A variety of DNA-based methods have been applied to identify genetic markers of attention deficit hyperactivity disorder (ADHD), but the connection to RNA-based gene expression has not been fully exploited.Using well defined cohorts of discordant, monozygotic twins from the Michigan State University Twin Registry, and case-controlled ADHD cases in adolescents, the present studies utilized advanced single molecule RNA sequencing to identify expressed changes in whole blood RNA in ADHD. Multiple analytical strategies were employed to narrow differentially expressed RNA targets to a small set of potential biomarkers of ADHD.RNA markers common to both the discordant twin study and case-controlled subjects further narrowed the putative targets, some of which had been previously associated with ADHD at the DNA level. The potential role of several differentially expressed genes, including ABCB5, RGS2, GAK, GIT1 and 3 members of the galactose metabolism pathway  are substantiated by prior associations to ADHD and by established mechanistic connections to molecular pathways relevant to ADHD and behavioral control.The convergence of DNA, RNA, and metabolic data suggests these may be promising targets for diagnostics and therapeutics in ADHD. Attention deficit hyperactivity disorder (ADHD) is the most common psychiatric disorder in childhood and adolescence, affecting roughly 5% of youth worldwide . AlthougPhysiologically, numerous avenues have been examined for a potential role in ADHD. One theory holds that ADHD involves changes in the glutaminergic neurotransmitter systems, a major excitatory pathway in the brain, however genome-wide SNP analysis has so far failed to identify glutamate receptor DNA variants as a significant association with ADHD . SeveralWhile a major focus has been on neurodevelopmental pathways, there are reasons to be cognizant of potentially important changes in non-neural endocrine systems. The strong linkage of ADHD with male gender has raised potential connections to steroid hormones, but it is difficult to exclude sex-linked genetic elements, and/or cultural perceptions related to the diagnosis of ADHD .It has recently been suggested that RNA sequencing may be informative for identifying ADHD-related biomarkers, based on positive findings in a small paired analysis in 3 multiplex cases . AdditioThe present studies employed cutting-edge RNA sequencing technology to very accurately quantify transcript levels across the entire transcriptome, for both coding, and non-coding transcripts. The RNA profiling approach was applied to stabilized whole blood samples drawn from two well-characterized cohorts: (a) children matched on ADHD and non-ADHD status and, (b) identical twins that were discordant for ADHD traits. The results provide a deep and broad map of RNA changes potentially related to ADHD, and can be combined with other ‘omic data, such as genome-wide association studies (GWAS), DNA methylation, and proteomic analysis to help identify new biomarkers, and secondarily, new clues to biological processes related to ADHD.Case-controls were selected from the Oregon ADHD Longitudinal Study Cohort , who independently assigned final diagnoses and comorbid disorders including ADHD, oppositional defiant disorder (ODD), and any lifetime mood disorder , as reported herein. History of seizures or head injury, parent–teacher rating discrepancy making diagnosis uncertain, psychosis, mania, current major depressive episode, Tourette’s syndrome, autism, and estimated IQ < 80 were a basis for exclusion in the present study.From 2144 volunteers, 850 eligible children were identified. The group used in the current study were a subset selected because they (a) clearly met Diagnostic and Statistical Manual of Mental Disorders-5 (DSM-5) criteria for ADHD or non-ADHD comparison group (rather than subthreshold), (b) had no prior history of psychiatric medication, (c) were Tanner stage 1 or 2 by parent report on the Pubertal Development Questionnaire, (d) were not a sibling of another child in the cohort; and, (e) were willing to give a blood sample. This process provided a set of non-twin case-controls  for whom Paxgene-stabilized, frozen blood samples were submitted for RNA sequencing. After RNA sequencing, 23 ADHD and 21 case controls were available, as described in Table Twins were recruited from the Michigan State University Twin Registry  about 1/3 of twin births are monozygotic, (b) ADHD has high heritability, so discordant pairs are rare. The final samples available for statistical analysis after clinical screening, RNA sequencing, and data quality checks is shown in Table ADHD was evaluated by the following measures: parent completed the Conners' Rating Scales-3rd Edition short form ; StrengtAt least a 3-symptom separation on the ADHD-RS or KSADS, with one twin below diagnostic threshold and never identified with ADHD; orAt least a 10 point T score difference on the ADHD-RS, orOne was previously diagnosed and treated and the other was not (and the untreated twin did not meet ADHD criteria by our measures), orAny combination of these criteria.Twins were considered discordant if they met the following criteria:Absolute difference between higher and lower scoring twin on an ADHD–RS (by parent) raw score (range 0–27) obtained at the same time as the RNA blood tube was collected, and rated over the past 6 months for (i) inattention, (ii) hyperactivity-impulsivity, and (iii) total. The total is reported here.For the fourth variable, each twin pair was ranked on “degree of discordance” based on considering all available data including multiple time points rating scales , the KSADs, and comorbid and substance use conditions. This was achieved by consensus rankings by JN and EB blind to the RNA data.To create a quantitative estimate of degree of discordance, the following four variables were created:For complete methods, see Additional file First strand cDNA synthesis was carried out with 50–100 ng RNA at 95 °C for 5 min using 50 ng/µL random hexamers  and 1 µL of 10 mM dNTP mix (Invitrogen #Y02256). 3′ Poly A tailing was achieved with terminal transferase (NEB #M0315). Cleaned samples were denatured and hybridized to the poly dT surface of SeqLL flow cells. Samples were sequenced using SeqLL’s True Single Molecule Sequencing (tSMS) technology that allows for RNA sequencing without requiring PCR amplification or library preparation ligation steps.Data output from the sequencer is in raw short read format (SRF) files. SRF files were processed using the HeliSphere Bioinformatics package, first converting to SMS format for alignment. SMS reads were trimmed for leading T homopolymers and were filtered for reads with a minimal length of 25 bases after trimming. Trimmed reads were aligned to the HG38 human genome (GRCh38) supplemented with the complete ribosomal repeat unit (GenBank Accession U13369.1) using the HeliSphere BASIC analysis pipeline.p value likelihood of a difference between groups adjusted for multiple testing using the Benjamini–Hochberg method. To constrain the size of the DEG list, it was predetermined to select the top 100 from each method and then combine them to achieve a single ranked list across methods. These were then ranked by the number of times a given DEG appeared in each of the 9 lists.From the aligned reads, a variety of analytical approaches were employed in order to identify differentially expressed genes (DEGs) which passed one or more filtering strategies. To minimize the impact of any one statistical method, the sample set was analyzed by 9 methods, deriving from 5 types of analysis: using the EdgeR Bioconductor package , 19 usinA second general strategy that has proven useful, due its simplicity and absence of assumptions about the distribution of RNAseq data, is to adjust the transcript counts only by correction for the transcript size and total informative reads. The RPKM calculation compensates for the size of the transcript, and for the total number of reads acquired. The number of reads aligned to each transcript are divided by the size of transcript in thousands of base pairs (per K), and then divided by the total number of informative reads obtained for that subject (in Millions), yielding RPKM. To further constrain the DEG list to transcripts with detectable expression, the total aligned read counts for the HG38 genome  were filtered to include transcripts present at > 0.01 RPKM in 70% of the samples of at least one diagnostic group, leaving ~ 95 K working transcripts for analysis  of gene expression over ~ 22 log2 orders of magnitude for more than 95,511 transcripts . Employing the 9 filters approach, 391 transcripts were ranked in the top 100 by at least one method. Of these, two transcripts were identified by all 9 methods, and 5 transcripts were identified by 8 of the 9 methods. Note that although this reduces the likelihood that their differential expression is dependent on only one analytical approach, it does not fully eliminate potential type I error because these are not completely independent methods Fig. . The fulABCB5 (↑1.9X in ADHD) is a member of the multi-drug resistance (MDR) family of transporters. ABCB5 has been mechanistically linked to glucose, phospholipid, and amino acid transport , and copCWC27 (↓1.4X) is a spliceosomal trans-peptidyl-isomerase that has been associated with retinal abnormalities and developmental disorders, including neurological defects, in children with mutations .IFI35 (↑1.4X) is an interferon-induced protein that acts as a ‘damage-associated molecular pattern (DAMP)’ , and thuAHNAK (↑1.3X) is a particularly interesting target because it is a giant 680 kD neuroblast differentiation-associated protein, that has been associated with a range of relevant neurological disorders including bipolar disorder , depressThe fifth transcript, RP11-35015.2 (↓1.6X) is a poorly annotated transcript that lies within intron 1 of the IGF1 receptor (IGF1R), and thus, difficult to more clearly understand.The complete list of 391 selected  is an ADP ribosylation factor-like GTPase 6-interacting protein, but is also known as JWA, a homologous gene of the glutamate-transporter-associated protein 3–18 (GTRAP3-18), and addicsin. ARL6IP5/JWA is expressed at high levels in the hippocampus and ARL6IP5/JWA knockout mice showed spatial cognitive potentiation and enhanced neurite growth in newborns . ConditiCCDC107 (↓1.9X) is closely related to CCDC132 and CCDC84 found in the case control study. While relatively little is known about these coiled coil family members, coiled coil helix proteins (e.g. Chchd2) have been implicated in ADHD-like mouse models .CCND1, cyclin D1 (↓2.9X), is related to CCNC and CCNL2 from the case control studies. While the cyclins are largely studied in relationship to cell cycle control, they can serve a variety of regulatory functions in cells.DBF4B, DBF4 Zinc Finger B (↑2.5X), has been extensively studied as an activator of the Mcm2-7 helicase, a partner to Cdc7 kinase, and thus important for the initiation of DNA replication. Potentially of interest, it has also been associated with autism spectrum disorders via a semaphorin 5A (SEMA5A) eQTL network .Dual specificity phosphatase 4  is a family member to DUSP6 from the case–control study, and has recently been described as a control element in the suprachiasmatic clock network via modulation of vasoactive intestinal