA (Buckwalter & Martin,
2006). In recent years, the number of patients diagnosed with varying degrees of knee OA has
increased due to the aging of the population and the obesity epidemic (Yuqing Zhang, 2010).
Among adults 60 years of age or older, the prevalence of symptomatic knee OA is approximately
10% in men and 13% in women. The risk of mobility disability - defined as the need for help to
walk or climb stairs- attributable only to knee OA is greater than that attributable to any other
medical condition in adults 65 years of age or older (Jo et al., 2017).
The burden of the disease has made knee OA a public health problem accentuated by the
gradual aging of the population (Michaud et al., 2006). Its high direct and indirect costs,
comorbidity and physical-labor disability (Rabenda et al., 2006), motivate the search for new
therapeutic options capable of delaying and reversing the progression of the disease.
Current approaches to the treatment of OA are predominantly palliative and are aimed at
reducing pain and recovering function. Analgesics are the most prescribed medications for
patients with OA. Recent studies suggest that the prolonged use of these could be associated with
the progression of knee OA and an increased risk of future arthroplasty (Buckwalter & Martin,
2006). Despite the urgency driven by the burden of the disease, no disease modifying
osteoarthritis drugs (DMOADs) have translated into treatments for patients and there are
currently no drugs that modify the natural course of the disease (Barry & Murphy, 2013). In
recent years, different forms of cell therapy, including the use of Mesenchymal Stromal Stem
Cells (MSCs), have shown efficacy in preclinical OA models where they appear to delay the
progression of the disease. (Diekman et al., 2013; Horie et al., 2012; J. M. Murphy, Fink,
Cellistem-F® 5
Hunziker, & Barry, 2003; “Scaffold-free adipose-derived stem cells (ASCs) improve
experimentally induced osteoarthritis in rabbits,” 2012). MSCs are easily obtained from various
tissues such as bone marrow, adipose tissue, synovial membrane, and others, and have a high
proliferation rate, chondral-differentiation capacity, and immunosuppressive activities (Imran
Ullah, 2015). Therefore, making them an ideal intervention for treating OA.
Stem cell therapy for knee OA is currently derived from fresh autologous or allogenic
sources, obtained via centrifugation of tissue or cell expansion and multiplication (Xing et al.,
2017). While this approach is effective for the symptomatic treatment of patients in a small case-
by-case basis, it is not efficient for larger scale applications because the process generates great
variability in the type, number and characteristics of the stem cells being produced (Reissis et al.,
2016).
Therefore, we propose a novel cryopreserved UC-MSCs therapy, that maintains original
MSCs’ properties and it’s efficient for larger scale application.

3. Preliminary Data
During the last decade, safety data has accumulated regarding the autologous and
allogenic use of MSC in both preclinical and case series as well as clinical trials, leading to
regulatory approval in Asia (Y.-B. Park, Ha, Lee, Yoon, & Park, 2016). Extensive local and
systemic infusion in a variety of cardiovascular, neurological or immune-mediated diseases have
not lead to adverse effects in the short and medium term (Lalu, McIntyre, & Pugliese, 2010;
Wakitani et al., 2011). Moreover, a systematic review concluded that the application of cultured
stem cells in joints appears to be safe (Peeters, Leijs, Reijman, van Osch, & Bos, 2013). No
anaphylactic reactions have been reported or any increase risk of infection. Because of their low
Cellistem-F® 6
immunogenicity, allogeneic MSC can be used without the need to test for histocompatibility
antigens (Franquesa et al., 2013). Moreover, in-vitro experiments and prolonged follow-up of
animals with immunodeficiency, have not shown an increase in tumorigenesis (Prockop & Oh,
2009). In humans, a systematic review of 1012 patients, including the meta-analysis of the
available controlled series, showed no significant adverse effects or increased incidence of
neoplastic pathology (Lalu et al., 2010).
Particularly in knee OA, published data by Varma et al (Varma, Dadarya, & Vidyarthi,
2010), Wong et al. (Wong et al., 2013) and Koh et al. (Koh & Choi, 2012) involving more than
100 patients treated with bone marrow-derived mesenchymal stem cells (BM-MSCs) or adipose-
derived mesenchymal stem cells (AD-MSCs), confirm a high degree of safety. More recently,
Orozco et al. (Orozco et al., 2014) studied the intra-articular injection of BM-MSCs and showed
no adverse effects compared to the active comparator, hyaluronic acid (HA).
Regarding MSCs’ efficacy for the treatment of knee OA, encouraging data have been
obtained in preclinical models. Murphy et al. (J. M. Murphy et al., 2003) in a surgical model of
OA (medial meniscectomy and anterior cruciate ligament section), demonstrated a decrease in
articular cartilage loss, fewer formation of osteophytes and less development of subchondral
sclerosis in goats treated with MSCs. Additionally, increases in joint cartilage thickness after
intra-articular administration of MSCs, evidenced by magnetic resonance imaging (MRI) and
histology have been reported (J. M. Murphy et al., 2003; “Scaffold-free adipose-derived stem
cells (ASCs) improve experimentally induced osteoarthritis in rabbits,” 2012).
In humans, Vangsness et. al. published a randomized, double-blind clinical trial showing
that the intra-articular administration of allogeneic MSCs has regenerative potential on the
meniscus, which is associated with pain and functional improvement (Vangsness et al., 2014).
Cellistem-F® 7
Moreover, a meta-analysis of seven published clinical studies from 2011 to 2014 that grouped
314 patients with knee OA treated with BM-MSCs or autologous AD-MSCs, demonstrated
improved joint function (Xia, Wang, Lin, & Li, 2015).
The Program for Translational Research in Cell Therapy, Clínica Universidad de los
Andes, Chile, recently concluded a randomized, double-blind, phase I-II controlled trial
comparing intraarticular (IA) injection of HA to IA injection of allogeneic umbilical cord
derived mesenchymal stem cell (UC-MSCs), in patients between 40 and 65 years of age, with
grade I-III knee OA, confirming the safety and efficacy of IA injection of allogeneic UC-MSCs
to improve symptomatic knee OA (manuscript in preparation).
While using fresh cells is effective for the symptomatic treatment of patients in a small
case-by-case basis, it is not efficient for a larger scale application, as it generates great variability
in the type, number and characteristics of the stem cells being used, and creates a logistic issue
with storage and transportation (Reissis et al., 2016). A method of preservation is critical for
both research and clinical application of stem-cell based therapies, as it is needed for the
transportation of cells between sites, as well as completion of safety and quality control testing
(Hanna & Hubel, 2009). In this sense, cryopreservation - the use of very low temperatures to
preserve structurally intact living cells and tissues living cells and tissues (Pegg, 2007)- offers an
efficient method for the preservation and pooling of MSCs to obtain the cell counts required for
clinical applications (Yong et al., 2015).
We propose a two-stage study to evaluate the properties (viability, cartilage differentiation
potential, in vitro anti-inflammatory effect) of cryopreserved UC-MSCs therapy, Cellistem-
Frozen® (Cellistem-F®), "in vitro" (Stage 1), and a subsequent phase I clinical trial to test the
Cellistem-F® 8
safety and estimate the effect size of Cellistem-F® to improve knee health in patients in patients
>55 years of age with grade I-III knee osteoarthritis (Stage 2).

