Viability of CD34+ cells and TNC microenvironment are the preclinical considerations in successful cord blood

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KKU Journal of Basic and Applied Sciences

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Viability of CD34+ cells and TNC microenvironment are the preclinical considerations in successful cord blood

transplantation

Harish C Chandramoorthy

^{a, b,}

*, Ahmed Al-Hakami

^a,b

, Ahmed Alrabai

^a

, Hussian Nasser Kariri

^a

, Abdulaziz Alamri

^c

, Ashish Kumar

^b

, Prasanna Rajagopalan

^d

a Center for Stem Cell Research, College of Medicine, King Khalid University, Abha, K.S.A.

b Department of Microbiology & Clinical Parasitology, College of Medicine, King Khalid University, Abha, K.S.A.

c Department of Medical Education, College of Medicine, King Khalid University, Abha, K.S.A.

d Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Khalid University, Abha, K.S.A.

Keywords: Total Nucleated Cells (TNC); Stem Cells; CD34+; Viability; Cord Blood Transplantations; Cord Blood Hematopoietic Stem Cells (CBHSC)

1.INTRODUCTION

Transplantation has become a key therapeutic intervention in modern medicine (1). Almost for four decades, it has saved thousands of lives and still a hope for millions in future. There has been tremendous development in the sources of stem cells, route of administration and timing of transplanting the cells in a clinical transplant setup (2). All of these factors majorly influence the success rate for a given disease or disorder.

However, along with these set factors, the preclinical processing of the stem cells irrespective of its sources

play role in the successive outcome of the transplantation (3). Cell number, other nucleated cells along with progenitor cell counts and viability of the harvested and post processed cells are crucial factors, which determines the success rates in a proper clinical transplant setting.

With the growing numbers of the transplantations worldwide and number of diseases treatable with hematopoietic stem cells are in rise, the success rates of these transplantation are varied and differ from country to country. There are no clear-cut literature sources on the selection preclinical parameters defined in many of the developing countries, where transplantation is attempted as part of medical tourism (4). The pursuit of Received: September 2018 / Revised: November 2018 / Accepted: December 2018 / Published: 25 December 2018

Abstract: Objective: Viability, total nucleated cell (TNC) and CD34+ cell counts of the progenitor stem cells are the major criteria for successful transplant outcome. The cell viability and TNC count varies due to collection and processing procedures results in altered transplant outcome .

Methods: Based on the hypothetical questions raised, the information on the type of transplant, source of cord blood stem cells, clinical condition, number of successful transplant outcome, viability, total nucleated cell count, age factor and flow up were collected from full research papers, case reports, follow up studies from standard journals and websites of transplant centers and analyzed .

Results: Many studies indicated the criteria of ≥ 95% CD34+ viability and a range of 5 to 5.5 X 108 TNC/kg body weight for successful transplant outcome. However, there was no uniformity in the establishment on the dose of cells. Many such studies claim that even low viability and cell numbers does not hinder success of the grafting and homing of stem cells. In the current investigation, we have analyzed a direct relationship between the cell viability and TNC/CD34+ count in the successful transplant outcome for non-malignant and malignant hematological disorders by cord blood transplantation.

Conclusions: There are very few controlled studies, which implies on the role and importance of the viability and TNC counts on the success of engraftment and homing. Largely many studies do not comment or even mention the viability percentage nor try to equate or compare their failure with the viability or cell number.