peptide signaling to ERK1/2 .FAM159A (↑1.7X) has counterparts FAM104A, FAM134B, FAM157C, FAM162A, and FAM213B as differentially expressed in the case–control study. Little is known about FAM159A, but FAM134B, aka RETREG1 reticulophagy regulator 1, has a substantial literature connecting it with various functions including autophagy, and sensory neuropathy in humans  and BordRARS2 (↓1.6X) is the arginyl-tRNA synthetase gene that has been associated with a spectrum of neurological disorders including myoclonic epilepsy, mental retardation, spasticity, and extrapyramidal features . PatientRN7SL454P (↓1.95X), has counterparts RN7SL423P and RN7SL687P as DEG in the case control cohort. It appears to be a small non-coding transcript, intronic to the dynein axonemal heavy chain 17 gene on chromosome 17 (DNAH17), but with no known function.SIK3.IT1 (↑2.1X) is salt-inducible kinase 3, with known relations to sleep and circadian rhythm, and to glucose and lipid homeostasis, steroidogenesis, and adipogenesis .SPICE1(↑1.4X) is a spindle and centriole-associated protein, which might relate to DBF4B and CCND1 in regards to cell cycle control. Computational screening identifies it as an aurora kinase substrate and it is known to cooperate with CEP120 in centriole elongation. Interestingly, SIK3 also interacts with aurora A, aurora B, and polo-like kinases, and SIK3 repression enhances the antimitotic effect of aurora inhibition . Likewisp = 0.318), it does suggest that these transcripts may merit further analysis as hypotheses in future studies.From the 385 DEG list compiled from 9 analyses of the discordant twins , but conversely, one cannot dismiss immune involvement as noted earlier. The potential role of inflammatory factors in ADHD has been raised over the years and is supported by various circumstantial data as recently reviewed , and recIn the same vein, IGHV3-74 is the variable region heavy chain transcript involved in antigen recognition by encoding IgM antibodies. While speculative, increased levels in the 2 cohorts could suggest some type of immune or autoimmune activity in ADHD.The regulator of G protein signaling RGS2 is increased in both cohorts. RGS2 has diverse actions including promoting the translation of stress-associated proteins ATF4 and CHOP via an eIF-2B inhibitory domain . Of poteOf potential interest is Park7 RNA (DJ-1), which is extensively investigated as related to early onset Parkinson’s Disease . Based oChanges in RNU1-14P, which is a small nuclear pseudogene, is quite difficult to interpret, as is the RP11-661A12 transcript, though the latter is potentially an upstream ORF or alternate 5′ exon for the zinc finger CCCH-type containing 3 (ZC3H3) transcript, which is involved in nuclear adenylation and export of mRNAs .An additional set of transcripts had very similar isoforms reported in the case control results, for example, MED7 vs MED6, CLIC2 vs CLIC1, JRK vs JRKL. While there is no assurance that these close family members perform similar functions, it is worth considering whether a similar pattern is reflected. A list of 66 of these overlapping transcripts was submitted for an unbiased analysis using pre-curated relationships between the gene products . Several plausible relationships are identified in a manner that could identify latent variables that might account for a substantial subset of the transcript variations. Statistically, the top pathway identified centered around the well-characterized Akt/Insulin/PI3kinase/NfKB axis, as shown in Fig. p value  filtering approach that has proven useful in prior biomarker studies [In the context of a hypothesis-generating, exploratory study such as this, the prior analysis using 9 DEG methods may risk missing biologically important pathways in favor of statistical rigor. The datasets were re-analyzed using an RPKM threshold of 0.01, and combined fold-change (> 1.5) and  studies , 67. ThiACP2 (↑2.0X twins) is a lysosomal acid phosphatase that is known to play a vital role in the removal of mannose-6-phosphate residues. ACP2 has known or suspected roles in several neurodevelopmental disorders, as emphasized by mutations in Acp2 causing severe cerebellar and neurodegenerative diseases . IntegraALKBH6 (↑2.8X twins) is potentially important because, while relatively little is known about it, by analogy to its homolog ALKBH5, it is likely to function as a methyl-N6-adenosine (m6A) demethylase . While tASPSCR1 (↑2.6X twins), is a UBX domain containing tether for SLC2A4, which has a known fusion protein to TFE3 that is involved in certain cancers. However, better known as TUG, it has important roles as an interactor with the glucose transporter GLUT4, with regulatory activity over insulin-regulated aminopeptidase (IRAP) and vasopressin secretion . While cCLYBL (↑2.6X twins) is citrate lyase beta-like transcript, which encodes a malate/ß-methylmalate synthase with known effects on Vitamin B12 levels . VitaminGAK (↑2.1X twins), cyclin G associated kinase, is potentially interesting in relation to ADHD. GAK (auxilin-2) has known involvement in synaptic function and neurological diseases , and is GALE (↑2.9X twins), UDP-galactose-4-epimerase, is one of 3–4 key enzymes in the synthesis and utilization of galactose, and changes in the other members of this family, especially GALT and GALK, were noticeably affected in the ADHD cases, with all 3 of these enzymes in the galactose processing pathway being elevated in the ADHD-affected twins Fig. .Fig. 6DiGIT1 (↑2.6X twins) was elevated > twofold in the ADHD subjects in both the discordant twin and case–control cohorts Fig. , and theSTAM2 (↑1.5X twins), signal transducing adaptor molecule 2, is a member of the endosome-associated ESCRT-0 complex that is highly expressed in neurons, especially in the cerebral and cerebellar cortex, hippocampus, and medial habenula . STAM2 rThe remaining targets identified in both cohorts are more difficult to interpret. ERCC6L2 (↑2.9X twins) is excision-repair like 2, which has known relevance in cancer, but is difficult to connect with ADHD. HDLBP (↑1.3X twins) is highly relevant to high density lipoprotein metabolism, but has only a tenuous connection to ASD by virtue of a 2q27 deletion that causes reduced expression of HDLBP and 2 other genes . IDS  bears some general similarity to the RN7SL454P target identified by the 9 filter approach, and discussed above. Both of these small nuclear pseudogenes are likely to have as yet unknown regulatory functions . SRP14,  fatigue . Among p fatigue , and so p values in our analysis.Prior exome sequencing of sporadic ADHD cases compared to sibling/parent triads identified ~ 8 interesting targets . Of thosAmong the strengths of the present approach are the unique and well-characterized ADHD cohorts. In particular, monozygotic, but discordant twins present a powerful genetic model for comparison, and here demonstrated some intriguing similarity in expression pattern with a case–control cohort. They open the possibility of understanding environmental influences while largely controlling for genotype. An important methodological detail is that the RNAseq analysis examines the expression pattern on a relatively high resolution scale to the level of transcript isoforms. Other common RNAseq analytic platforms tend to aggregate expression to ‘gene level’ expression as a single transcript, which has the effect of masking changes in alternatively spliced transcripts. The case of GIT1 is an excellent example of where very specific changes in one splice variant might have high relevance to the disorder in question.Limitations to the present study principally derive from the observational nature of the studies, a necessarily small samples for MZ discordant twins, and the necessity to use peripheral blood RNA, as opposed to a tissue more proximal to the presumed neural influences on ADHD. Further, the mRNA profiling gives us a very comprehensive view of the transcriptome, albeit at a specific point in time, and without strict control of the mental or physical state of the participants. Of course, causality is indeterminate: we cannot evaluate, for instance, whether changes in the galactose pathway ‘cause’ ADHD, or somehow result from the increased activity or altered diet or other behaviors of the children. A third option, which must be considered, is that both ADHD and galactose changes could result from changes in a different pathway, or from coincidental differences in ADHD teens, such as diet, drugs, or activity. While we can exclude ADHD medications as a source of variations herein, it is difficult to exclude other type of nutritional or nutraceutical differences.Technically speaking, the RNAseq approach is intrinsically limited by the known genome and transcriptomes that are used to align and interpret the reads. Every RNA profiling method has unique ‘gaps’ and biases that can influence the outcomes, and thus it should not be surprising if a different RNAseq method identified other different differentially expressed transcripts. Further, the RNAseq of whole blood allows for the possibility that the types of cells present in blood at the time of sampling differ from patient to patient, or group to group. Because blood cell counts were not available on the subjects at the time of the blood draw, we were unable to identify such differences or adjust for them. Additionally, a valuable approach to understanding this large data set would be to conduct co-expression analysis, which is a logical next step that might reveal systematic changes not apparent from the current analysis –100.While the present results therefore should be seen as preliminary, the nature of this work is largely unprecedented and therefore it is valuable to note that several patterns were identified that are suggestive as hypotheses for further investigation. Collectively, the results affirm some prior targets, such as GIT1, that were identified by DNA-based technologies, as also relevant to ADHD at the RNA level. Several new pathways are brought to light as potentially productive ground for further exploration. Based on a variety of lines of evidence, it would be quite unlikely if there is a single etiologic cause for ADHD, and the present results demonstrate that none of the RNA transcript changes were observed to occur in all of the youth. It is interesting to speculate that changes in GAK or GIT1, which showed quite strong changes in some, but not all subjects, could indicate particular genetic/epigenetic subtypes of ADHD. The cohort sizes obviously were not powered for a detailed subtype analysis.An intriguing future direction would explore the possible role of the galactose pathway in ADHD, either as a modulator of core energy sensing via the insulin/AKT/NFkB/mediator pathway Fig. , or as aThese results are the most extensive discordant MZ study of RNAseq expression in ADHD. The results, while preliminary, suggest several interesting hypotheses for further study.Additional file 1. Enrollment details.Additional file 2. Transcript IDs by 9 DEG methods for case/control study.Additional file 3. Transcript IDs by 9 DEG methods for discordant twin study.Additional file 4. Case control T-test 524 transcripts.Additional file 5. Discordant twins paired T-test 505 transcripts.Additional file 6. Discordance DEG correlation.Additional file 7. Detailed methods.Additional file 8. DEG Concordance between methods.