4. Study Scheme
General Study Outline
The first year of the study will be dedicated to testing Cellistem-F® properties in vitro.
The second and third year will encompass a phase I clinical trial for Cellistem-F® (Figure 1).

Phase I Clinical Trial “3+3” Design with Dose Escalation to determine Maximum Tolerated
Dose (MTD)
For the phase I clinical trial, a “3+3” design with dose escalation will be implemented (Le
Tourneau, Lee, & Siu, 2009). MTD is defined as the highest dose at which one or zero patients,
out of a cohort of six patients, experience DLT, defined as grade three or greater in the Common
Terminology Criteria for Adverse Events v4.0 (CTCAE) (“Protocol Development | CTEP,” n.d.),
within four weeks of initial treatment. The first cohort of three patients will be treated at a low
starting dose Cellistem-F® (10 x 10
6
cells = 3cc; dose K). Patients will be monitored for four
weeks. If none of the three patients experience a DLT, a second cohort of three patients will be
treated at the next higher dose level (20 x 10
6
cells = 3cc; dose K+1). However, if one of the first
three patients experience a DLT, the second cohort will be treated at the same dose level. If
necessary, a third cohort of three patients will be enrolled after four weeks of cohort 2 initial
treatment. The dose escalation will continue until at least two patients among a cohort of six
patients experience a DLT. MTD will be considered the highest dose at which one or zero out of
the six patients experienced DLT within for weeks of initial treatment (Le Tourneau et al., 2009)
Cellistem-F® 9
(Figure 2); and the recommended dose for the phase II trial will be defined as the dose level just
below the toxic dose level

Phase I Clinical Trial: 52 weeks’ follow-up plan
We will evaluate the safety and estimate effect size of Cellistem-F® for the symptomatic
treatment of OA, at weeks 4, 12, 24, 36 and 52 post-intervention. Safety will be assessed through
participant self-report and results will be summarized using counts and proportion of severe
adverse events experienced by patients during the first year of treatment. Effect size will be
measured by documenting change in general knee health using the Western Ontario and
McMaster Universities Arthritis Index (WOMAC) score. The score will be applied to each
patient at every follow-up consultation to calculate the overall median change in patient’s
assessment of knee health (Figure 3) at the end of the study compared to baseline. A change of
0.5 in the WOMAC standardized median difference (SMD) units will be considered clinically
meaningful (Vega et al., 2015).

5. Study Design and Methods
5.1 Stage 1: In vitro evaluation of Cellistem-F®
Primary Aim
To evaluate the viability of in vitro cryopreserved UC-MSCs (Cellistem-F®)
compared to fresh UC-MSCs (Cellistem ®).