young transplant surgeons to assess these parameters at the stage of training and/or a commoner, who needs to understand the treatment modality and intricacies before opting for a transplantation are misled either to go for or restrain form transplantation. In last two decades, there has been more flexibility with choosing the source of stem cells (5, 6). Cord blood hematopoietic progenitor stem cells or simply cord blood hematopoietic stem cells (CBHSC) has been an excellent source especially with pediatric transplantation for non-malignant and malignant hematological disorders (7). There are public and private cord blood banks to store the post processed umbilical cord blood from the newborn collected pre or post placental delivery. The concept of the cord blood banking is very successful in the developed countries, overtaken by the developing countries. Transplantation with the stem cells for the sports medicine and cosmetics have become more prominent in the developing countries though there is no solid evidence for complete reversal of the symptoms. These issues of failures, which are contemplated with the established transplantation options for many hematological diseases remains debatable (8, 9, 10, 11). This issue are one side of the lacunae in understanding the transplant outcome while the other issues are the published literature sources about the success of the transplants wither case-by-case or retrospective data for a particular period. Many of these literatures are silent about the preclinical parameters of the cell source, concentrations, viability (12, 13). Further even with the peer reviewed sources there no uniformity in the terms of cellular dose, viability and number of units. Therefore in the current investigation, we sought to address the logic points that affects the transplant outcome like investigating the direct relationship between the cell viability, TNC, CD34⁺ progenitor cell count and viability post cryostorage in malignant and non-malignant disease conditions. There are few literature sources, which comment on viability alone irrespective on the cell dose or cryopreservation status (14) as an important factor for transplant success.

Further, since cord blood banking in United States is roughly three decades old, much of the literature talks on public cord blank donations and use of them in transplantation. Little is known on the impact of viability on the long-term preservation and the transplant outcome or its relationship (15) of privately stored cord blood units. We therefore assessed literature evidence to address whether; there is a phenomenal difference between the long-term cyrostored cord blood stem cells verses relatively fresh in the transplant outcome. Lastly the difference observed in the literatures were due to different clinical transplant setups between developed and developing world and their priorities in choosing the preclinical parameters like TNC count, CD34⁺ progenitor cells and viability as vital factors for successful transplant outcome.

2.MATERIALSANDMETHODS

The current study is retrospective and based on the hypothetical questions raised. Materials of the our study were full research articles, individual case reports, follow up studies and review articles from standard peer

reviewed literature sources like PUBMED and ISI Thomson Reuters (16), MEDLINE (17), SCOPUS (18) etc. Further success stories from authenticated web sites on transplantations, pamphlets and write-ups from various cord blood banks formed other assistive literatures (19, 20, 21, 22, 23, 24).

Inclusion Criteria

The articles having information on TNC count, CD34+

cells, viability, information on source of the stem cells as fresh frozen or long time cryopreserved, post thaw viability for CD34+ cells, follow up of the case for at least 15 months.

Exclusion Criteria

The information from non-peer reviewed journals, articles lacking on cell dose, viability information, source of stem cells as fresh frozen or cryopreserved for longer periods, very few days follow up after transplantation etc were excluded from the analysis.

Analysis of the literature evidence are based on the disease conditions like malignant or non-malignant, TNC count (dose/bodyweight), CD34+ cell count (dose/bodyweight), viability, cryopreservation status and number of follow up in years and re occurrence. The data collected based on the geographic contents like North America, South America, Europe, Africa, Asia and Australia. Later these data were divided into developed and developing world with North America, Europe and Australia along with Japan as developed world while South America, Africa, Asia and East European countries forming the developing world. Age was included as an additional factor while assessing the transplant outcome.

Studies without follow up or just very short follow up post transplantations was not considered for the assessment, since we do not know the long-term success of such cases.

Statistical Analysis

Since the data were not uniform, ranges were set based on the standard literature for the TNC count; CD34+ cells and real time count of the cells expressed were taken for the analysis. Viability was represented as percentage (%).

The cord blood banks, number of CBTs and viability were expressed in %, while the cell concentration in millions. Student’s t test (unpaired) with Welch’s correction was used to compare the significance.

Differences between groups of various combinations of count Vs viability were analyzed by one-way ANOVA using Duncan’s post-hoc test, and p values < 0.05 were considered significant.