Globally, 1 in 11 adults have diabetes mellitus, and 90% of the cases are type 2 diabetes mellitus. Insulin resistance is a central defect in type 2 diabetes mellitus, and although multiple drugs have been developed to ameliorate insulin resistance, the limitations and accompanying side effects cannot be ignored. Thus, more effective methods are required to improve insulin resistance.In the current study, db/m and db/db mice were injected with human umbilical cord-derived mesenchymal stem cells (HUC-MSCs) via tail vein injection, intraperitoneal injection, and skeletal muscle injection. Body weight, fasting blood glucose, and the survival rates were monitored. Furthermore, the anti-insulin resistance effects and potential mechanisms of transplanted HUC-MSCs were investigated in db/db mice in vivo.The results showed that HUC-MSC transplantation by skeletal muscle injection was safer compared with tail vein injection and intraperitoneal injection, and the survival rate reached 100% in the skeletal muscle injection transplanted mice. HUC-MSCs can stabilize localization and differentiation in skeletal muscle tissue and significantly ameliorate insulin resistance. Potential regulatory mechanisms are associated with downregulation of inflammation, regulating the balance between PI3K/Akt and ERK/MAPK signaling pathway via PTEN, but was not associated with the IGF-1/IGF-1R signaling pathway.These results suggest HUC-MSC transplantation may be a novel therapeutic direction to prevent insulin resistance and increase insulin sensitivity, and skeletal muscle injection was the safest and most effective way. Diabetes mellitus is a life-long metabolic disease with high morbidity and mortality rates, and DM reduces the patients’ quality of life due to acute and chronic complications , 2. TypeInsulin resistance (IR) and relative insulin deficiency are the pathological consequence in T2DM, and these conditions are concurrent and interacting conditions. Insulin has two different functions: firstly, hepatic glucose output or glucose uptake and utilization can be inhibited or impaired by insulin. As a result, insulin levels continued to rise chronically; secondly, it begins to fail for the pancreatic β-cells regulating a hyperinsulinemic state , 5. CompWith the development of research technologies, epigenetics studies showed that stem cells are pluripotent, and this is achieved via cellular reprogramming techniques. Thus, alternative therapeutic approaches allowed for new advances and improved understanding of stem cell therapy . At presIn the present study, the treatment and anti-IR effects of HUC-MSCs were further investigated in db/db mice. The multiple targeted mechanisms of HUC-MSCs in vivo were also investigated and may serve as potential treatments for IR in T2DM.Mouse insulin (EMD Millipore), TNF-α and IL-6 (Abcam) ELISA kits, anti-DNA (cat. no. AC30-10) monoclonal antibody , D-Luciferin, sodium salt D-Fluorescein sodium salt , LUC-labeled lentiviral particles , RIPA lysis buffer, nitrocellulose membranes, and an SABC (rabbit IgG)-POD kit were obtained from Beijing Solarbio Science & Technology Co., Ltd. A blood glucose meter and test strips were acquired from ACCU-CHEK . TRIzol® was purchased from Invitrogen . Antibodies against PI3K, P85, phospho-PI3K P85 (cat. no. Y607), insulin receptor β (IR), p-IR (cat. no. Y1185), PTEN, p-PTEN (cat. no. S380+T382+T383), glucose transporter 4 (cat. no. GLUT4), and IGF-1R were obtained from Abcam. Antibodies against Akt, p-Akt (ser 473), ERK, and p-ERK (1/2) (Thr202/Tyr204) were obtained from Cell Signaling Technology, Inc. Anti-GAPDH, anti-mouse IgG, and anti-rabbit IgG were obtained from Cell Signaling Technology, Inc. Other reagents used were of analytical grade.Six-week old female db/db mice (18–20 g) were purchased from Model Animal Research Center of Nanjing University. All mice were housed individually with a 12-h light/dark cycle at 23 ± 2 °C and a humidity of 55 ± 10%, with free access to water and food. The mice were allowed to adapt to these conditions for 1 week prior to beginning the experimental procedures. The animal procedures were approved by the Animal Care and Use Committee on the Ethics of Animal Experiments of Taizhou University of Science and Technology. All related facilities and experimental procedures were performed according to the guidelines described by the Technical Standards for the Testing & Assessment of Health Food (2003).HUC-MSCs were provided by Jilin Tuohua Bioengineering Co. Ltd. (Siping). HUC-MSCs were immediately obtained from healthy mothers during routine term elective cesarean section births. Fully informed consent was obtained several weeks prior to delivery. Ethics approval was obtained from the Ethics Committee of the Siping Center Hospital . HUC-MSCs were isolated and propagated, as previously described .n = 10), db/m+HUC-MSCs , db/m+HUC-MSCs , and db/m+HUC-MSCs ; db/db (n = 15), db/db+HUC-MSCs , db/db+HUC-MSCs (tail vain injection), and db/db+HUC-MSCs . A total of 1 × 107 HUC-MSCs (passage 4) were resuspended in 0.7 ml saline and administered by IP/IV; or 1 × 108 (passage 4) in 2 ml saline were administered by local injection IM for the db/m+HUC-MSCs and db/db+HUC-MSCs groups. The other groups were given an equivalent volume of saline as the db/m and db/db groups, and both groups were fed a normal chow diet. To determine the effect of HUC-MSCs, survival rate, weight, blood glucose, ALT, AST, Cre, BUN, γ-GT, and the levels of serum insulin were measured at the mentioned times.The db/db mice were divided into four groups: db/m . Subsequently, the mice were imaged using an in vivo imaging system . In vivo imaging was performed prior to sacrifice.HUC-MSCs were infected with luciferase-lentivirus  to establish HUC-MSCs-luc cell stably expressing luciferase. The HUC-MSCs-luc skeletal muscle transplantation db/db mice were anesthetized using isoflurane and injected intraperitoneally with 100 μl of 15 mg/ml IR calculated as follows: HOMA IR = serum insulin mmol/l × blood glucose mmol/l/22.5 .A portion of the extracted skeletal muscle was immediately fixed in PBS mixed with 4% paraformaldehyde and embedded in paraffin. The sections (4 μm in thickness) were stained with hematoxylin and eosin (H&E). Histological analysis was performed using a light microscope .Skeletal muscle sections were subjected to signal-direct IF staining of human DNA , followed by incubation with Alexa Flour 488-conjugated secondary antibodies . Nuclei were counterstained with Hoechst . All sections were scanned, and images were acquired using a laser scanning confocal microscope .2O2 solution to block endogenous peroxidases. Antigen retrieval was performed using 0.1 M sodium citrate (pH 6.0) for 60 min. Sections were incubated with anti-PI3K, anti-GLUT4, anti-p-IR, anti-p-PTEN , or anti-p-ERK  antibodies overnight at 4 °C, and a horseradish peroxidase-conjugated secondary antibody and diaminobenzidine substrate were added sequentially. Following hematoxylin counterstaining and dehydration, the sections were mounted and observed using a Leica M4000B photomicroscope .After de-paraffinization, the sections were incubated with a 3% HThe concentrations of TNF-α, IL-6  and serum insulin (EMD Millipore) were determined using specific ELISA kits.−ΔΔCq method. The sequences of the primers used are as follows: 18S-forward 5′-ACTCAACACGGGAAACCTCAC-3′, and reverse, 5′-TCGCTCCACCAACTAAGAACG-3′; c-fos-forward, 5′-GGAATTAACCTGGTGCTGGATTG-3′, and reverse, 5′-GAACATTGACGCTGAAGGACTAC-3′; c-myc-forward, 5′-CTATCACCAGCAACAGCAGAG-3′, and reverse, 5′-ACATAGGATGGAGAGCAGAGC-3′; JNK-forward, 5′-ACATAGGATGGAGAGCAGAGC-3′, and reverse, 5′-CATTGACAGACGGCGAAGAC-3′; GSK-3β-forward, 5′-CACCGCTCCTTCCTTCCTTC-3′, and reverse, 5′-GACTCCTCTTCCTAACCACCTG-3′; FoxO1-forward, 5′-CAGCCTTGAGCAGCCTAATG-3′, and reverse, 5′-AGACTGGGAAACACCGATGG-3′; GLUT4-forward, 5′-ACGGATAGGGAGCAGAAACC-3′, and reverse, 5′-CAGCACAGGACACTCATCTTC-3′; PTEN-forward, 5′-AGAGATTGGCTGCTGTCCTG-3′, and reverse, 5′-TGGTTAAGTCATTGCTGCTGTG-3′; IRS-1-forward, 5′-AGCAGCAGTAGCAGCATCAG-3′, and reverse, 5′-TTACCGCCACCACTCTCAAC-3′; IGF-1-forward, 5′-TATGGAGATGGGAGGGTTTCAG-3′, and reverse, 5′-GTAGGCACAGCATTCGTTAGG-3′; mTOR-forward, 5′-GCAGCAACAGTGAGAGTGAAG-3′, and reverse, 5′-CAAGGAGATAGAACGGAAGAAGC-3′.Gene expression of c-fos, c-myc, JNK, GSK-3β, FOXO1, GLUT4, PTEN, and IRS-1in skeletal muscle section was analyzed using RT-qPCR. TRIzol® reagent was used to extract RNA, and cDNA was synthesized using a reverse transcription kit . qPCR containing was performed using a SYBR Premix EX Taq™ cDNA with specific gene primers, and genes were amplified using a 7300PULAS system . The relative expression of each gene was determined and normalized to the expression of 18S and calculated using the 2Protein samples from skeletal muscle section were resolved using 8 or 10% SDS-PAGE and transferred to PVDF membranes (EMD Millipore). Membranes were blocked in 5% nonfat dry milk and subsequently incubated with primary antibodies against p-PI3K/PI3K, p-AKT/Akt, p-IR/IR, p-PTEN/PTEN and IGF-1R , p-ERK/ERK1/2, and GAPDH  overnight. Horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG  were used as the secondary antibodies. PVDF membranes were developed using an Image-ProPlus system.t test. P values were adjusted for multiple comparisons using Bonferroni correction. Analyses were performed using GraphPad Prism version 7 . P < 0.05 was considered to indicate a statistically significant difference.Data are represented as the mean ± standard deviation and analyzed using a one-way ANOVA or a two-tailed unpaired Student’s To evaluate the potential capacity of HUC-MSCs and the effect of administration route, FBG, weight, and survival rates were assessed. The HUC-MSCs were administered by three clinically relevant routes: IV, IP, and IM. These different transplantation routes resulted in different FBG levels, but did not have any effect on weight in the db/db mice. Although administration of cells using IV was the best method for controlling FBG, IV administration resulted in the lower survival rate of 71.9%. HUC-MSC transplantation by IM also reduced the FBG, and the survival rate was 100%. Thus, IM transplantation was deemed to be the safest and most effective method of clinical application of HUC-MSCs.In addition, HUC-MSC transplantation did not result in injury to the liver and kidneys in mice. The levels of ALT, AST, γ-GT, Cre, and BUN in serum were measured 72 h after HUC-MSC transplantation Table . The resTo facilitate longitudinal cell tracking in vivo imaging, HUC-MSCs were infected with luciferase-lentivirus (CMV-Luciferase-PGK-Puro) to establish HUC-MSCs-luc cell stably expressing luciferase. The results showed that HUC-MSCs stably colonized in the skeletal muscle following IM transplantation. Although fluorescent signals were detected in the bladder region 1.5 h after transplantation, these signals were absent after 2 h . However, HUC-MSC implantation did not significantly alter the HOMA IR in the db/m mice . In addition, the mRNA expression levels of PTEN exhibited similar changes as the changes in the protein expression levels (Fig. To further investigate the effect of PTEN on PI3K/Akt and Erk/MAPK signaling in db/db mice with HUC-MSC transplantation, the effect of PTEN in skeletal muscle tissues was determined. The results showed that the protein expression levels of PTEN and p-PTEN were decreased in db/db+HUC-MSC mice compared with db/db mice (Fig. els Fig. c.Fig. 6P < 0.01; Fig. To determine whether HUC-MSC transplantation improved insulin sensitivity was associated with the IGF-1/IGF-1R pathway, the expression of IGF-1 and IGF-1R in skeletal muscle tissue of db/db+HUC-MSC mice was assessed. Only the expression of IGF-1 mRNA was reduced by HUC-MSC implantation compared with db/db mice (IR is a central defect in metabolic syndromes such as T2DM and serves a critical role in the progression from pre-T2DM to T2DM. Despite the development of multiple drugs to ameliorate IR, the limitations of the accompanying side effects are still notable and should not be ignored. Therefore, development of a more effective method to improve IR is required. Recently, there has been a focus on the link between HUC-MSC transplantation and IR . The preMSCs, as a source of seed cells, have garnered increasing attention in the field of stem cell therapy. At present, a large number of studies have discussed the clinical efficacy of stem cell therapy –23; howeLow-grade chronic inflammation can be found in adipose tissue, skeletal muscle, liver, pancreas and the hypothalamus, correlated with nutrient overload . HoweverIn addition, IR induced the development of obesity and T2DM, which is correlated with damage of PI3K/AKT pathway in various tissues of the body, following IR aggravates the PI3K/AKT pathway into a vicious circle . Upon inNumerous studies found that MAPK-dependent signal transduction is crucial for an array of metabolic events and inappropriate MAPK signaling is associated with the development of T2DM and metabolic syndrome . HUC-MSCAccumulating evidence has suggested that the PI3K/Akt and ERK/MAPK pathways are activated due to a loss of phosphatase and PTEN function , and PTENevertheless, IGF-1R serves a significant role in insulin signaling. IGF-1 and IGF-1R are known to maintain insulin sensitivity in different tissues . IR seemTogether, these results demonstrate that IM HUC-MSC transplantation is the safer than IV or IP administration, and IM administration resulted in 100% survival rate of mice. HUC-MSCs can stabilize localization and differentiation in skeletal muscle tissue and significantly ameliorate IR, and this was mediated by PTEN-mediated regulation of the balance between PI3K-Akt and Erk/MAPK signaling pathways. Furthermore, HUC-MSC implantation also downregulated the expression of inflammatory markers in the skeletal muscle tissue. Therefore, HUC-MSC transplantation may be a potential therapeutic direction for preventing IR and increasing insulin sensitivity.