Hypothesis
Cellistem-F® viability does not differ to that of fresh UC-MSCs, in vitro.
Cellistem-F® 10
Outcome: Viability
To evaluate stem cell viability, the Annexin / 7-Aminoactinomycin D (7-AAD)
technique will be used (Zembruski, Stache, Haefeli, & Weiss, 2012). Cellistem-F® and
fresh UC-MSCs will be stained using Annexin 7 / 7AAD kit (BD-Bioscience) following
the manufacturer's instructions (“PE,Annexin,V,Apoptosis,Detection,Kit,I,RUO - 559763
| BD Biosciences-Europe,” n.d.). Negative percentages for Annexin and 7AAD will be
determined and analyzed using flow cytometry (FACS CANTO II equipment located in
the facilities of the Biomedical Research Center (CIB) of the Universidad de Los Andes,
Chile). We define equal viability to fresh UC-MSCs as ≥85% negative percentage for
Annexim and 7AAD in Cellistem-F®.

Secondary Aim
To evaluate in vitro cartilage differentiation potential of Cellistem-F®, compared
to standard fresh UC-MSCs.

Hypothesis
Cellistem-F® trombospondina 2 secretion levels in cryopreserved samples do not
differ to that of fresh UC-MSCs, in vitro.

Outcome: Cartilage differentiation potential
To determine the differentiation potential of Cellistem-F® versus fresh UC-
MSCs, a differentiation test of chondrogenic lineage will be carried out using a
standardized protocol established by the Cells for Cells Laboratory (Gonzalez-Lorca, P et
Cellistem-F® 11
al, 2015). Cellistem-F® will be thawed, counted and left at a final concentration of
30,000 cells in 10 μl. The thawed cells will be then seeded in a "microdrop" to establish
the 3D conditions necessary for chondrogenic differentiation. Induction medium will be
added to the chondrogenic lineage consisting of DMEM High glucose, ITS (insulin /
transferrin / selenic acid), 0.1μM dexamethasone, 1mM sodium pyruvate, 170μM
ascorbate-2 phosphate and 10 ng / mL human TGF-β3. After 3, 7, 14 and 21 days. The
potential for cartilage differentiation will be evaluated by quantifying chondrogenesis
marker expression (Collagen II, Sox II and Aggregan, and collagen I inhibition) using
RT-qPCR (H. Li et al., 2011) and safranin O staining (Schmitz, Laverty, Kraus, &
Aigner, 2010). An expression of >50% for each marker will be considered a positive
result (Gómez-Leduc et al., 2016).

Tertiary Aim
To evaluate anti-inflammatory effect of Cellistem-F® towards cartilage in vitro,
compared to fresh UC-MSCs.

Hypothesis
Cellistem-F® inhibition of interleukin 10 and TNF 𝛼 does not differ to that of
fresh UC-MSCs, in vitro.

Outcome: Anti-inflammatory effect
The effects of thawed vs. non-thawed UC-MSCs on inflammation will be
evaluated by co-cultivating the cells with cultures of macrophages derived from blood
Cellistem-F® 12
monocytes. Blood from Cell for Cells Laboratory volunteer bank will be collected, and
monocytes will be isolated using a monocyte separation kit according to the expression of
the CD14 marker following the manufacturer's instructions. Monocytes will be matured
into macrophages by culturing them in the presence of 40ng / ml of M-CSF (RYD). After
5 days, macrophages that adhere to the plate will be collected and matured in 1ug / ml of
LPS and cultured in the presence of thawed Cellistem-F® and fresh UC-MSCs. After 24
hours, the supernatant will be collected and TNF 𝛼 , IL-10, and macrophage phenotype
will be evaluated according to the expression of activation molecules (CD80, CD83,
CD86 and HLA-DR) and the expression of the associated molecules to anti-inflammatory
macrophages CD206. An expression of >50% will be considered a positive result
(Gómez-Leduc et al., 2016).