3.RESULTSANDDISCUSSIONS

Cord Blood Banks and Cord Blood Transplantations In the last two decades, the number of the cord blood transplantations (CBT) have increased. More number of such transplantations for both malignant and non- malignant hematological diseases are reported with success (5). The growing rate of cord blood banks in the developing countries and parts of the Europe especially private cord blood banks will outnumber the established

banks in US or Western Europe (25). This report was in accordance to our observation on the current number of projected cord blood banks in developing countries, which is 43% compared to the 57% of the developed or western world (Fig 1a). In few years from now, the number of the cord blood banks in the third world countries will outnumber other places though there is no watch or control on their promises for cure or successful transplantation services. We further do not know the status of the accreditation, which poses a great hurdle in understanding the universality of the cellular parameters followed for successful or established transplant services.

Our current analysis shows around 23% (Fig 1b) of the accredited cord blood banks in the developing world; far less compared to the total number of the operating units (26). However, 77% (Fig 1b)of the cord blood banks both private and public (almost the entire public establishment and few of the private cord blood banks offering public services) is accredited to one or many of the apex bodies regulating the stem cell transplantation and research in US, Europe, Japan and Australia (26, 27,28, 29, 30).

The CBTs done from 2005 to 2014 clearly (Fig 1c) shows rise in number of cases, which used to be in few hundreds every year before 2005 in western world, there is no clear cut published data available in developing countries before 2005 except for few reports (31, 32, 33, 34, 35,).

In all these reports, the successful transplantations are measured by the post-transplant survival of TNC and marrow engrafting retuning near normal, while in few studies extent to report periodical follow-up through checkups indicating the reversal of the pathology to normalcy.

TNC/CD34⁺/Viability are basic preclinical parameters that determines the success of transplantations Our aim was to assess the transplant strategies like preselection of the cord blood stem cell units, HLA match,

adequate number of TNC and CD34⁺ cells and post thaw high viability of the cryopreserved units. Many studies both laboratory and clinical trials have revealed the importance of the adequate progenitor cells along with other nucleated cells and high viability to create a micro environment favoring the survival of the progenitor cells in the host (36, 37, 38). On the analysis of the published peer reviewed literature sources and data from the official sites of the accredited banks and policy making bodies, the average TNC counts ranged between ~ 8 X 10⁸ cells to 10 X 10⁸ (Fig 2a). There was not much difference observed in these numbers from the data’s of the developing world as their counts ranged within these numbers for at least CB units received form accredited units. Similarly, we did not observe much deviation in the concentration of CD34⁺ cells (Fig 2b) between the developed and developing countries. The average cell concentration was ~ 15 X 10⁶ to 18 X 10⁶ cells. In fact we observed slightly higher number of CD34⁺cells in the CB units of developing countries as reported in many publications may be due to practice of CB collection at the site of labor even before the placenta has delivered against post placental delivery observed in developed countries (39, 40, and 41). The TNC viability at the time of harvesting was around 90% in both developing and developed countries (Fig 2c). The CD34⁺cell viability given mostly were from cryopreserved units with few exceptions, like the fresh CB units transiently stored for the few days before transplantation showed relatively higher viability. The lower range of the reported viability was more than 80 to 85%. None of the literature reported low viability rather they did not mention anything in general about the viability of the CD34⁺cells.

Viability of the TNC/CD34⁺ determines the success of transplantations

There were some studies, which did not mention viability or negative transplant outcome as previously discussed.

Figure 1. Cord Blood Banks and Average CBT.

(a) The distribution of projected cord blood banks from the developed and developing world, which will be active in next decade. (b) The actual distribution of accredited cord blood banks in developed and developing countries. (c) Average number of CBTs done in developing and developed world from 2005 – 2014 from the published literature sources.

Figure 2.TNC/CD34+ Count. (a) Average TNC

count ranging about 8X10

⁸

to 10 X 10

⁸

in both

developing and developed countries. (b) Average

CD34

⁺

count about 17X10

⁶

to 19 X 10

⁶

cells in

developing and developed countries.