Human umbilical cord mesenchymal stem cells (HUC-MSCs) present in the umbilical cord tissue are self-renewing and multipotent. They can renew themselves continuously and, under certain conditions, differentiate into one or more cell types constituting human tissues and organs. HUC-MSCs differentiate, among others, into osteoblasts, chondrocytes, and adipocytes and have the ability to secrete cytokines. The possibility of noninvasive harvesting and low immunogenicity of HUC-MSCs give them a unique advantage in clinical applications. In recent years, HUC-MSCs have been widely used in clinical practice, and some progress has been made in their use for therapeutic purposes.This article describes two aspects of the clinical therapeutic effects of HUC-MSCs. On the one hand, it explains the benefits and mechanisms of HUC-MSC treatment in various diseases. On the other hand, it summarizes the results of basic research on HUC-MSCs related to clinical applications. The first part of this review highlights several functions of HUC-MSCs that are critical for their therapeutic properties: differentiation into terminal cells, immune regulation, paracrine effects, anti-inflammatory effects, anti-fibrotic effects, and regulating non-coding RNA. These characteristics of HUC-MSCs are discussed in the context of diabetes and its complications, liver disease, systemic lupus erythematosus, arthritis, brain injury and cerebrovascular diseases, heart diseases, spinal cord injury, respiratory diseases, viral infections, and other diseases. The second part emphasizes the need to establish an HUC-MSC cell bank, discusses tumorigenicity of HUC-MSCs and the characteristics of different in vitro generations of these cells in the treatment of diseases, and provides technical and theoretical support for the clinical applications of HUC-MSCs.HUC-MSCs can treat a variety of diseases clinically and have achieved good therapeutic effects, and the development of HUC-MSC assistive technology has laid the foundation for its clinical application. HUC-MSCs are self-renewing and multipotent. They can continuously proliferate and differentiate under specific conditions into one or more cell types that constitute human tissues and organs. They affect immune responses and can be easily harvested, separated, cultured, expanded, and purified. HUC-MSCs retain the stemness after multiple passages and expansion. The surface antigens of HUC-MSCs are not prominent, the rejection of transplanted cells is insignificant, and the matching requirements are not strict, facilitating their use in allografts –3.At present, HUC-MSCs are used in the treatment of various diseases. They have several distinct properties essential for their therapeutic applications. (1) Differentiation: the generation of differentiated cells by HUC-MSCs promotes tissue regeneration and improves tissue function , 5. (2) Currently, HUC-MSCs are used to treat more than ten types of diseases, and major therapeutic breakthroughs have been achieved with these cells. In this review, we will summarize the progress in the application of HUC-MSCs during the last 5 years, with the objective of guiding further research and clinical applications Fig. .Fig. 1CDiabetes is a group of metabolic diseases characterized by hyperglycemia resulting from insufficient insulin secretion, impaired response to insulin, or both . ClinicaDiabetic complications, such as diabetic foot, diabetic nephropathy, diabetic wound ulcers, and diabetic retinopathy, are frequently the primary causes of disability and death. In the clinical treatment of diabetic foot, HUC-MSCs could be targeted to the ulcer and increase the formation of vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF). These factors could promote the epithelialization of ulcerated tissue by stimulating the release of cytokeratin 19 from keratinocytes and extracellular matrix formation –19. All When HUC-MSCs have been applied in the treatment of diabetic nephropathy, they exerted a therapeutic effect by reducing the expression of inflammatory cytokines, increasing the number of Sertoli cells, and upregulating the expression of their proteins and enhancing the expression of anti-apoptotic proteins in the kidney . ExperimIn summary, HUC-MSCs have the following characteristics: (1) targeting inflammatory tissues and differentiation into functional islet-like cells to block the activity of inflammasomes and exert anti-inflammatory effects; (2) targeting the ulcer tissue and promote the release of cytokines, and then promote the epithelialization of ulcerated tissue, eventually resulting in partial or complete healing of the ulcer; and (3) potential to differentiate in vitro into functional cells and being transplanted in vivo, providing a satisfactory therapeutic effect. Therefore, we believe that when HUC-MSCs are used therapeutically, they need first to target damaged or inflamed tissues, and then differentiate into differentiated cells or secrete immune regulatory factors. HUC-MSCs can also be induced in vitro to differentiate into functional cells and subsequently be used for transplantation to produce adequate therapeutic outcomes.Hepatitis, cirrhosis, and liver cancer are common liver diseases, and fibrosis is the common pathway underlying the development of multiple chronic conditions of the liver. The activation of hepatic stellate cells (HSCs) is a critical element of the etiology of hepatic fibrosis , which cWe believe that HUC-MSCs could treat liver diseases based on the following properties: (1) HUC-MSCs inhibit proliferation and promote apoptosis of HSCs, delaying or even reversing liver fibrosis and fibrosis-related liver diseases; (2) HUC-MSCs release exosomes that can reduce the expression of NLRP3 inflammasomes and decrease the level of pro-inflammatory factors, thereby achieving an anti-inflammatory effect; and (3) HUC-MSCs can reduce the level of ALT and AST, suppress the infiltration by neutrophils, decline oxidative stress and apoptosis of liver cells, and protect against oxidative and ischemia-reperfusion injury of the liver. At present, initial steps in the field of the application of HUC-MSCs for the treatment of liver diseases have been made, but in-depth research on the underlying mechanism of action remains to be performed. For example, the optimal time for transplantation, the administration method, and the effective dosage need to be determined. In addition, side effects of HUC-MSC transplantation must be considered. Resolving these issues and obtaining a better understanding of the biology of HUC-MSCs, transplantation of these cells will certainly gain a broader application in the treatment of liver diseases.Systemic lupus erythematosus (SLE) is an autoimmune inflammatory disease of the connective tissue involving multiple organs. SLE affects prevalently young women. In most patients, traditional therapies for SLE can manage the condition but are associated with a high rate of adverse reactions, such as infection, ovarian failure, malignant tumors, osteoporosis, and other diseases, seriously affecting the patient’s quality of life. The immunoregulatory function of HUC-MSCs had been widely employed for the treatment of various autoimmune diseases, particularly in cases of severe and refractory SLE that had failed to respond to pharmacologic therapy, and some beneficial effects have been obtained.The treatment of SLE by HUC-MSCs is safe and effective . The oveThe findings discussed above point to two possible mechanisms by which HUC-MSCs can treat SLE: (1) inhibition of the proliferation of T and Tfh cells, upregulation of Treg cells, maintaining the Th1/Th2 balance, and decreasing the level of TNF-α and IL-17 and (2) regulation of the expression of certain miRNAs. Several in vitro and in vivo studies have demonstrated the immunomodulatory properties of HUC-MSCs, providing basic science support for the application of these cells in clinical practice. Although the current clinical application of the research on HUC-MSCs begins to materialize and shows good prospects, the possibility of excessive immunosuppression by HUC-MSCs creates the risk for infection and tumorigenesis. The possibility of this type of adverse effects necessitates further research and in-depth discussion.Arthritis is an inflammatory disease affecting joints and surrounding tissues. Its etiology is complex and mainly related to an autoimmune reaction, infections, and trauma. Traditional treatments do not effectively solve the problem of the lack of immune tolerance mechanisms and are burdened by obvious side effects. The use of stem cells became a new therapeutic strategy for this disease. HUC-MSCs have been shown to effectively treat arthritis by differentiating into osteoblasts ; inhibitHUC-MSCs also have a chondroprotective effect, which is considered to depend on the reduction of inflammation, which delays cartilage destruction. At the same time, HUC-MSCs inhibited the expression of MMP-13, collagen type X α1 chain, and cyclooxygenase-2, and enhanced the proliferation of chondrocytes, while osteoarthritis chondrocytes promoted HUC-MSCs to differentiate into chondrocytes , 48. AddIn summary, HUC-MSCs can treat arthritis through the following mechanisms: (1) differentiation into cartilage or osteoblasts to repair the cartilage and regenerate the knee cartilage; (2) the release of soluble molecules such as cytokines, growth factors, and immunomodulatory factors to exert an immunomodulatory effect; and (3) inhibition of the proliferation of immature dendritic cells (DC) and natural killer (NK) cells, suppression of cytokine cytotoxicity, induction of macrophage differentiation from pro-inflammatory M1 phenotype to anti-inflammatory M2 phenotype, and secretion of IL-10 and nutritional factors. These properties of HUC-MSCs reduce inflammation and promote tissue repair. In addition, HUC-MSCs can inhibit the proliferation of T cells and B cells, and the immunomodulatory properties of HUC-MSCs significantly weaken the progress of osteoarthritis . TherefoThe incidence of death and disability in cerebrovascular diseases such as brain injury and stroke is high. Traditional drug therapies do not provide satisfying results, and the sequelae of the damage can be severe, indicating that an effective treatment for stroke and other diseases is not available. It has been shown that the motor and nerve function scores in patients treated by HUC-MSC transplantation were improved , implyinHUC-MSCs and their exosomes treat cerebrovascular diseases primarily by inducing an anti-inflammatory effect through the downregulation of inflammation-related genes and reduction in the level