Methods
Isolation of MSCs derived from the umbilical cord under good manufacturing standards
(GMP)
UC-MCSs will be donated by Cells for Cells Laboratory, obtained following the
GMP criteria. The plastic materials and reagents used are GMP grade (“Good
Manufacturing Practices: Supplementary Guidelines for the Manufacture of
Investigational Pharmaceutical Products for Clinical Trials in Humans. WHO Technical
Report, Series No. 863 , 1996, Annex 7,” n.d.) or have the certification that ensures
traceability in the production process. To comply with the standards for pre-clinical
studies and international standards of quality management for cell therapies described by
the International Society for Cellular Therapy (ISCT) and Foundation for the
Cellistem-F® 13
Accreditation of Cellular Therapy (FACT), informed consent and serological analysis of
four umbilical cord donors will be requested. The donated biological material (placenta)
will be processed in the quarantine room of the Cells for Cells Cell Therapy Productive
Laboratory, which has a certification for the number of particles ISO 7 or Class 100000
(350000 particles <0.5um) and regimes of air according to ISO 14644 standards that
ensure asepsis during the process. In addition, this room will be isolated from the
production room by a pressure differential> 15KPa. For the production of master cell lots
(LCM), isolation will be performed through the explant method according to the
previously described protocol (Gonzalez P. et.al 2015). The obtained cells will be seeded
in a 75 cm
2
T-Flasks to allow their adhesion and clonal expansion in alphaMEM,
enriched with 10% Gibco FBS (ThermoScientific®), penicillin (100 U / ml),
streptomycin (100 μg / ml) (P / S), supplements that ensure asepsis throughout the
procedure. After reaching an 80% confluence, cell cultures will be expanded to
approximately 2x10
8
cells. During this process, the cultures will be analyzed
microbiologically twice for presence of anaerobic and aerobic pathogens as well as to
rule out the presence of mycoplasmas. Finally, about 100 vials-inoculum containing
2x10
6
cells in passage three will be packaged to subsequently be frozen controlled under
serum-free conditions at a rate of -1 ° C / minute and cryopreserved at -190 ° C in liquid
nitrogen tanks. After 6 months of freezing and in compliance with the standards for
manufacturing of minimally manipulated products, a quality control will be carried out,
including: sterility tests (microbiological and, mycoplasma), endotoxin,
immunophenotyping for the expression of CD73 mesodermal antigens, CD90, CD105, as
well as the absence of CD45 and CD34 antigens and their ability to differentiate
Cellistem-F® 14
adipocytes, chondrocytes and osteoblasts, according to international ISCT criteria, as
well as viability. Likewise, each LCM will be evaluated for the secretion of
Thrombospondin 2 (TSP2) (Krady et al., 2008), a marker that has been associated with
the promotion of chondrogenic differentiation produced by MSCs through its paracrine
action.

Cryopreservation of MSCs in doses programmed for OA
After the preparation and approval for quality assurance of the master cell batch
that secretes the highest amount of TSP2, we will proceed to a large-scale production of
the doses for OA (20x10
6
). This consists of expanding the cells in devices called Cell
Factory, which have a working surface of 6320 cm
2
. This allows to obtain up to 80x10
6
cells from 12x10
6
seeding cells. The cells are cultured in aMEM culture medium
supplemented with 10% platelet lysate and P / S (Humanized culture medium). At the
time of harvest, the cells will be treated with TrypLE, a solution of modified GMP-grade
trypsin, to be collected by centrifugation. After centrifugation, the cells will be washed
with 1X PBS and resuspended in 5 cc in cryopreservation solution previously cold at 4°C.
The cell suspension will be transferred to 15mL COP vials (cyclic olefin polymer),
resistant to ultra-low temperatures and inert to cellular material. The vials will be
transferred to -80ºC for a gradual freeze and after 24 hours, the vials will be transferred to
the liquid nitrogen tank until the request for therapy. At the time of treatment, the cells
will be thawed, washed and re-suspended in suspension vehicle (saline solution + 5% AB
+ plasma) prior to transfer to the medical center and injection.

Cellistem-F® 15
Statistical analysis
Our goal is to test the hypothesis that the outcome medians in the cryopreserved
vs. non-cryopreserved samples are equal. We will use a Wilcoxon Rank Sum test for the
comparison of medians, considering the small experimental groups with non-normal
distributions (“Lesson 39: Tests of the Equality of Two Means | STAT 414 / 415,” n.d.).
Because we are testing for equality, a value of p >0.05 will be considered as significant to
reject the null hypothesis that outcome medians are not equal (equivalence bound = ±
0.2). The results will be expressed as the median and interquartile range (IQR).

5.2 Stage 2: Phase I Clinical Trial
Aim 1: Establishing Maximum Tolerated Dose
To determine the maximum tolerated dose (MTD) of Cellistem-F® for the
treatment of grade I-III knee OA, in patients age >55 years.

Hypothesis
Based on previous studies done using fresh UC-MSCs (manuscript in
preparation), we hypothesize that Cellistem-F® MTD for the treatment of grade I-III
knee OA in patients >55 years of age is 20x10
6
cells = 3cc.

Outcome
MTD will be defined as the highest dose at which one or zero patients, out of a
cohort of six patients, experience dose-limiting toxicity (DLT), defined as grade three or
Cellistem-F® 16
greater in the Common Terminology Criteria for Adverse Events v4.0 (CTCAE)
(“Protocol Development | CTEP,” n.d.), within four weeks of initial treatment.

Aim 2: Safety
To evaluate the safety of Cellistem-F® for the symptomatic treatment of grade I-
III knee OA, in patients >55 years of age.

Hypothesis
Patients aged >55 years, with grade I-III knee OA and treated with Cellistem-F®,
will experience no severe adverse events (SAEs), within the first year of initial treatment.

Outcome
SAEs are defined as any untoward medical occurrence that results in death or is
life-threatening, requires inpatient hospitalization or prolongation of existing
hospitalization (“Protocol Development | CTEP,” n.d.), or results in persistent or
significant disability (“Protocol Development | CTEP,” n.d.), within the first year of
initial treatment.

Aim III: Estimate effect size for future Phase II Clinical Trial
To estimate the effect size of Cellistem-F® to improve knee health in patients >55
years of age with grade I-III knee OA.