We further do not know the success of the transplantations, since no follow up or the paper itself did not assess the long follow up to ensure the success of stem cells to home/graft and differentiate into the matured functional adult cells (42). Therefore the success of the transplantation varied with numbers of the TNC or CD34⁺ cell counts however, the viability in all these reposts were higher (43, 44, 45). It is further evident that low cell concentration of the TNC count (Fig 3a) showed lower success rate somewhere between 70 to 75%. These were from the data obtained, which had generally higher CD34⁺ count. On contrast, lower number of CD34⁺ cell count always corresponded to the success rate between 50 to 55% irrespective of its high or low TNC count (Fig 3b).

Though the average success rate based CD34⁺ and high viability is about 65 to 70% (Fig 3b) depending upon the type of the hematological disorder for which the transplantation requested (46, 47). Since in our investigation we did not segregate malignant with non- malignant diseases, the success rate reported here is slightly higher taking into account of the survival measured is only for a short period of time over 15 months post transplantation, but not exceeding beyond 3 years.

Viability is the key for successful engraftment confirming the success of transplantation

The disparity in the cell count between TNC and CD34⁺ clearly show that, for a successful CD34⁺ progenitor engraftment, there must be a considerate number of TNC microenvironment to sustain the sterile environment created by radiation or chemotherapy (Fig 4 a, b).

Therefore, we assessed the various combinations of TNC and CD34⁺ cells keeping the viability at constant and or changing the viability and keeping the high CD34⁺ count as constant (Fig 4 c). The intention of these assessments was to observer as what is the preclinical condition that needs a check while receiving the CB units for a successful transplantation. From the data analyzed, we

find low TNC/high CD34⁺, all high TNC/CD34⁺/viability and low TN/high CD34⁺/viability showed around 80%

engraftment leading to transplant success (Fig 4c).

Clearly, other groups like high TNC/low CD34⁺, high TNC/ highCD34⁺, high TNC/ low CD34⁺/ medium viability showed success around 40 % except the group low TNC/CD34⁺ showed around 20% success revealing count of progenitors also plays a crucial role in the transplant success. The observations shown here are combined data from various hematological disorders and age groups with majority from pediatric age group (48, 49). However, the engraftment potential may vary with the severity of the disease, type of disease, HLA matching and transplant setup, individual’s response, which needs to be understood here. Out of all odds, viability stood as important deciding factor with adequate number of CD34⁺ cells and TNC.

4.CONCLUSION

Our analysis shows there is a growing number of CBT every year. We do observer large number of success stories on CBT. However, we do not have common protocol to know the preclinical conditions to select CB unit from the expanding public and private cord blood banks in developing world. Current analysis reveals CB units with higher viability and high CD34+ cells with adequate TNC will be perfect for a successful transplantation.

ACKNOWLEDGEMENT

We render our sincere thanks to Dr. Mohamed Eajaz Ahmed Sharif, Department of Physiology, College of

Figure 4 Different ranges of TNC and CD34⁺ cells and various combinations in the transplant success and engraftment.

(a) Average TNC counts and % success. (b) Average CD34

⁺

cells and its % success rate. (c) The various combinational showing viability along with high TNC and CD34

⁺

cells are major factor in the transplant success.

Figure 3 Viability of TNC and CD34⁺ Cells. (a)

The viability of TNC during processing is shown

which reflects proper processing of the samples

in both developing and developed countries. (b)

The viability of post thaw of CD34

⁺

cells

reflecting proper cryopreservation in developing

and developed countries.

Medicine, King Khalid University, Abha, Saudi Arabia for his support in literature collection and student’s guidance. and Mr. Muhammad Abid, Department of Medical Education, College of Medicine, King Khalid University, Abha, Saudi Arabia for his statistical guidance and help in the data analysis.

COMMON ABBREVIATIONS TNC – Total Nucleated Cells

CBHSC – Cord Blood Hematopoietic Stem Cells CBT- Cord blood Transplantations

HLA – Human Leucocyte Antigen CB- Cord Blood

CD - Cluster of Differentiation

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