of pro-inflammatory factors while promoting the release of VEGF and neovascularization. Moreover, HUC-MSCs and HUC-MSC-ex increase the level of BDNF and glial cell-derived neurotrophic factor, which protect neurons and enhance their function. Due to the biological characteristics of HUC-MSCs and the uniqueness of cerebrovascular diseases, different transplantation pathways can affect the number and spatial distribution of HUC-MSCs that home to the brain parenchyma, thereby affecting the therapeutic effect of these cells. Therefore, the route of delivery of HUC-MSCs for the treatment of cerebrovascular diseases has become the focus of current research.Heart diseases are the major cause of mortality worldwide, with approximately 20 million people aged 30–70 years dying from the disease every year. At present, the disease tends to affect younger individuals. The available treatments include heart transplantation, surgical interventions, and pharmaceutical therapies. Surgical treatment is typically associated with complications and generally is not recommended unless the condition is severe. Even if the patients survive and the condition improves, a long-term maintenance treatment is necessary. HUC-MSCs offer a relatively safe and effective alternative therapy for heart diseases.HUC-MSCs have been shown to treat and relieve various cardiovascular diseases, including myocardial infarction, heart failure, myocardial ischemia, and myocarditis. These cells promote cardiac tissue regeneration and angiogenesis, inhibit inflammation , and sigHUC-MSCs can also upregulate the level of superoxide dismutase (SOD) and glutathione (GSH), reduce the concentration of malondialdehyde (MDA) in infarcted myocardium, and reduce oxidative stress and extracellular matrix (ECM) remodeling . HUC-MSCIn summary, HUC-MSCs perform a therapeutic function in heart-related diseases by the following mechanisms: (1) differentiation into cardiomyocytes to improve heart function, (2) differentiation into vascular endothelial cells to promote angiogenesis and blood supply, (3) improvement of cardiac performance by inhibiting myocardial cell apoptosis, (4) anti-inflammatory and anti-fibrotic activity through paracrine effects, and (5) regulating the expression levels of miRNAs, lncRNAs, and circRNAs involved in cardiac repair. HUC-MSCs have broad prospects for clinical application in the treatment of cardiac diseases.However, several issues related to the application of HUC-MSCs in cardiac therapies remain to be investigated, such as the timing, quantity, and administration mode of transplanted cells; the mobilization and homing of the cells; and the safety and long-term outcomes of cell transplantation. Therefore, in-depth studies of the specific mechanism underlying the therapeutic effects of HUC-MSCs for cardiovascular diseases are necessary to enable the future use of these cells to treat heart-related diseases and restore cardiac function.Spinal cord injury (SCI) is a cross-sectional injury of the spinal cord caused by trauma, inflammation, and other factors. SCI results in impairment or loss of motor, sensory, and other nerve functions below the site of injury. Once SCI takes place, particularly in cases of traumatic injury, the patient should be rescued as soon as possible to maintain blood volume and prevent neurogenic shock. After resuscitation, the patient should be given drug therapy to repair damaged nerve fibers and maintain spinal cord stability to prevent further nerve injury. However, the effects of pharmaceuticals are very limited, and the adverse effects of corticosteroids are significant. Given this backdrop, HUC-MSCs, with their strong proliferation, differentiation, and self-renewal potential, attracted the interest of scientists. HUC-MSCs represent a new treatment strategy for spinal cord injury, which is effective and engenders few side effects. Thus, treatment with HUC-MSCs is expected to become an alternative therapy for SCI.Clinical research showed that HUC-MSC treatment of patients with SCI could restore intestinal and bladder function and significantly improve sensation, movement, and self-care ability, as indicated by higher American Spinal Injury Association scores and daily life activity scores , 70. SevIn summary, HUC-MSCs can treat SCI cord injury mostly by two mechanisms: (1) reduction of the apoptosis of spinal cord neurons and (2) suppression of the secretion of inflammatory cytokines IL-6, IL-7, and TNF-α secretion, resulting in the inhibition of the inflammatory response at the injured site, promotion of neuronal regeneration, and reduction in the formation of glial scars. Although rapid progress has been made in the use of HUC-MSCs for the treatment of SCI, their clinical application continues to face many problems. Future efforts should focus on the standardization of various HUC-MSC technologies, identification of the mechanisms of neural repair, a better understanding of signaling molecules and conduction pathways, and comprehensive assessment of different treatment protocols.At present, there are not many instances of the use of HUC-MSCs to treat the respiratory system. Typical diseases include acute lung injury, bronchial asthma, and chronic obstructive pulmonary disease. For respiratory diseases, HUC-MSC treatment significantly elevated functional scores and increased patient survival . IntrapuHUC-MSCs have been used in the treatment of lung diseases for more than 10 years. HUC-MSCs protect lung tissue primarily through anti-inflammatory and anti-fibrotic activity, immune regulation, and paracrine mechanisms. HUC-MSCs also indirectly treat lung diseases by regulating the expression of microRNAs. In short, these cells provide an important therapeutic effect and have a wide range of applications in the treatment of respiratory diseases.A recent study reported the use of HUC-MSCs to treat the coronavirus disease-19 (COVID-19) . CurrentAntiviral therapy utilizing HUC-MSCs has provided certain therapeutic effects, but this application of HUC-MSCs only begun to be studied. Understanding involved mechanisms or their combinations, adverse reactions, usage and dosage, and treatment methods is minimal. Clinical treatments should proceed with extreme caution, and scientific research needs urgently to be developed.HUC-MSCs reduced the incidence of the graft versus host disease (GVHD) and relieved its clinical symptoms by immunomodulatory effects. Specifically, HUC-MSCs increased the number of B lymphocytes, Treg cells, and the Th1/Th2 ratio and decreased the number of NK cells in patients affected by GVHD . In autiMost of the HUC-MSCs currently used clinically are provided by stem cell banks that collect, prepare, and store stem cells on a large scale. Stem cell banks are also called “Life Banks.” The HUC-MSC cell bank is one of a wide variety of stem cell banks. At present, there are few dedicated HUC-MSC cell banks, and in most cases, HUC-MSCs are supplied by comprehensive stem cell banks. In 2004, the world’s first stem cell bank that stored HUC-MSCs was opened in the UK. The National Institute for Biological Standards and Control (NIBSC) was responsible for the operation and storage of embryos, fetuses, adult tissues, and various stem cell lines, including HUC-MSC lines. In April 2006, the first Chinese HUC-MSC cell bank was established in Tianjin, with a storage capacity of tens of thousands of copies. It has established standardized protocols of stem cell isolation, identification, culture, expansion, storage, supporting technologies, and quality control, and the cell bank developed the first domestic technical standard for HUC-MSCs and fulfilled the ISO 9001:2008 quality management system requirements . In 2011An HUC-MSC cell bank could provide high-quality stem cell “seeds” from legal sources for the clinical application, and many companies and hospitals have the ability to build HUC-MSC cell banks. However, there are no uniform quality control standards in stem cell banks, which will directly affect the outcomes of clinical treatment. This issue represents the biggest problem of stem cell banks and should be resolved as soon as possible.The tumorigenicity of transplanted HUC-MSCs, which will affect the therapeutic effect, is one of the current concerns. The experiments on tumorigenicity safety conducted by Jun-Won Yun and coworkers in mice did not detect tumors related to HUC-MSCs . Also, cThe therapeutic effects of HUC-MSCs are dependent on the number of their generations in culture, HUC-MSCs at less than 10 passages have better cardiogenic differentiation ability, while at passages 11–20, the differentiation towards nerve cells is evidently enhanced . In addiCurrently, mesenchymal stem cells are a hot research subject in the field of regenerative medicine. This article analyzes five functions of mesenchymal stem cells relevant to clinical applications and their therapeutic effects in eleven types of diseases, including liver and respiratory system disorders.Before 2006, the research of stem cells was mainly directed at the identification of biological characteristics of stem cells. In 2006, the International Association for Cell Therapy (ISCT) proposed a unified definition of mesenchymal stem cells, which became the identification standard used worldwide. Only cells that meet the following three criteria simultaneously can be classified as mesenchymal stem cells: (1) anchorage-dependent growth, (2) expression of certain specific antigens (markers) on the cell surface, and (3) ability to differentiate into adipocytes, osteoblasts, and chondrocytes. In subsequent years, great progress has been made in the use of HUC-MSCs for clinical application. An increasing number of relevant studies have been conducted, and the research methods have matured. The comparison of the therapeutic effects of HUC-MSCs with those of traditional treatments demonstrates that HUC-MSCs not only improve survival but also significantly ameliorate various clinical symptoms of the disease, markedly improving patients’ quality of life . Since HHUC-MSCs bring new hope to the future of regenerative medicine. Further in-depth research and clinical applications of mesenchymal stem cells will certainly benefit mankind in the near future. Mesenchymal stem cells have a remarkable potential to differentiate, strong proliferation capacity, and low immunogenicity. Their use is not limited by moral and ethical restrictions, and they are easy to prepare on an industrial scale. They may become pluripotent stem cells with the best prospects for broad clinical applications.Additional file 1: Table S1. Application of HUC-MSCs in clinical treatment.


Objective: We determined the reliability of The Kiddie-Schedule forAffective Disorders and Schizophrenia for School-Age Children–Present andLifetime (K-SADS-PL) for screening and diagnosing ADHD in children.Method: K-SADS-PL was administered to 2,074 children in thecommunity. Psychometric properties, factorial structure, and clinical validityof K-SADS-PL in screening or diagnosis of ADHD were examined.Results: Internal consistency was excellent for items in thescreening interview  and diagnostic supplement . Thestandardized coefficients for items in the screening interview were acceptable(0.59-0.85), while fit indices for single factorial structure reached acceptablelevels. Screening items were associated with high sensitivity  and specificity  for diagnosis ofADHD in the supplement. The test-retest and interinformant reliability asmeasured by intraclass correlation coefficient was good for most of the items.Conclusion: This large study shows that K-SADS-PL can bereliably used to screen and diagnose ADHD in children in Kenya. ADHD is one of the most common neuropsychiatric disorders in childhood that canpersist into adulthood. According to the Global Burden of Disease, ADHD wasresponsible for 500,000 disability adjusted life years (DALYS), which is 0.2% of allmental health disorders . In spitDiagnostic and Statistical Manual of Mental DisordersDSM-5  version is popular for epidemiological andclinical studies of neuropsychiatric disorders in children, as it is regarded as agold standard for We evaluated the psychometric properties, reliability, and validity of ADHD module ofK-SADS-PL in screening and diagnosing ADHD in epidemiological studies conducted inAfrica. In particular, we aimed to examine the item reliability (includingstandardized coefficients/loadings); compute fit indices for the one-dimensionK-SADS-PL; assess polychoric correlations, sensitivity/specificity/positivepredictive values (PPV)/negative predictive values (NPV) of the screen interviewswith diagnostic supplements; and determine test-retest and interraterreliability.This study was conducted on a rural area of Kenya in a demographic surveillancearea . FieldwoAll children aged 6 to 9 years living within demographic area, who form a totalpopulation of about 28,000 were eligible. This current study was nested in alarge epidemiological study which aimed at screening about 15,000 childrenrandomly selected from the 28,000 children aged 6 to 9 years within thedemographic. The RAND command of MySQL was used to randomly select theeligible children. The age group of 6 to 9 years was chosen because this is whenmost neurodevelopmental disorders such as ADHD become apparent. In this baselineepidemiological study, 11,223 children were successfully screened withneurodevelopmental disorders screening tool (NDST), and 2,162 invited forfurther clinical and neuropsychological assessments including administration ofthe K-SADS-PL. During piloting, NDST screened ADHD with a sensitivity of 89.2% and a specificity of 94.8% .DSM-5 criteria: (a) predominantly inattentive subtype; (b)predominantly hyperactive-impulsive sub-type; (c) combined type; and (d) otherspecified ADHD; all of which manifests either in the past (>6 months ago) orthe present (≤6 months to present).ADHD module of K-SADS-PL is a semistructured interview with two components: ascreening interview and diagnostic supplement . The scrhttp://www.who.int/substance_abuse/research_tools/translation/en/).A panel approach was used to harmonize the translation. A team made up of adevelopmental psychologist, epidemiologists, and professionals  fluent in English, Swahili, and familiar withthe local culture were members of this harmonization team. All the tools werepiloted to test their appropriateness in assessing neurodevelopment and adaptedaccordingly before use in the epidemiological survey. Translation, adaptation,and administration of K-SADS-PL was supervised by a developmental psychologist(A.A.); child and adolescent psychiatrist (J.P.O.); linguists fluent in English,Kiswahili, and Mijikenda; and neuroepidemiologists (S.K. and C.N.).The ADHD module of K-SADS-PL was translated into the local language, Kiswahili,through a standardized forward and back translation process according tointernational guidelines (K-SADS-PL was administered by assessors trained by a consultant child andadolescent psychiatrist (J.P.O.), who actively supervised the assessors on aweekly basis so that difficulties in rating items and making decisions ondiagnosis could be resolved. The child and adolescent psychiatrist hasreliability approval for administering and supervising administration ofK-SADS-PL from an approved K-SADS-PL trainer .Trained administrators of the tool first introduced the purpose of the K-SADS-PLto the parents and the child, including explaining the scoring requirements tomove from screening interview to diagnostic supplement. Questions were asked toboth the parent and the child, with the assessor integrating their responses.Although ADHD module of K-SADS-PL requires parent–child participation, we reliedmostly on the parent’s or caretaker’s accounts of the child’s behavior. In thescreening interview, a response was rated as either absent (coded as 1),subthreshold levels (coded as 2), or threshold levels (coded as 3). While in thediagnostic supplement, ratings were done on a scale of 0, 1, or 2, with 2representing presence of ADHD symptoms. Opposition defiant disorders, anxietydisorders, and other neuropsychiatric disorders (not reported in this study)were also considered for differential diagnosis when diagnosing ADHD.Two weeks after initial screening with ADHD module of K-SADS-PL, 29 children wererandomly selected for readministration of the screening interviews of the toolto examine the test-retest reliability. Another 20 children with and withoutADHD in the supplement ADHD module of K-SADS-PL were examined through directclinical interviews and/or recorded videos by a child and adolescentpsychiatrist who was blinded to the initial diagnosis made by trainedadministrators of K-SADS-PL.Written informed consent for the study was obtained from parents of children whoparticipated in this work.psych package of R, with their CIs estimated bybootstrapping for 1,000 iterations. Reliability measured by ω presents betterestimates than that of Cronbach’s alpha (α) when the tool or test hasasymmetrical items, violation of normality introduced by small samples, ormissing tau-equivalence (congeneric)  orR statistical software (version 3.4.0 (2017-04-21)). The internal consistency orinteritem correlation was computed as MacDonald’s Omega (ω), using thegeneric) , and so irr package in RR as intraclass correlationcoefficients, specifying consistency types for test-retests, agreement types forinterrater reliability, and one-way random effect to allow the estimates to varyacross the participants. We also examined the overlap of ADHD with oppositionaldefiant disorders and anxiety disorders, using proportions and tetrachoriccorrelations and hypothesized that overlap will be more substantial in theformer  than in the latter.Using structural equation models that assumed a linear trend for thethree-ordinal responses of items of K-SADS-PL, we generated the standardizedcoefficients or loadings associated with responses from the screening interviewand from the diagnostic supplement, while also investigating the fit indices associated with a one-dimensional factorialstructure of the ADHD module of K-SADS-PL . We ensuInitial screen with ADHD K-SADS-PL module was done on 2,074 children, which is~95% of those approached, following initial screening with NDST in Stage I. Ofthe 2,074 children aged 6 to 9 years, 1,084 (52.3%) were males. Those whoreached the threshold in the initial screening were 385/2,074 (18.5%); responsefor individual questions is shown in The item reliability coefficients for the screening stage were excellent. The items reliabilitycoefficients for the supplement stage were excellent as well . The item reliability coefficients for K-SADS-PL screening itemswere comparable for males  and females .The standardized coefficients associated with each item of the screening ADHDmodule of K-SADS-PL ranged from 0.59 to 0.85, with an overall mean of 0.75  see . Most ofFit indices for the one-dimensional structure ) and two negative correlations (ρ = −0.17 for hyperactive/impulsive typeand ρ = −0.17 for the unspecified ADHD type) . The scrReaching a threshold score of 3 points in the relevant subsection of thescreening component was associated with very high sensitivity  and specificity  for diagnosis of ADHD in thesupplement stage, with NPV being higher than PPV, as expected of diagnostictools . Asexpep < .001), except for impulsivity (p < .001), except forimpulsivity  and so were moststandardized coefficients and fit indices for a single factorial structure.Screening interview ratings correlated well with those of the diagnostic supplement,with very high sensitivities (~97%) and specificities (~95%). Additionally, most ofthe items had an acceptable test-retest and interrater reliability (up to 0.77). Asexpected, the ADHD symptoms showed little overlap with anxiety disorders (1%), andhigh overlap with oppositional defiant disorders (25%).The findings in this study are similar to studies in other cultures. For instance,similar interrater reliability (0.77 in our study vs. 0.75) and positive screen rate(15.8% in our study vs. 15.2%) in the Korean study . The proThe internal consistency is an important measure of the relatedness of the individualitems used to assess or diagnose ADHD . In ourItem scale reliability (as measured by standardized coefficients) was withinacceptable ranges for all items in the screening interview. This is important intest administration as it means the questions are capturing what they are intendedto measure. However, standardized coefficients were much lower in the diagnosticsupplement, which is less detrimental as its administration is dependent on reachinga threshold on the screening interview, whose standardized coefficients were allacceptable. In the screening interview, impulsivity had the lowest standardizedcoefficients, implying that this term is either (a) not fully understood in thisregion or (b) not a common manifestation of ADHD in this area. Our findings tendedto support the former possibility as polychoric correlations with unspecified ADHD(symptoms not fully described/understood by parents/children) were largest withscreening for impulsivity.We performed polychoric correlations to examine how well ratings for the screeninginterview domains related with those of the diagnostic supplement, and two screeningdomains showed consistent significant correlations with all ratings of thediagnostic supplement. These were “difficulty sustaining attention” and “easilydistracted” which may be the traits of ADHD that are best understood by parents inthis community. The directionality (positive or negative) of these correlationsdepends on the broad categories of ADHD in the supplement (either hyperactivity orattention-deficit) the screening item of interest is designed to assess. Forexample, “difficulty sustaining attention” assesses the ADHD category“attention-deficit” and not “hyperactivity,” and so is positively correlated withthe former category of ADHD and negatively with the latter. However, to improvesensitivity of obtaining reliable estimates of ADHD in epidemiological studies,other two screening items should also be included as they correlated with at leastone ADHD type from supplement.The sensitivities and specificities of positivity to any screening interviewquestions are very high, implying that these questions may be useful in identifyingchildren requiring clinical assessment and care particularly in LMIC, whereresources are so limited that full administration of the ADHD module of K-SADS-PLcan be logistically intensive. High sensitivities are also important in detectingADHD in epidemiological studies. Sensitivity may depend on the number of questionsin a tool or their ability to accurately describe the symptoms of a condition ofinterest such as ADHD. Low sensitivity can result in false negatives and therebymissed opportunities for management and targeted care, which can be avoided byensuring that a tool has adequate items that accurately describe the symptoms, andthat the questions are properly adapted during translation for use in the localpopulation. Nevertheless, all screening questions should be asked, as individualquestions had higher specificities as expected. The sensitivities may depend on thecorrect diagnosis of ADHD at the supplement; fortunately, interrater reliabilityagainst a child psychiatrist was good for most items. Test-retest reliabilityscreening interview questions, as measured by ICC, showed that most of the questionswere stable over time and may persist. Although low test-retest ICC may suggest thatADHD symptoms were transient, those for impulsivity warrants further work, as itsinterrater reliability was also low.This is the largest study in Africa to evaluate the utility of K-SADS-PL in screeningand diagnosing ADHD, having involved over 2,000 children. The participants wererandomly drawn from the general population as part of an epidemiological survey, andis thus far much more representative than hospital samples. In addition topsychometric properties and validity, the article additionally examines internalconsistency and the factorial structure of ADHD module of K-SADS-PL, which were notreported in most previous studies. However, the study has a small overlap withanxiety disorders, may lead to slight misclassification limitations, which could beaddressed by screening for both conditions. The Child and Adolescent Psychiatristwas not able to interact directly with many children receiving K-SADS-PL and had toreview recorded videos of these children. This is understandable since thepsychiatrist practices in the United Kingdom, and travelled severally to the studyarea to train the administrators of K-SADS-PL.We conclude that K-SADS-PL can be reliably used in rural settings in Africa to screenand diagnose ADHD in children. Neuropsychological assessors and primary health careworkers can be trained and supervised by child and adolescent psychiatrists toadminister K-SADS-PL in rural Kenya. Further work would still be needed to improvecommunity understanding of ADHD symptoms such as impulsivity.


Low birthweight (LBW) infants are at higher risk of mortality and morbidity  during their life. Due to the high incidence of (pre-) eclampsia in Haiti, LBW infants are common. We assessed the anthropometric growth (weight and length) and neurodevelopmental delay in LBW and normal birthweight (NBW) infants born at an obstetric emergency hospital in Port au Prince, Haiti, between 2014 and 2017.Infants were followed at discharge and 3, 6, 12, 15, 18, 21 and 24 months of corrected gestational age. At each visit they underwent a physical checkup . At 6, 12, 18 and 24 months they underwent a neurodevelopmental assessment using the Bayley Scale III . We modelled the trajectories between birth and 24 months of age of NBW compared to LBW infants for weight, length, and raw scores for Bayley III assessments using mixed linear models.In total 500 LBW and 210 NBW infants were recruited of which 333 (46.7%) were followed up for 24 months  and 150 died (LBW = 137 and NBW = 13). LBW and NBW babies gained a mean 15.8 g and 11.4 g per kg of weight from discharge per day respectively. The speed of weight gain decreased rapidly after 3 months in both groups. Both groups grow rapidly up to 6 months of age. LBW grew more than the NBW group during this period (22.8 cm vs. 21.1 cm). Both groups had WHZ scores <− 2 up to 15 months. At 24 months NBW babies scored significantly higher on the Bayley scales for gross motor, cognitive and receptive and expressive communication skills. There was no difference between the groups for fine motor skills.LBW babies that survive neonatal care in urban Haiti and live up to 24 months of age, perform similar to their NBW for weight, length and fine motor skills. LBW babies are delayed in gross motor, cognitive and communication skills development. Further research on the clinical significance of these findings and long term implications of this neurodevelopmental delay is needed.The online version contains supplementary material available at 10.1186/s12887-021-02605-3. Low birthweight (LBW) in neonates is defined as a birth weight of less than 2500 g by the World Health Organization . LBW claThe survival rates of LBW infants have improved with improved clinical management. However, this group of infants remains at risk of higher mortality and morbidity during the neonatal period (i.e. within 28 days after birth) and thereafter. LBW is known to be associated with subsequent health issues such as poor anthropometric growth in childhood and higher incidence of non-communicable disease in adulthood such as hypertension, stroke, diabetes, and hypercholesterolemia and with long term neurological problems  –7. SeverHaiti is the poorest country in the Americas with recent estimates that 59% of Haitians live under the national poverty line . The prePublic health structures (or organisations) able to manage long term care for infants with morbidities resulting from their prematurity or LBW are extremely limited in Haiti. Assessing neurodevelopmental outcomes in these infants may help clinical decisions to be made thoughtfully for neonatal care and ongoing support in humanitarian settings. We undertook a prospective cohort study to describe and compare the anthropometric growth (in terms of weight and length) and neurodevelopment between LBW and normal birthweight (NBW) infants born at CRUO. We hypothesized that growth and neurodevelopment outcomes would be poorer in the LBW group compared to the NBW group within 24 months after their birth.CRUO was an obstetric specialty hospital providing care for complicated pregnancies, deliveries and neonates when it was needed. It was opened in 2013 and closed in July 2018.A pediatric healthcare team  provided neonatal care at CRUO. Midwives and nurses were trained in basic neonatal life support (NLS). Neonatal care included treatment for the following morbidities: hypothermia , hypoglycemia , respiratory problems , infections (antibiotics), apneas of prematurity (caffeine), jaundice (phototherapy) and gastrointestinal problems . For well or improved LBW/premature neonates a well-functioning Kangaroo Mother Care (KMC) ward was available. Establishing breastfeeding was supported in all gestations and expressed breast milk given through a variety of means . Routine neonatal care was available to all neonates born at CRUO, including the administration of vitamin K for those with bleeding disorders and the administration of hepatitis B, polio and Bacillus Calmette–Guérin (BCG) vaccinations to all healthy/recovered neonates prior to discharge from the hospital.We conducted a prospective cohort study of infants born in CRUO hospital with participants followed up from birth and post discharge at 3, 6, 12, 15, 18, 21 and 24 months corrected for gestational age.All neonates born in the hospital between October 2014 and May 2015 were eligible for inclusion in the study. They were excluded if presenting with any major and obvious congenital malformations at birth . They were also excluded if they were abandoned by their parents or caretakers at the hospital or if their parents or caretakers did not consent to inclusion in the study.Infant recruitment was done by the study team with the parents and/or caretakers of the neonates. The study team was alerted each time a new baby was born and then explored the options for inclusion into the study. In order to maintain a feasible number of babies to be seen during follow up visits, the team included no more than 10 infants per day.Evaluation of LBW was done through measurements at birth, but also upon admission to the neonatal unit. The weight at birth was measured using a digital Seca scale. Gestational age of the neonate was measured initially in the emergency department or delivery room either by fundal height, ultrasound or by history given by mother. After birth, the gestational age was additionally confirmed using the Ballard score for those babies who were admitted to the NICU or pediatric ward. The correction for gestational age was done by subtracting the measured gestational age at birth from 40 weeks . The difference was then added to the babies’ true date of birth to estimate what the date of birth would have been at term. This corrected birthdate was used to schedule follow up visits. We defined low Apgar at birth as any neonate that scored below seven at one, five and 10 min.All infants recruited into the study had their medical charts and their mother’s medical charts reviewed prior to discharge. At discharge, 3, 6, 12, 15, 18, 21 and 24 months of corrected gestational age, the infants underwent a physical checkup . For the visits of 6, 12, 18 and 24 months they also underwent a neurodevelopmental assessment using the Bayley Scale III .During follow up visits the following medical conditions revealed by physical examination were treated by the study team: simple infections not requiring hospital admission, uncomplicated malaria, clinically suspected gastrointestinal helminthic infections or feeding difficulties . Any suspected medical condition that required inpatient paediatric, orthopaedic or surgical services, were referred to specialised care when possible . If between follow up visits study participants were sick, they could also consult the study team for free medical care or referral when possible.We conducted weight assessments using a Salter scale until the infants were able to stand by themselves. Thereafter we used Seca 725 electronic weight scales. Length measurements at birth, at discharge and at six months were done using flexible, non-stretch tape measures. All further length measurements were conducted using a wooden infant length board. Infants were placed on their back and their length was measured from the top of their head to the sole of their feet. Length measurements with a measuring tape are not standard practice for six-month-old infants, therefore we calibrated these measurements to the equivalent wooden infant board measurement using a corrective factor derived from a new set of 40 infants (20 LBW and 20 NBW) for whom we took measurements at discharge, three and six months using both the measuring tape and measuring board approach. A linear regression model was used to estimate the corrective factor.Neurodevelopment was measured at four time points  using the Bayley Scales of Infant and Toddler Development . The Bayley III is used to determine developmental delay but has never been validated for the Haitian population. The test comprises of three index scores: 1) motor skills 2) cognitive skills and 3) language. In children under two years of age, the motor scale score consists of assessment of fine motor skills  and gross motor skills . The cognitive score assesses play skills  and information processing . The language score assesses both receptive  and expressive communication skills .Data was collected by a team of pediatricians (three) and pediatric nurses (three) over a period of three years. All chart review and medical visits were documented in structured questionnaires for those visits. Data were transferred from paper format to electronic databases (password protected) which had been created in Epi Data 3.1  by a specifically trained study nurse. We also used the software Psychmotor Corp for the entry of data around the Bayley III assessments.All pediatricians and pediatric nurses were trained on the administration of the Bayley scales by a certified Bayley scale trainer. This training took place for one week prior to the start of the study and a refresher training (plus more advanced modules) were covered one year after the start of the study. In order to minimize differences in scoring between the teams of pediatricians and pediatric nurses, repeated quality control sessions were done at the start of each follow up round. One team would film the child under evaluation on all Bayley scale aspects. The other two teams would then repeat the Bayley scale assessments (blinded from the other teams) by reviewing the film footage. Discrepancies were discussed with the Study Coordinator and a pediatrician from CRUO (not part of the study team) to agree on best scoring practices. This was repeated for up to five infants at the start of each follow up visit round.https://www.openepi.com/Menu/OE_Menu.htm).As there is very limited published evidence using a similar cohort of infants in a low resource setting, establishing reliable assumptions for the calculation of a sample size to estimate growth and neurodevelopment trajectories was challenging. Similar studies carried out in high resource settings generally include thousands of babies from birth cohorts, which was not a feasible option in our setting. Without existing evidence from Haiti, we therefore based our assumptions for mortality in LBW (and sub-categories of LBW) and NBW infants on data collected at CRUO during 2013 and on studies on mortality in infants born in Nigeria, Kenya and Tanzania –20. We uIn order to be able to have a sufficient sample size to identify additional risk factors within the LBW infants discharged from CRUO we assumed that the ‘exposure’ occurred in different categories of LBW  and the outcome was mortality. During 2013 in CRUO, 1047 infants were born that were < 2.5 kg  and the remainder were normal weight (therefore a ratio of 1:1 for LBW and NBW). Out of this group of infants, 445 (42.5%) weighed between 1.75–2.5kgs, and 602 (57.5%) weighed < 1.75kgs (i.e. 1045 VBLW and ELBW combined). If we consider the first group as LBW and the second group as VLBW and ELBW, then the ratio of non-exposed to exposed would be 0.74 (around 3 non-exposed to 4 exposed). We assumed: 1) two-sided significance level of 95%; 2) power of the study is 80%; 3) ratio of unexposed/exposed is 0.75; 4) expected relative risk is 3; 5) expected proportion of outcome in unexposed group is 5%. For the outcome of mortality in sub-categories of LBW we calculated 191 infants in the exposed group (VLBW and ELBW infants) and 143 infants in the unexposed group (LBW excluding VLBW and ELBW infants) for a total of 334 infants. Assuming a default rate in the LBW group of approximately 35% (assuming 334 infants remain in the study) and 25% in the NBW group (assuming 160 remain in the study) we aimed to enroll 500 LBW infants and 210 NBW infants.We summarised the characteristics of the study population using number and percentage for categorical variables and mean and standard deviation for continuous variables. We compared differences of all sociodemographic variables for the infants between the NBW and LBW groups using Chi-square tests for categorical variables and two independent t-test for continuous variables. We calculated weight gain (in grams) per day per kilogram (kg) of last weight measurement for each infant enrolled in the study. Z-scores for weight-for-height (WHZ) were calculated using the WHO standards and described for the NBW and LBW for each visit to determine the level of malnutrition in the study population.The primary outcomes were the growth trajectories between birth and 24 months of age of NBW compared to LBW infants in terms of weight (grams), length (centimeters), and raw scores for Bayley III assessments for motor, cognitive and communication skills. For the weight, length and raw scores of the Bayley III assessments’ trajectories, we fitted a linear mixed model to estimate the length, weight and Bayley III assessment raw scores of each individual infant in the NBW and LBW group. Thus, the weight, length and Bayley III assessment raw scores at each follow up visit of the individual were the dependent variables.We modelled the trajectories by considering time since birth as a series of linear spline components . We chosWe included interaction terms between the LBW and NBW groups and the spline terms in order to reflect the shape of the curves for weight, length, cognitive and language skills, fine and gross motor skills for each group (NBW or LBW). We assumed a random effects covariance structure that allowed each child to have their own latent intercept and linear spline growth trend. The models were also adjusted for sex (supplementary information). We did not adjust the models based on the prematurity of individual babies as we know gestational age has limited precision in this setting (where it was estimated using numerous methods).p values were also calculated from the linear mixed model results in the same way.The estimated values of a child’s length, weight and raw Bayley III scores and 95% confidence intervals (95%CI) were calculated from the linear mixed regression models at each six months of age from birth to 24 months of age (depending at which age they would be recorded). Estimated values were made by birthweight group (NBW vs LBW), and sex of the infant when relevant. In some cases, the could not be calculated where data were too sparse for that particular time point. Estimated values and 95%CI of the outcome variables at specific time points  were calculated from the results of the relevant linear mixed models using linear combinations (contrasts) of the parameter estimates and associated variance-covariance-matrix. Absolute differences between outcomes at specific time points and p-values [Data cleaning was conducted using STATA13 and data analysis using R (version 3.6.2) . The ‘emp-values , 24.We enrolled 710 infants in the study between October 2014 and May 2015; 500 were LBW and 210 were normal weight. Of the 500 LBW infants that were enrolled in the study, 206 (41%) were followed up to the 24-month period. Between enrollment and the last follow-up visit, 137 (27%) died and 157 (31%) were lost to follow up (LTFU). Of the 210 NBW infants enrolled in the study, 13 died (6%) and 70 were lost to follow-up (33%) and 127 (60%) were followed up to the 24-months’ time-period , the majority  died before the neonates were discharged from the hospital during a time in which the pediatric unit was battling a hospital-acquired outbreak of sepsis caused by a multi-drug-resistant Klebsiella pneumoniae [Out of the total deaths n = 170),p < 0.001). According to the prematurity categories, 16 NBW babies (8.2%) were born with late or moderate prematurity   Table . The meaWe identified 22 infants with suspected cardiopathy (LBW = 16 and NBW = 6), 10 with macrocephaly (LBW = 9 and NBW = 1), six with microcephaly (LBW = 3 and NBW = 3), seven with a suspected chromosomal abnormality (LBW = 5 and NBW = 2) and three infants with club foot (LBW = 2 and NBW = 1). There were no significant difference between the LBW and NBW for any of these. Three infants with suspected cardiopathy  and two with a chromosomal abnormality (both LBW) died during the study.p > 0.05 for all categories).The most commonly less severe morbidities were skin-related  and flu-like illness syndrome . Other commonly identified morbidities included acute respiratory infections , anemia , gastrointestinal symptoms , suspected urogenital infection  and conjunctivitis . There were no significant differences in morbidity rates between LBW and NBW groups (The mean weight at birth for the LBW group was 1981 g (SD:460.1) and for the NBW group 3009 g SD:407.2). At 24 months the mean weight of the LBW and NBW groups was 10,912 g (SD:1438.0) and 11,464 g (SD:1388.8), respectively . The difference in mean length between the LBW and NBW decreased by the 24 month follow up period (83.4 cm vs. 85 cm)  to 36.8 (95%CI: 36.2–37.4) between 6 and 24 months of age for NBW babies and from 17.4 (95%CI: 16.9–17.9) to 35.8 (95%CI: 35.4–36.3) for the LBW babies (Table The estimated raw scores for cognitive skills from 12 to 24 months of age increase from 39.2 (95%CI: 37.9–40.5) to 56.3 (95%CI: 54.9–57.6) in NBW babies and from 38.1 (95%CI: 37.1–39.2) to 53.9 (95%CI: 53.0–54.9) in LBW babies  to 23.8 (95%CI: 23.2–24.4) in NBW babies and from 11.7 (95%CI: 11.2–12.2) to 22.4 (95%CI: 22.0–22.8) in LBW babies  was close to that observed in extremely LBW babies included in a recent study in South Africa . It is aThe Bayley III scale is a protocolized manner of measuring the neurodevelopment of our study cohort. Across all categories , NBW babies were estimated to have higher raw scores compared to LBW babies for the duration of the study period. An unexpected finding was that NBW and LBW babies were estimated to perform similarly at all age groups for fine motor skills, but larger differences existed for the estimated gross motor skills in the same age groups. We are unable to explain this observation but hypothesize that it might have to do with the sequence in which Haitian children acquire gross and motor skills (which might differ in other countries). Larger differences between NBW and LBW were estimated for cognitive and communication skills, with the largest proportional differences estimates for expressive communication skills (and NBW babies performing better in these categories). This suggests that the cognitive development and communication of LBW babies in Haitian society are delayed. This is not unexpected and has been well described in other studies .One unexpected finding from this study was that male babies had consistently higher estimates for weight, length, cognitive skills and communication skills in both the NBW and LBW groups. In contrast, most studies report that female infants consistently outperform their male counterparts in both development and mortality. Two recent studies (one in Slovenia and Croatia and one in South Africa), showed that females consistently outperformed male infants in Bayley III cognitive and communication skills , 35. AlsThis study faced numerous limitations. The unexpected high mortality in the LBW baby group due to the hospital acquired outbreak in the neonatal care unit, probably meant that a large proportion of the most vulnerable LBW babies did not survive for the remainder of the study period. The results of this study therefore cannot be extrapolated to all LBW babies, as the study group likely represents the ‘stronger’ babies that were enrolled in the cohort. Also, partially due to this high mortality, we did not achieve the original calculated study sample size. Considering the narrow confidence intervals obtained from our estimates, we do not think this impacted on the power of our study. We were unable to reliably adjust for prematurity in the models for physiological growth and neurodevelopment as gestational age measurements in Haiti were done through a variety of methods and were not standardized. This limitation is well acknowledged in similar studies in low resource settings as distinguishing between LBW, SGA and prematurity (or a combination of these) where unreliable gestational age measurements is difficult (41). There are no validated Bayley III scales for Haiti, thus we were unable to compare the scores of our study cohort to a standardized population and the comparison could only be done between the NBW and LBW groups. The sample size requirements to validate the Bayley III scales for the Haitian population were too large to be feasible to explore in the current study. We also we unable to calculate reliably the intra and inter-reliability between study teams for their neurodevelopmental assessments. Even though we minimized differences by conducting quality control checks and discussing discrepancies, we could not account for these in our final analysis. Finally, for the Bayley III scales regression models, we only had three or four follow up visit data from the study cohort. This limited the ability to introduce more knots into the regression models which might have led to the current estimates being quite crude estimates of neurodevelopment in Haitian infants. Even so, we are confident that the differences estimated between the LBW and NBW are reliable.LBW babies that survive any inpatient neonatal care in Haiti and live up to 24 months of age, will almost catch up to their NBW baby peers in terms of weight, length and fine motor skills. There does appear to be a delay in the LBW group in terms of gross motor, cognitive and communication skills development. Even though such delays are known to cause cognitive and behavioral problems in the future in other settings, the precise impact of these delays in the Haitian context would merit further research. Also, validating neurodevelopmental tools such as the Bayley scales for the Haitian pediatric population would be of added value, to better understand the clinical significance of delays observed. We will also further explore the current study data to identify specific maternal or hospital related interventions that impact positively on growth and neurodevelopment of LBW babies in order to better guide clinical management of LBW infants in low resource contexts.Additional file 1: Figure S1: Weight measurements in grams by NBW and LBW babies over 24 month follow up period. Figure S2: Weight gain (g per day per kg from previous visit) in the NBW and LBW babies over 24 month follow up period. Figure S3: Height measurements in cm for NBW and LBW babies over 24 month follow up period. Table S 1 Weight change per day (in g) from the previous visit by group (NBW vs LBW). Table S2 Estimated weight from linear mixed models and 95% confidence intervals (in grams) at birth, 6, 12, 18 and 24 months for NBW and LBW infants adjusted by age and sex. Table S3 Estimated length from linear mixed models and 95% confidence intervals (in grams) at birth, 6, 12, 18 and 24 months for NBW and LBW infants adjusted by sex. Table S4 Estimated Bayley Scales III raw scores from linear mixed models and 95% confidence intervals for gross motor skills, fine motor skills, cognitive skills, receptive communication skills and expressive communication skills in female infants. Table S5 Estimated Bayley Scales III raw scores from linear mixed models and 95% confidence intervals for gross motor skills, fine motor skills, cognitive skills, receptive communication skills and expressive communication skills in male infants. Table S6 Prediction models for outcome weight and length with predictor of age. Table S7: Prediction models for outcome weight and length with predictor of age and sex. Table S8: Prediction models for outcome weight and length with predictor of age, sex and prematurity. Table S9: Prediction models for cognitive, receptive communication and expressive communication skills with predictor of age. Table S10: Prediction models for cognitive, receptive communication and expressive communication skills with predictor of age and sex. Table S11: Prediction models for gross and fine motor skills with predictor of age. Table S12: Prediction models for gross and fine motor skills with predictor of age and sex. Table S13: Prediction models for gross and fine motor skills with predictor of age, sex and prematurity. Table S14: Reference list of the R packages used in the data analysis.