Cellistem-F® 17
Hypothesis
Treatment with Cellistem-F® improves baseline knee health by >0.50 (Vega et
al., 2015) standardized median difference (SMD) in knee health measured by the Western
Ontario and McMaster Universities Arthritis Index (WOMAC) score, at baseline vs. 52
weeks in patients >55 years of age with grade I-III knee OA.

Outcome
The outcome is median change in the patient’s assessment of knee health at
baseline vs. 52 weeks after intervention. Median change in patient’s assessment of knee
health will be measured by calculating the WOMAC score SMD, computed as baseline
value minus value at weeks 52 after intervention, divided by the median absolute
deviation (MAD) of the baseline value {WenxianHuang:fi}. SMD is used as an estimate
of the effect size. The correlation between effect size and magnitude of the change will be
quantified as: 0 = null, 0.20 = small, 0.50 = medium, 0.8 = large (Vega et al., 2015). The
WOMAC is one of the most widely utilized self-reported measures of health-related
quality of life that includes lower extremity symptoms and function. It has been used over
a period of almost 30 years in many different contexts and patient populations, and there
are abundant data regarding its utility and measurement properties (“Western Ontario &
McMaster Universities Osteoarthritis Index (WOMUOI),” n.d.). It is available in over 65
alternate language forms and, for our purpose, has been validated in Spanish (Escobar,
Quintana, Bilbao, Azkárate, & Güenaga, 2002; Villanueva, del Mar Guzman, Javier
Toyos, Ariza-Ariza, & Navarro, 2004). For more details visit
Cellistem-F® 18
http://www.rheumatology.org/I-Am-A/Rheumatologist/Research/Clinician-
Researchers/Western-Ontario-McMaster-Universities-Osteoarthritis-Index-WOMAC

Study Design
For the phase I clinical trial, a “3+3” design with dose escalation will be
implemented (Le Tourneau et al., 2009). The first three patients will be treated at a low
starting dose Cellistem-F® (10 x 10
6
cells = 3cc; dose K). Patients will be monitored for
four weeks. If none of the three patients experience a DLT, another three patients will be
treated at the next higher dose level (20 x 10
6
cells = 3cc; dose K+1). However, if one of
the first three patients experience a DLT, three more patients will be treated at the same
dose level. The dose escalation will continue until at least two patients among a cohort of
six patients experience a DLT. MTD will be considered the highest dose at which one or
zero out of six patients experiences DLT within for weeks of initial treatment (Le
Tourneau et al., 2009). DLT will be monitored for four weeks, after which a second
cohort of three patients will initiate treatment based on the results obtained in cohort one.
If necessary (Figure 2.) a third cohort of three patients will be enrolled after four weeks of
cohort 2 initial treatment.
Our second aim will evaluate the safety of Cellistem-F® for the symptomatic
treatment of OA, at weeks 4, 12, 24, 36 and 52 after intervention. Safety will be measured
by monitoring the number of severe adverse events experienced by patients during the
first year of treatment.
For our third aim, effect size will be measured using the Western Ontario and
McMaster Universities Arthritis Index (WOMAC) score to assess change in knee health.
Cellistem-F® 19
The questionnaire will be administered by trained personnel to each patient at every
follow-up consultation. All patients will be followed for a total 52 weeks after initial
treatment. Results will be calculated as the medain change in the patient’s assessment of
knee health at baseline vs 52 weeks after intervention.

Treatment Description
Cellistem-F ®, 10 x 10
6
cells (dose K), in 3ml of saline with 5% plasma.

Recruitment, Screening and Baseline Assessment
Target Population: Patients with grade I-III knee OA
Source Population: Primary health care patients referred to the National
Institute of Geriatrics, Santiago, Chile, during the year
2019
Study Population: Patients age >55 years, with grade I-III knee OA, with or
without meniscal rupture
Inclusion Criteria: Primary health care patients with knee OA referred to the
National Institute of Geriatrics, Santiago, Chile
Patient with knee OA - Kellgreen I, II or III in knee
radiography (annex 3)
Age >55 years
Willingness to participate in the study for one year
Willingness to sign informed consent
Cellistem-F® 20
Exclusion Criteria: Skin wound or lesion in the studied knee (at time of
intervention)
Concomitant inflammatory arthritis
Unable to come to follow-up visits.
Active infections including HIV, HBV and HCV
Fever one day before or same day of the procedure
Use of corticosteroids
Use of anticoagulants
Immunodepression
Active neoplasia or in the past 5 years
Use of drugs or alcoholism
Intra articular infiltration of the treated knee in the past 180
days
History of severe allergy or anaphylactic shock

Recruitment:
Patients with knee OA referred from primary health care for treatment will be
recruited by rheumatologists from the National Institute of Geriatrics, Santiago, Chile
using a convenience non-probabilistic sampling approach. Those interested in
participating will be referred to an interview with the researchers at the Clínica
Universidad de los Andes, Santiago, Chile where the objectives and procedures of the
research will be explained. Those complying with inclusion and without exclusion
criteria will be recruited and included in the final sample.
Cellistem-F® 21
Sample Size
As a “3+3” design with dose escalation will be used, a maximum of nine patients
will be required (Le Tourneau et al., 2009). However, considering an attrition rate of 15%
based on previous studies, the sample will be inflated to eleven. The same patients will be
invited to participate in the phase II part of the study. Based on previous recruiting
experience for similar interventions, we estimate to enroll eight participants per month,
with an approximated accrual time of six weeks.
With only eleven patients, the study will be underpowered for a phase II trial.
Hence, we will use this opportunity and the 52 weeks of follow-up to estimate effect size
to power a phase II trial.

Data Management Plan
Data will be captured on paper records and then entered into an electronic system
for analysis (REDCap™). At the end of each study visit, a research nurse will review data
collected while the participant is present to ensure data completeness. Dual data entry by
two study personnel will be used for transcribing study information from the paper
records into the electronic system. Any discrepancies identified will be reconciled by
consensus between the two data entry personnel.
All paper records will be kept in locked cabinets in locked offices to prevent
unauthorized access. Electronic records will be de-identified and saved by research
number. Electronic data will be entered into password protected and encrypted electronic
systems to ensure that only authorized personnel have access and study data remains
Cellistem-F® 22
protected. The project manager will maintain a password-protected linkage file to link
research numbers to patient names.

Data Analysis Plan
Statistical analyses will be conducted in STATA and adhere to Good Statistical
Practice in Clinical Research. Descriptive statistics will be used to summarize patient’s
baseline characteristics. Means and 95% confidence intervals will be reported.
A Wilcoxon Signed Ranked test will be conducted to compare median changes in
the WOMAC score from baseline to follow-up. Descriptive statistics will be used to
summarize the data obtained to evaluate the number of adverse events reported by
patients at each time point. Means and 95% confidence intervals will be reported. 2-sided
p values will be calculated.

Missing Data
Missing data will not be imputed, as imputation methods are less reliable in
smaller sample sizes. Outliers will be assessed individually to see if they are still within
reasonable ranges or if they are the result of recording errors or laboratory errors.

Interim Monitoring of Aim 1 Outcome Measure (MTD)
Early termination will be determined by the PI and the Ethics Committee of the
Universidad de Los Andes. The interim analysis will be conducted when at least three
participants are evaluable, which is expected to be two weeks after initial treatment. At
Cellistem-F® 23
this interim analysis, treatment will be evaluated via DLT, using the O’Brien-Fleming
flexible group sequential method. Early termination will be also considered if:
• Two or more of the first three participants experience treatment-related DLT, within
52 weeks of initial treatment. The acceptable percentage of patients to experience
severe adverse events is one out of six patients.
The final analysis will be conducted when all participants have completed the last visit at
52 weeks from treatment.

Baseline and Follow-Up Measurements

Selection Treatment Follow Up Final Visit

Visit 1 Visit 2 Visit 3 Visit 4 Visit 5 Visit Visit 6

Week -2, -1 Week 0 Week 4 Week 12 Week 24 Week 36 Week 52
Inclusion/exclusion criteria x

Informed consent x

Current Medication x x x x x x x
Evaluation of adverse events x

x x x x x
Weight and Height x

Vital signs x x x x x x x
Physical examination x x x x x x x
Detailed examination of the knee x x x x x x x
Review of previous knee x-rays x

Review of previous knee MRIs x

Order New MRI of treated knee x

x

New MRI of treated knee x

x
Order Blood sample and Urine sample for next visit x

x

Blood sample - blood count, VHS, creatinine, CRP,
SGOT, SGPT, b-HCG
x

x
Blood sample - HIV, HBV, HCV x

x
Urine sample x

x
WOMAC instructions x

Cellistem-F® 24
WOMAC x

x
Intervention

x

30 minutes’ observation after intervention

x

Last visit closing procedure

x

Description of instruments used to assess the different evaluations to be carried out
during the duration of the protocol:
• Clinical history: the information will be recorded in extensive during the initial
evaluation, including surgeries, treatments and previous Rx of the knee, and in the
following controls will focus on the detection and registration of AD.
• Physical examination: will be performed for all study visits, in addition to a detailed
examination of the knee under study including: loss of muscle mass, range of motion,
stability, edema, heat, redness, sensitivity, joint effusion.
• Pharmacological record: weekly record of taking analgesic medications verified by a
contact of the coordinating nurse, to ensure no drug interference with Cellistem-F®.
• WOMAC Questionnaire (Western Ontario and McMaster Universities Osteoarthritis
Index) Instructions for filling out the form will be given at the screening visit before
filling out the form for the first time. The evaluation of the WOMAC will be done
prior to the physical examination and other painful or stressful procedures such as IA
injections or blood draw (Appendix 1).

Intervention
Experimental maneuver; VISIT 2: Day 0
The first three participants in the study will receive one of the following
treatment:
Cellistem-F® 25
• Cellistem-F®, dose of 10 x 10
6
cell, in 3cc of SF + 5% Plasma, via IA knee injection.
The IA puncture will be performed by a trained specialist in an ad-hoc room, using a
sterile technique, after cooling the skin with local ice and under ultrasound guidance.

Infiltration technique
The procedure will be explained to the patient. In the supine position, with the
extended limb, the insertion point of the 1.5 cm needle proximal to the upper edge of the
patella and 1.5 cm posterior to the patellar border of the patella (superior external portal
of the arthroscopic procedures) is located. The skin is cleaned with a cotton swab with
70% denatured alcohol. The syringe, with a needle of diameter # 21G, is directed parallel
to the articular surface of the patella. Intra-articular and extra synovial location is
confirmed under ultrasound. The procedure will be carried out under ultrasound view,
ensuring that the content is placed intra-articular.

Terms of use of concomitant analgesic medication
From day 0 the patient will be asked to suspend the consumption of any analgesic
medication of daily or chronic use (paracetamol, NSAIDs or opiates).
The PI or IC may indicate the use of acetaminophen at the time of infiltration IA
for a maximum duration of 48 hrs. From this moment on, the patient can use rescue
analgesics (paracetamol, NSAIDs or opiates) (according to the prescription given by the
PI or IC) and must be registered weekly in a booklet that will be delivered to the patient.
Additionally, patients will be contacted weekly by the coordinating nurse for the
surveillance and registration of analgesic drugs.
Cellistem-F® 26

Terms of the use of non-pharmacological therapy
Kinesiology, acupuncture or homeopathic medication during the period of the
study will not be allowed.

Adverse Event
All adverse events (AE) will be registered. AE will be classified according to the
Common Terminology Criteria for Adverse Events v4.0 (CTCAE) scale classification
(“Protocol Development | CTEP,” n.d.):
• Adverse event: An adverse event is defined as any unfavorable occurrence in a
human subject, including signs, symptoms or disease, temporarily associated with the
use of the study treatment, regardless of the causal relationship between the treatment
of the study and the adverse event.
• Severe adverse event: The occurrence of one or more of the following is considered
as severe adverse event (SAE):
- Death of some subject
- Result with vital risk
- It results in a hospitalization of the patient
- Disability / Significant disability that prevents ADL
- Medical event that does not meet the criteria previously stated, but requires
medical or surgical intervention to prevent the death of the subject.
In case of presenting an SAE the evaluator will contact the person in charge of the study
to evaluate the measures to be taken and continuity of the protocol.
Cellistem-F® 27
Severity of the adverse event
The adverse effects are defined according to the following scale and / or the NCI
CTAEC scale.
1. Mild adverse event: does not require intervention, it does not interfere with the
activities of daily life (ADL)
2. Moderate adverse event: requires some degree of local or non-invasive intervention,
with moderate impact on ADLs
3. Severe adverse event: according to the EAG criteria

Unforeseeable events
Any event that involves a risk for the subjects is defined as a non-foreseeable
problem. In general, any experience or result that meets the following criteria:
• Unexpected in terms of its nature, severity or frequency for the procedure or
population described in the protocol.
• Related or possibly related or not to the intervention
• That according to the researchers put subjects at greater risk, physical, psychological,
economic or social than budgeted.

Data Safety Monitoring Board
During the course of the study, the PI will be responsible for the supervision and
good practice of the study, ensuring that the rights of patients are respected, as well
clinical trial registration and handling of information and materials necessary for the
procedures included in the protocol and manual of procedures.
Cellistem-F® 28
In order to monitor the safety of the study, an external data safety monitory board
will be established to which all AEs will be reported. The PI and the clinical investigators
will be responsible for preparing and sending this report to the external Board on a
quarterly basis. The external Board has the following faculties:
• Suspend or interrupt the protocol based on security analysis
• Recommend changes in the study design based on safety facts
• Define an adverse effect in severe or not when there is a difficulty to categorize it as
such

Ethical Protection of Patients
Institutional Regulatory Committee
The research protocol will be presented to the Ethics Committee of the
Universidad de Los Andes for approval. Any change in the protocol will be evaluated and
submitted in the second instance to a new evaluation by the same committee.

Regional Committee
In parallel, the protocol will be submitted for review and approval to the Ethics
Committee of the Metropolitan Health Service, under which the center responsible for the
study is hosted.

Informed consent
The IC will be necessary for the participation of the subjects in the study. In this,
the subjects will be assured of the confidentiality of the data obtained, as well as the
Cellistem-F® 29
complete coverage of any event secondary to the participation in the study. In the
consent, the different procedures, examinations and controls to be carried out during the
study are detailed.

Confidentiality
The confidentiality of patient data will be ensured in the IC and will be the
responsibility of both the clinical evaluators and the sponsors of the study. This
confidentiality will cover both the data and the examinations to which subjects submit.

Cellistem-F® 30
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7. Figures

Figure 1. General Study Outline. The first year of the study will be dedicates to the evaluation of
Cellistem-F®. The second and third year will encompass phase I clinical trial for Cellistem-F®.

Development of
Cellistem-F ®
Phase I Clinical Trial
Stage 1: first year
Stage 2: second and
third year
Cellistem-F® 34

Figure 2. Phase I Clinical Trial “3+3” Design with Dose Escalation. For the phase I clinical trial,
a “3+3” design with dose escalation will be implemented (Le Tourneau et al., 2009). The first
three patients will be treated at a low starting dose Cellistem-F® (10 x 10
6
cells = 3cc; dose K).
Patients will be monitored for four weeks. If none of the three patients experience a DLT,
another three patients will be treated at the next higher dose level (20 x 10
6
cells = 3cc; dose
K+1). However, if one of the first three patients experience a DLT, three more patients will be
treated at the same dose level. The dose escalation will continue until at least two patients among
a cohort of six patients experience a DLT. MTD will be considered the highest dose at which one
or zero out of six patients experiences DLT within for weeks of initial treatment (Le Tourneau et
al., 2009). DLT, dose-limiting toxicity; MTD, maximum tolerated dose.

Dose K
3 cases
DLT = 0
Dose K+1
Add 3 more cases
DLT ≤1
Dose K+1
Add 3 more cases
DLT ≤1
DLT = K+1
DLT ≥2
DLT = K
DLT ≥2
Dose K
Add 3 more cases
DLT ≤1 DLT ≥2
Discontinue
Study
DLT = 1
Dose K
Add 3 more cases
DLT ≤1 DLT ≥2
Discontinue
Study
DLT ≥ 2
Discontinue
Study
Cellistem-F® 35

Figure 3. Phase I Clinical Trial: 52 weeks’ follow-up plan. DLT will be monitored for four
weeks, after which a second cohort of three patients will initiate treatment based on the results
obtained in cohort one. If necessary, a third cohort of three patients will be enrolled after four
weeks of cohort 2 initial treatment. Our second and third aims will evaluate the safety and
estimate effect size of Cellistem-F® for the symptomatic treatment of OA, at weeks 4, 12, 24, 36

Cohort 1 (n=3)
WOMAC
IA injection of Cellistem-
F®

Participants assessed for eligibility (n)
Included (n)
Cohort 1 (n=3)
Evaluation of DLT

Enrollment Intervention
Follow-up
Time 0

4 weeks

12 weeks

24 weeks

52 weeks

Excluded (n)
36 weeks

Cohort 1 (n=3)
Evaluation of SAE

Cohort 1 (n=3)
Evaluation of SAE

Cohort 1 (n=3)
Evaluation of SAE
WOMAC

Cohort 1 (n=3)
Evaluation of SAE

Start treatment in
cohort 2 (n=3) based
on result on cohort 1

Cellistem-F® 36
and 52 after intervention. Safety will be measured by monitoring the number of severe adverse
events experienced by patients during the first year of treatment. Effect size will be measured
using the WOMAC score. The score will be applied to each patients’ in every follow-up
consultation to calculate the mean change in patient’s assessment of knee health. DLT, dose-
limiting toxicity; CTCAE, Common Terminology Criteria for Adverse Events v4.0; MTD,
maximum tolerated dose; WOMAC, Western Ontario and McMaster Universities Arthritis Index

Cellistem-F® 37
8. Appendix 1

Abstract (if available)

Abstract Hip and knee Osteoarthritis (OA) are among the leading causes of global disability. Current treatment focuses on symptomatic relief and relies on the use of analgesics and anti-inflammatory drugs. However, the unsatisfactory effects and side effects associated with traditional OA drugs warrant a continued search for potential new interventions. Recent evidence points towards the safety and efficacy of Mesenchymal Stromal Stem Cells (MSCs) for the treatment of knee OA, and its disease modifying potential. We propose a two-stage process to evaluate an existing standardized, cryopreserved version of umbilical cord (UC)- MSCs therapy, Cellistem-Frozen® (Cellistem-F®) using in vitro studies to measure viability, differential potential to cartilage and anti-inflammatory effect, (Stage 1), and a subsequent phase I clinical trial to test the safety and estimate effect size of Cellistem-F® among patients >55 years of age with grade I-III knee osteoarthritis (Stage 2).

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Asset Metadata

Creator Orrego Garbin, Francisca Javiera (author)

Core Title Cryopreserved umbilical cord mesenchymal stem cells therapy for the treatment of knee osteoarthritis: in-vitro evaluation and phase I clinical trial protocol

School Keck School of Medicine

Degree Master of Science

Degree Program Clinical, Biomedical and Translational Investigations

Publication Date 02/15/2020

Defense Date 01/26/2018

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag clinical trial,cryopreservation,disease modifying drug,in vitro,knee,mesenchymal stem cells,OAI-PMH Harvest,osteoarthritis,stem cell therapy,stem cells,Treatment,umbilical cord,umbilical cord mesenchymal stem cells

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Patino-Sutton, Cecilia (committee chair), Vangsness, C. Thomas, Jr. (committee member), Wilson, Melissa (committee member)

Creator Email fran.orrego.g@gmail.com,orregoga@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-474318

Unique identifier UC11267183

Identifier etd-OrregoGarb-6043.pdf (filename),usctheses-c40-474318 (legacy record id)

Legacy Identifier etd-OrregoGarb-6043.pdf

Dmrecord 474318

Document Type Thesis

Rights Orrego Garbin, Francisca Javiera

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA